Plant Light StressSharon A Robinson, University of Wollongong, New South Wales, Australia
If plants are exposed to light intensities that exceed their photosynthetic capacity, there is
the potential for photodamage. Plants have therefore evolved a number of
photoprotective strategies that allow them to dissipate excess energy and prevent damage
to the photosynthetic apparatus.
Introduction
Light provides the energy source for photosynthesis and isessential for all plants and ultimately for animal life onplanet Earth. However, excess light can be detrimental toplants, damaging the photosynthetic apparatus and, inextreme cases, causing photooxidation of chlorophyll. Thechallenge facing photosynthetic organisms is therefore tooptimize light interception for photosynthesis whileminimizing the potential for damage. Plants have evolveda number of strategies to tailor light absorption to thecapacity for its utilization in photosynthesis. These rangein scale from the whole plant to the molecular level.
The energy to drive photosynthesis comes from thevisible spectrum (400–700 nm) and excess visible light willbe the focus in this article. However, solar radiation alsocontains ultraviolet (UV) radiation, which is absorbed byplants and can be very damaging, especially at themolecular level (for review, see Jansen et al., 1998).Terrestrial plants have evolved strategies to protectthemselves from UV radiation; and many of the mechan-isms described below, which reduce visible light intercep-tion, are equally protective against UV light.
The light levels to which plants are exposed varyconsiderably, for example there are spatial differencesdue to latitude, elevation, varying levels of air pollutionand shading by overhanging vegetation, as well astemporal changes at the diurnal and seasonal level.
What is Excess Light?
Excess light is determined as light energy that exceeds theplant’s requirements for photochemistry and which mustbe dissipated safely to avoid damage. The absolutequantity of excess light depends on the photosyntheticcapacity of the plant. Plants adapted to growth at high lightlevels will have high photosynthetic capacities, and thresh-olds for excess light in these plants will be greater than inthose adapted to low light with correspondingly lowphotosynthetic capacities (Figure 1). It is worth noting thatin most C3 plants photosynthesis is saturated at approxi-mately 25% of full sunlight, suggesting that even undernormal conditions plants are forced to deal with excess
photons. Plants are usually able to cope with normal,diurnal fluctuations in light levels and can adapt toseasonal changes over time. Sudden increases in lightpresent the greatest challenge to plants, for example thelow-light to high-light transition that occurs when a treefall gap is created in a rainforest.Often plants experience excess light because an addi-
tional environmental or biotic stress reduces their photo-synthetic rate and therefore the threshold for excess light isreduced (Figure1).Drought conditions or high temperaturestress can result in stomatal closure,which limits the supplyof carbondioxide and therefore reduces the photosyntheticrate. Cold temperature stress reduces photosynthesis bylowering enzymatic rates within the Calvin cycle. Lowlevels of nutrients and infection by viruses can also reducephotosynthetic potential. Whenever the quantity of lightabsorbed by the plant is surplus to that which is requiredfor photosynthesis, the plant must engage protectivestrategies to reduce the potential for photodamage. Thesestrategies (Table 1) can be divided into those that operate toreduce light absorption by the leaf (external photoprotec-tive mechanisms) and those that act within the leaf toprevent absorbed light causing damage within the chlor-oplast (internal photoprotective mechanisms).
Protection against Excess Light
External photoprotective mechanisms
The first line of defence against excess light is to reduce theabsorptionof photonsuntil theymatch the requirement forphotosynthesis. This type of mechanism is employed byplants growing in continuous high-light environments.Plants growing in full sun (sun-plants) typically have leaveswith reduced surface area (small, narrow leaves). Lightinterception can also be reduced by vertical orientation ofthe leaf, as seen in many Eucalyptus species. Anotherstrategy is increased leaf reflectance due to surfacewaxes orhairs. In the succulent plantCotyledon orbiculata cuticularwax reduces absorption by up to 50% (Robinson et al.,1993). These mechanisms reduce the absorption of lightacross the spectrum, thus protecting against excess visibleand UV light. Reduced leaf area and increased reflectance
also reduce heat load and water loss by plants and aretherefore common features of xeric plants. Although thesefeatures are very effective at reducing light interception,they are usually determined during leaf development andare therefore not reversible. This permanence means theyare only suitable for constant high-irradiance environ-ments.
For plants exposed to fluctuating light levels a morerapid and reversible protective mechanism is necessary.Plants that grow under dense forest canopies are normallyexposed to very low light and their leaves are adapted tomaximize light absorption. However, they may alsoexperience the sudden increase in light associated withsunflecks (bright sunlight breaking through gaps in thecanopy). Several understorey plants move their leaves toavoid these intense periods of light. Leaves of Oxalis,Siratro and Omalanthus have been shown to fold fromhorizontal to vertical, within minutes, in response to asudden increase in light intensity. Once the sunfleck haspassed the leaves slowly return to the horizontal position(Watling et al., 1997, Ludlow and Bjorkman, 1984).
Internal photoprotective mechanisms
Once light enters the leaf it can be absorbed by thephotosynthetic pigments in the thylakoid membranes ofthe chloroplast. Higher plants contain two types ofphotosynthetic pigments (chlorophylls a and b) and arange of carotenoid pigments (xanthophylls, carotenes,etc.). Light energy absorbed by carotenoids is passed ontochlorophyll molecules.Once light energy reaches reaction centre chlorophylls it
is utilized by one of three competitive processes that can bedifferentiated by means of chlorophyll fluorescence,namely:
. assimilatory and nonassimilatory photochemistry(photochemical quenching, qP);
. dissipation as heat (nonphotochemical quenching, qNor NPQ); and
. chlorophyll fluorescence.
The amount of energy used for assimilatory versusnonassimilatory photochemistry varies depending on theavailability of light and carbon dioxide. The photorespira-
AssimilatoryOxygenase
photorespirationCarboxylase reaction
NonassimilatoryPhotochemistry
Mehlerreaction
Heat dissipation – xanthophyll cycle
Photochemistry
Photoprotection
Photodamage
Sun-plant in full sun
Sun-plant experiencingdrought stress
Shade-plant inunderstorey
Shade-plant exposed tosunfleck
Shade-plant exposed tofull sun in newly formedtree-fall gap
Formation ofdissipative
PS II centresPhotodamage
0 600 1200 1800 2400
Available light (µmol photons m–2 s–1)
Figure 1 Schematic representation of the utilization and dissipation of available light energy by photochemical or photoprotective mechanisms in sun-and shade-plants exposed to various conditions. Photodamage occurs only when these photochemical and dissipative processes are exhausted. Therelative magnitude of each process is illustrated for a sun-plant growing in full sun, the same plant exposed to drought stress, a shade-plant growing in lowlight in the understorey of a rainforest, a shade-plant exposed to a transient sunfleck and a shade-plant exposed to full sun as a result of a tree-fall gap.
Plant Light Stress
2
tory reactions allow electron transport to proceed andphotons to be utilized under conditions where carbondioxide levels limit assimilatory photochemistry. In plantsexposed to water stress, for example, the Rubiscooxygenase reaction becomes increasingly important asthe severity of the water stress increases (Figure 1b). It hasbeen suggested (Osmond and Grace, 1995; Grace andLogan, 1996) that these nonassimilatory photochemicalreactions could account for a large proportion of the excessphotons absorbed by plants.
Dissipation as heat – the xanthophyll cycle
When ambient light is not excessive, the bulk of lightenergy will be used for photochemistry. However, oncelight levels approach the photochemical capacity, a greaterproportion of the incoming energy will be dissipated asheat. A special group of carotenoids, the xanthophyll cyclepigments (violaxanthin, antheraxanthin and zeaxanthin),are important in this process.Under excess light conditionsviolaxanthin (V) is de-epoxidated to antheraxanthin (A)and zeaxanthin (Z) (Figure 2). The de-epoxidation reaction
is favoured by low pH and the presence of ascorbate(conditions that are likely under excess light conditions),while the epoxidation reaction is more likely under lowlight (higher pH optimum). The presence of this xantho-phyll cycle within the thylakoid membrane, and itssensitivity to prevailing light conditions, means that itcan provide a rapid and effective response to excess lightconditions.High levels of zeaxanthin in leaves correlate well with
increased levels of heat dissipation (measured as non-photochemical quenching) and the ability of plants towithstand excess light conditions. In high-light plants thexanthophyll cycle pigmentsmake up a larger proportion ofthe carotenoid pool than in shade-plants. In addition,under excess light conditionsmost of the xanthophyll cyclepool is converted to zeaxanthin (Bjorkman and Demmig-Adams, 1994). The exact mechanism by which zeaxanthindissipates energy is disputed but there is widespreadacceptance that this molecule is involved in quenchingexcess energy within the thylakoid membrane, eitherdirectly or indirectly. The direct quenching theory (Owens,1994) relies on the fact that the energy levels of the first
Table 1 Mechanisms by which photosynthetic organisms achieve photoprotection and the photodamage events which occuronce such photoprotection is exceeded
Photoprotective strategy Examples
External photoprotection(scattering and reflection of radiation)
Internal photoprotection(management of absorbed light)
Assimilatory electron transport (photosynthetic carbon reduction)b
Nonassimilatory electron transport (photorespiration, Mehler reaction)e
Increases in xanthophyll cycle pigmentsd,f
Antioxidants (scavenge reactive O2 species)g,h
Formation of photoinactivated PS II centresi
Photodamage Chlorophyll bleachingj,k
Cell deathj,k
Regeneration potentiall
a Robinson et al. (1993).b Bjorkman and Demmig-Adams (1994).c Ludlow and Bjorkman (1984).d Watling et al. (1997).e Osmond and Grace (1995).f Demmig-Adams et al. (1996).g Grace and Logan (1996).h Jansen et al. (1998).i Anderson et al. (1998).j Jones et al. (1998).k Lovelock et al. (1994).l Ball et al. (1991).
Plant Light Stress
3
excited (S1) states of the three xanthophyll molecules aredifferent. In violaxanthin the S1 state is higher than that ofchlorophyll, suggesting that V probably acts as anaccessory carotenoid, passing energy to chlorophyllmolecules and therefore promoting photochemistry. How-ever, in zeaxanthin the S1 state is reduced such that it canaccept energy from the chlorophyll S1 state, allowing Z toact as a quencher of energy. The energy passed fromchlorophyll to Z is then lost as heat (see Demmig-Adamset al., 1996). The indirect method (for review see Hortonet al., 1996) proposes that heat dissipationoccurs as a resultof protonation and subsequent aggregation of the light-harvesting complex II (LH-II) polypeptides, which allowthese antennae complexes to become quenching LH-IIs. Inthis model Z promotes aggregation and formation ofquenching LH-IIs rather than acting as a direct quencher.The formation of such quenching LH-II complexes is alsosupported by recent time-resolved fluorescence studies(Gilmore, 1997).
Zeaxanthin-dependent heat dissipation depends on theactual presence of protons within the thylakoid membranein addition to the requirement of low pH for the formationof Z from V. This means that heat dissipation occurs onlywhen Z is present and when there is a build-up of protonssignalling an inability of the electron transport chain todeal with the light energy available. The additional protonrequirement means that nonphotochemical quenching canrespond quickly to changes in light and can be switched offrapidly as light levels decline, e.g. after a sunfleck. Ifnonphotochemical quenching were linked solely to Zpresence, then the relatively slow epoxidation of Z to Vwould determine the responsiveness (Watling et al., 1997).
This reliance of zeaxanthin-dependent nonphotochem-ical quenching on protons also highlights the importanceof nonassimilatory electron transport in maintainingthylakoid membrane energization and therefore promot-ing xanthophyll-mediated photoprotection under condi-tions when assimilatory photochemistry is reduced(Osmond and Grace, 1995).Studies by Grace and Logan (1996) suggest that in some
plants these photoprotective mechanisms are not sufficientto utilize all excess energy and photoreduction ofmolecular oxygen can still occur. Plants grown underhigh-light conditions showed high levels of antioxidantssuch as ascorbate, glutathione and a-tocopherol, whichwould scavenge reactive oxygen species. In addition thereis also an increase in the activities of the enzyme systemsthat maintain these antioxidants in the reduced state.
Photoinhibition and Photodamage
When plants dissipate energy by xanthophyll-mediatedphotoprotection there is a reduction in the quantumefficiency of photosynthesis since a smaller proportion ofphotons are used for photochemistry. Under most condi-tions the photosynthetic maximum (Pm) remains high andthe quantum efficiency increases rapidly once light levelsare reduced. These rapidly reversible photoprotectiveprocesses are part of the plant’s normal photosyntheticresponse. In severe cases where exposure to excess lightenergy is prolonged or combined with additional stresses,photoinhibition may occur. Under these conditions bothquantum efficiency and Pm are reduced and the down-
OHO
OOH
Violaxanthin
Antheraxanthin
OHO
OHDe-epoxidationFavoured under excessive light
pH optimum 5.1(requires ascorbate, NADPH)
Fast reaction(occurs within minutes)
EpoxidationFavoured under limiting light
pH optimum 7.5(requires O2, NADPH)
Slow reaction(occurs within hoursbut can be delayedby additional stress)
ZeaxanthinHO
OH
Figure 2 The xanthophyll cycle present in the thylakoid membranes of higher plants consists of three carotenoid pigments, the diepoxide violaxanthin,the monoepoxide antheraxanthin and the de-epoxidated form zeaxanthin. The enzymatic de-epoxidation is a fast reaction and is favoured underconditions of excess light. The reverse reaction is much slower and occurs when leaves are returned to normal light levels.
Plant Light Stress
4
regulation of the photosynthetic apparatus is sustainedeven after return to low light levels. This chronicphotoinhibition (Osmond and Grace, 1995) involvesgeneration of nonfunctional photosystem II (PS II)reaction centres, which accumulate in the stacked mem-brane regions and promote heat dissipation. It appearsthat the high oxidizing potentials that are generated withinthe PS II reaction centres during the light reactions areinherently damaging, with the D1 core protein beingparticularly susceptible to degradation (Anderson et al.,1998). D1 protein turnover occurs as part of the normalphotosynthetic process, but if the rate of synthesis of newD1 protein is lower than the rate of protein degradationthen photoinactivated PS II centres will accumulate.Recovery of photosynthesis from chronic photoinhibitionwill occur once plants are returned to normal irradiance,provided that conditions are suitable for protein synthesis.
Photodamage (photooxidation of chlorophyll and celldeath) only occurs when plants are exposed to extreme orprolonged stress and is relatively rare in nature. Undermost circumstances the photoprotective processes de-scribed above are sufficient to cope with a range ofenvironmental stresses. Photodamage tends to occur whenplants are exposed to extreme environmental stress such aswhen the leaves of understorey shade-adapted plants aresuddenly exposed to prolonged sunlight as a result of a treefall gap, or when excess light is combined with extremes oftemperature (Ball et al., 1991; Lovelock et al., 1994; Joneset al., 1998).
Summary
Photoprotection describes a suite ofmechanisms that forman integral part of the plant’s photosynthetic strategy.Under most environmental conditions these processesallow plants to copewith a range of light levels and preventexcess photons from causing damage to the photosyntheticapparatus.
References
Anderson JM, Park Y-I and Chow WS (1998) Unifying model for the
photoinactivation of Photosystem II in vivo under steady-state
photosynthesis. Photosynthesis Research 56: 1–13.
Ball MC, Hodges VS and Laughlin GP (1991) Cold-induced photo-
inhibition limits regeneration of snow gum at tree-line. Functional
Ecology 5: 663–668.
Bjorkman O and Demmig-Adams B (1994) Regulation of photosyn-
thetic light energy capture, conversion and dissipation in leaves of
higher plants. In: Schulze ED and Caldwell MM (eds) Ecophysiology
of Photosynthesis. Ecological Studies, vol. 100, pp. 17–47. Berlin:
Springer-Verlag.
Demmig-Adams B, Gilmore AM and Adams WW III (1996) In vivo
functions of carotenoids in higher plants.FASEBJournal 10: 403–412.
GilmoreAM (1997)Mechanistic aspects of xanthophyll cycle dependent
photoprotection in higher plant chloroplasts and leaves. Physiologia
Plantarum 99: 197–209.
Grace SC and Logan BA (1996) Acclimation of foliar antioxidant
systems to growth irradiance in three broad-leaved evergreen species.
Plant Physiology 112: 1631–1640.
Horton P, Ruban AV and Walters RG (1996) Regulation of light
harvesting in green plants. Annual Review of Plant Physiology and
Long SP, Humphries S and Falkowski PG (1994) Photoinhibition of
photosynthesis in nature. Annual Review of Plant Physiology 45: 633–
662.
Niyogi KK (1999) Photoprotection revisited: genetic and molecular
approaches. Annual Review of Plant Physiology and Plant Molecular
Biology 50: 333–359.
Plant Light Stress
5
reviews
The role of xanthophyll cycle carotenoids in the protection of photosynthesis o,o. o
William W° Adams, Hm
The use of solar energy in photosynthesis depends on the ability to safely dissipate excess energy. The key dissipation process employed by plants in their natural en- vironment is mediated by a particular group of carotenoids. Multiple levels of control allow adjustments in energy dissipation activity in response to changing levels of light stress in the natural environment. Recent advances in the understanding of the photophysics, biochemical regulation and ecophysiology of this essential photo- protective process are reviewed.
A lthough global food chains depend on the use of solar energy by photosynthetic organisms, the absorption of sunlight in excess of what can be used for photo-
synthesis poses a serious threat. Excess energy can result in photo-oxidative damage to the photosynthetic apparatus as well as to a range of other essential cell components. Recent research has made rapid progress in the identification and characterization of a ubiquitous process that safely dissi- pates the potentially destructive excess energy.
Research development over the past decade Early research identified a dissipation process that was
proposed to protect the photosynthetic apparatus (reviewed in Ref. 1). This process could be induced by lowering the pH within the photosynthetic membrane, a condition triggered by excess light. Since the process was characterized in- directly from changes in chlorophyll fluorescence emission, it was termed high energy-dependent or pH-dependent fluorescence quenching.
HO / , ~ Low l / r-xcess light ] / light Antheraxanthin /
k u OHpoxidation De-epoxidation ~ % ~ ~ ~
HO / Low "~ ( Excess . light )
HO
Fig. 1. Scheme of the xanthophyll cycle and its regulation by excess or limiting light. Stepwise removal (de-epoxidation) of two oxygen functions (the epoxy groups) in violaxanthin results in a lengthening of the conjugated system of double bonds from nine in violaxanthin to ten in antheraxanthin to 11 in zeaxanthin. De-epoxi- dation occurs within minutes. Epoxidation occurs within minutes to hours, but can take days under additional stresses.
A connection was subsequently made between energy dissipation and the carotenoids of the xanthophyll cycle (Fig. 1)2, 3. Under excess light, violaxanthin is converted rapidly via the intermediate antheraxanthin to zeaxanthin, and this reaction is reversed under low light levels (reviewed in Refs 4-7). This reversible sequence of two in- dependent reactions is termed the xanthophyll cycle and is present throughout the plant kingdomS, 8. The carotenoids participating in this cycle are the only carotenoids present in the photosynthetic membrane that undergo very rapid, light-triggered concentration changes. Although the bril- liant work of Yamamoto 4 and HageP had characterized how the biochemistry of the xanthophy]l cycle is regulated by excess or limiting light (see below), the functional role of the cycle had remained elusive until recently.
Numerous correlations were shown subsequently be- tween increases in the level of zeaxanthin and increases in energy dissipation as estimated from chlorophyll fluor- escence quenching2, 9 (reviewed in Refs 3 and 10), leading to
the proposal that zeaxanthin mediates the harmless dissipation of excess energy as heat (Fig. 2). Zeaxanthin was proposed to catalyze the removal of the same energized form of chlorophyll (excited singlet chlorophyll) that gives rise to chlorophyll fluorescence, which is the same form of chlorophyll that is used for photosynthesis (Fig. 2) 3 . The xanthophyll cycle seemed to be ideally suited to interfere at this sensitive step, allowing efficient use of energy under low light followed by a rapid switch to dissipation under excess light.
Resistance to this proposal of a causal link between zeaxanthin and energy dis- sipation arose from the lack of a mecha- nism allowing a simple energy transfer from the light-harnessing chlorophyll to zeaxanthin. Contemporary theory 11 assumed that a transfer of energy fi"om excited singlet chlorophyll to zeaxan- thin was thermodynamically imposs- ible. Subsequently, the question of an
involvement of the xanthophyll cycle in energy dissipation, as well as the photophysics of these carotenoids, received intense interest resulting in new insights into the mecha- nism, regulation, ubiquity and environmental response of this essential energy dissipation process.
Advances over the past eight years include the confir- marion of a causal link between the combined presence of zeaxanthin (and antheraxanthin) and a low pH within the photosynthetic membrane on one hand and photoprotective energy dissipation on the other. These fndings revealed that high energy-dependent/pH-dependent and xanthophyll cycle-dependent dissipation are one and the same process in vivo (Figs 2 and 3)7,~0,12-15. Recently, strong evidence has been provided demonstrating the essential involvement of the xanthophyll cycle in energy dissipation in the chloro- plast under physiological conditions 16-~8. In addition, new developments in the photophysics of carotenoids have revealed that a simple and direct downhill energy transfer from excited singlet chlorophyll to zeaxanthin is thermo- dynamically possible (Fig. 4a)~5,19. Consequently, this area of research is attracting very strong and expanding interest.
Role of the xanthophyll cycle in energy dissipation Much recent debate has centered on whether energy
dissipation can be induced by low pH alone or whether the presence of zeaxanthin (and antheraxanthin 9) is obligatory for dissipation to occur6,12-15, 20. It has been speculated recently 7 that these differing views might be explained by the devel- opment of alternative forms of dissipation when xanthophyll cycle-dependent dissipation is prevented. A series of impor- tant experiments has now clearly established that under physiological conditions, the key energy dissipation process is obligatorily and stoichiometrically dependent on the pres- ence of zeaxanthin and antheraxanthin9,16-1s, 2~. For example, energy dissipation was shown to be inducible in darkness by protonation of zeaxanthin-containing chloroplast membranes but not in zeaxanthin-free chloroplasts ~6. In addition, an el- egant experiment where zeaxanthin was allowed to be re- converted to violaxanthin under excess light led to a loss of energy dissipation activity in spite of the continued pres- ence of a low pH ~7. Furthermore, a survey of energy dissi- pation in leaves of a wide variety of plant species in the field concluded that excess absorbed light is invariably dissipated via xanthophyll cycle-dependent energy dissipation 22. It is thus concluded that zeaxanthin and pH act synergistically and are both required for energy dissipation in vivo.
Site of energy dissipation within the photochemical apparatus
Proposed energy-dissipation sites within the photochemi- cal system initially included the photochemical reaction centers as well as the light-collecting, chlorophyll- and carotenoid-binding antenna complexes of photosystem II (discussed recently in Refs 12 and 15). The antenna system is increasingly thought to be the key site of energy dissi- pation under physiological conditions12,14,15, 20. Dissipation of excess energy in the antennae would thus protect the photochemical reaction centers from overexcitation. The antennae are layered around the photosystem II centers with chlorophyll a-binding core antennae being most closely associated with the centers, followed by the inner, minor components and the outer, major components of the chloro- phyll a- and b-binding, light-harvesting antennae (see Refs 7, 15 and 20). Whereas the major, peripheral complexes bind
/a/o ~ ~ - Chl + photosynthesis
(b)
~Chl
H+/Zeaxanthin
L_~ Chl + heat
+ photosynthesis
Fig. 2. Depiction of conditions where (a) all or (b) only part of the sunlight absorbed by chlorophyll (Chl) within a leaf can be used for photosynthesis. In the latter case, the remainder of the absorbed light is dissipated safely in a photo- protective process that depends on the presence of zea- xanthin as well as a low pH within the photosynthetic mere- brane. The same energized form of chlorophyll (i.e. excited singlet chlorophyll, 1Chl*), is either used for photosynthesis or loses its energy in the form of heat.
the majority of the chlorophyll molecules, the minor, proxi- mal antennae bind only a small fraction of the total chloro- phyll but are enriched with the xanthophy]l cycle compo- nents 7 - these particular components of the antenna system of photosystem II, the minor, proximal antennae, are emerging as key sites of dissipationT,12,2o, 23. This view is also supported by several recent studies ~4-27 of the response of photosynthetic systems in which the levels of these various antenna proteins are altered (i.e. chlorophyll b-deficient mutants and plants grown under intermittent light). H~rtel and Lokstein 25 reported recently that the lack of the minor, proximal antennae in plants grown under intermittent light inhibits a key component of energy dissipation.
It has also been shown that plants deficient in the major, peripheral light-harvesting complexes exhibited a drastically slowed reconversion of antheraxanthin and zeaxanthin to violaxanthin subsequent to high light exposures 24. Based on such observations, the proposal was made that the major, peripheral light-harvesting complex may actually carry the epoxidase function 2s. However, several alternative possi- bilities for a maintenance of high levels of antheraxanthin and zeaxanthin subsequent to light stress have also been proposed (cf. 'combinations of environmental stresses').
Regulation of energy dissipation in the antennae by the proton gradient
The pH within the photosynthetic membrane exerts a dual function in regulating energy dissipation (Fig. 3). First,
2 2 January 1996, Vol. 1, No. 1
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pH-decreases in the lumen of the membrane induce the bio- chemical conversion of violaxanthin to antheraxanthin and zeaxanthin under excess light, as was so elegantly charac- terized more than two decades ago by Yamamoto 4 and Hager s. In addition, a low pH within certain membrane domains 21 (see below) is necessary to facilitate the xantho- phyl] cycle-dependent energy dissipation process itself ~2.
the proposal of a novel model t9 (see also Ref. 15) in which the xanthophyll cycle conversions constitute a 'molecular gear shift' from violaxanthin (with nine conjugated carbon-car- bon double bonds) to antheraxanthin and zeaxanthin (with ten and 11 carbon-carbon double bonds, respectively; see Fig. 1), which could potentially accept energy from chloro- phyll and then dissipate this energy as heat (Fig. 4a).
Involvement of structural changes in antenna proteins The protonation of proteins of the antenna system has been
proposed to cause structural changes of these proteins necess- ary to induce energy dissipation (Fig. 3). Initially, this propo- sal involved the peripheral or bulk light-harvesting complexes 14. More recently, protonation of the minor, proximal antenna proteins has been proposed to be a key step in the induction of energy dissipationT,12,~4,15,20, 23. The role of these protonation- induced structural changes may be to bring chlorophyll and zeaxanthin (or antheraxanthin) molecules into the close proximity required for an efficient energy transfer between these two molecules (Fig. 4a)~2,~8, ~9. Alternatively, zeaxanthin (and antheraxanthin) may induce structural changes necess- ary for a protonation-induced loss of energy directly from chlorophyll in the form of heat (Fig. 4b) 6,7,12-15,2°.
New insights into the photophysics of the xanthophyll cycle carotenoids
Major advances have been made recently in the photo- physics of carotenoids, opening up the possibility of a simple downhill transfer of energy from excited singlet chlorophyll to zeaxanthin (Fig. 4a) 12,~,~9. In essence, zeaxanthin could act much like a lightning rod, receiving the excess energy from chlorophyll and then dissipating it harmlessly. Carotenoids are known to be able to lose excitation energy relatively rapidly in the form of heat. In contrast, chloro- phyll has a high probability of passing energy on to the ever- present oxygen (0 2 ) leading to the potential for photo-oxidative damage. Energy transfer to zeaxanthin would thus prevent this dangerous transfer of energy to 0 2. However, direct experi- mental evidence for this attractive possi- bility is needed in order to identify the molecular mechanism of xantho- phyll cycle-dependent energy dissi- pation in vivo.
The important work of Frank ~9 and Owens 1~ has shown that the properties of most higher plant carotenoids are distinctly different from those of lower photosynthetic organisms. In bacteria and certain algal groups, carotenoids that are structurally different from those of higher plants have been shown to act as accessory light-harvesting pigments that pass excitation energy down to chlorophylls ~1 (see also Refs 12 and 15). In contrast, most higher plant carotenoids (those with ten" or more conjugated carbon-carbon double bonds) have now been shown to possess a pre- viously poorly characterized, low-lying energy level that can instead accept energy from the excited sing]et state of chlorophyll. This analysis has led to
Environmental responses Diumal responses to changes in the light environment
In nature; changes in the level of excess light, pH, and the levels of antheraxanthin and zeaxanthin often occur in parallel, such as in sun-exposed sites where diurnal increases and decreases in excess light over hours are closely tracked by increases and decreases in the levels of antheraxanthin and zeaxanthin in leaves as well as their levels of energy dissipation (cf. Fig. 6) 1°,12,29,3°. In contrast, extremely rapid and pronounced fluctuations in excess light can be experi- enced by leaves in the understory of a forest during a series of sunflecks 30. Such fluctuations on a timescale of seconds are matched by equally rapid changes in the level of energy dissipation. Under these conditions, high levels of anthera- xanthin and zeaxanthin are maintained subsequent to the first sunfleck. In this case, rapid fluctuations in pH are likely to modulate energy dissipation against a background of continuously high levels of antheraxanthin and zeaxanthin.
The biochemical conversions among the xanthophyll cycle components as well as the control of their engagement in energy dissipation via pH provide an elegant control mechanism for regulating a plant's energy balance in re- sponse to how much excess light is absorbed. This regulation insures that, when light is low and limiting to photosynthesis, no competing dissipation of energy occurs. On the other hand, it allows a rapid switch to effective energy dissipation whenever excess light is absorbed.
H + A n t ' a n n ~ Ascorbate
Fig. 3. Depiction of the regulation of the biochemistry of the xanthophyll cycle as well as the induction of xanthophyll cycle-dependent energy dissipation by pH. The de-epoxidation to antheraxanthin (A) and zeaxanthin (Z) (from violaxanthin, V) requires a low pH in the lumen of the thylakoid, as well as reduced ascorbate. In addition, a low pH of certain domains within the membrane, together with the pres- ence of zeaxanthin or antheraxanthin, is required to induce the actual energy dis- sipation. This dissipation takes place within the light-collecting antenna complexes, particularly the minor, proximal antennae.
January 1996, Vol. 1, No. 1 2 3
reviews
(a)
15000
10000 - -
5000 - -
[ O - - E
- - S 1
S 0
$1
~.----~ Heat
S o o
(b) IJJ
15000
10000 - -
5000 - -
0 - -
- - S 1
S 0
Chl
H +
~ S 1
Heat
, S O
Chl Chl
Fig. 4. The two possible principal paths for the conversion of excitation energy to heat during xanthophyll cycle-depen- dent energy dissipation. (a) Direct singlet-singlet energy transfer from the excited singlet state (S 1) of chlorophyll (Chl) to that of zeaxanthin (Z), followed by loss of excitation energy from zeaxanthin as heat. (b) Internal conversion within chlorophyll from the S I state to the ground state (S 0) resulting in the loss of excitation energy from chlorophyll as heat. This latter process in (b) would be induced by the pres- ence of zeaxanthin, and under low pH, and could involve formation of chlorophyll-chlorophyll dimers, introducing additional energy levels and thus allowing thermal de-excit- ation (cf. Ref. 20).
Z A 1% 6 % ~
Shade Sun
29°/~
26%
Summer Winter
9%
High N Low N
Fig. 5. Differences in zeaxanthin (Z) and antheraxanthin (A) contents of leaves as a result of the acclimation Of leaves to greater levels of light stress. Greater levels of light stress are experienced by sun-grown (sun) versus shade-grown (shade) periwinkle (Vinca minor) leaves 22,32, by Douglas fir (Pseudotsuga menziesii) needles during a cold winter versus a warm summer season 37, or by spinach (Spinacia oleracea) leaves grown with ample (high N) versus limiting (low N) supply of nitrogen (N) in the soiP 2. The total areas of the pie charts reflect the concentrations of the xanthophyll cycle components violaxanthin (V), antheraxanthin and zeaxan- thin, relative to chlorophyll. The size of the largest pie chart corresponds to 241mmol V + A + Z per mmol chlorophyll in Douglas fir needles in the winter. Data from Refs 22, 32 and 37.
Acclimation to sun and shade Compared to shade-grown leaves, sun-acclimated leaves
possess not only a higher capacity for the use of light in photosynthesis but also for rapid increases in xanthophyll cycle-dependent energy dissipationlO,31,32. Sun-grown leaves typically exhibit a larger total pool size of the xanthophyll cycle components33, 34 as well as a greater ability to convert this pool to antheraxanthin and zeaxanthin rapidly under high light (Fig. 5)31, 32 .
Combinations of environmental stresses Al l additional environmental stresses that lower a plant's
photosynthetic rate, such as water stress, nutrient stress, or temperature stress, increase the degree to which absorbed
light can be excessive, increasing the need for energy dissi- pation 34. A wide variety of environmental stresses has now been shown to induce increases in the levels of xantho- phyll cycle-dependent energy dissipation12,3o, 34. As is the case for shade-sun acclimation, increases in the ratio of the total pool of the xanthophyll cycle carotenoids to chlorophyll, as well as the maximal degree of conversion to zeaxanthin and antheraxanthin, are also observed under combinations of stress factors (Fig. 5). In addition to these adjustments in the levels of zeaxanthin and antheraxanthin, changes in how the necessary pH gradient is generated under stress conditions have also been reported recently 35. Under low- temperature stress, a larger pH gradient can be generated by a combination of changes in the proton permeability of
2 4 January 1996, Vol. 1, No. 1
reviews
the photosynthetic membrane, as well as in the ATPase activity 35. This leads to an increased pH gradient and thus en- hanced xanthophyll cycle-dependent energy dissipation for a given light intensity at low temperatures 35-37, and compen- sates for the low rates of photosynthetic use of absorbed light.
Exposure to combinations of stresses in the field can also lead to a maintenance of high levels of zeaxanthin and antheraxanthin throughout the day and night (Fig. 6) 37. For example, the diurnal transient changes in the levels of antheraxanthin and z.eaxanthin under favorable conditions in the summer are in stark contrast to the high zeaxanthin and antheraxanthin levels persisting throughout cold days during the winter in many species (Fig. 6). It has also been proposed that the maintenance of high levels of xanthophyll cycle-dependent energy dissipation may be responsible for the lasting depressions in the efficiency of energy conversion in photosystem II that can be observed in overwintering plants on cold days in the field (Fig. 6) 37. The overall efficiency of energy conversion in photosystem II would be expected to re- main low as long as high levels of energy dissipation persist in the antennae, since less of the absorbed light is delivered to the photochemical reaction centers. The same proposal had been previously made for the effects of combined high light intensity and either water stress .38 or salinity stress34, 39.
From these studies, it is clear that the kinetics of the re- conversion of zeaxanthin or antheraxanthin to violaxanthin are extremely dynamic and can be slowed considerably by additional environmental stresses. As a consequence, xantho- pbyll cycle-dependent energy dissipation can be the under- lying mechanism not only of rapidly reversible components of energy dissipation but also of slowly reversible ones.
The means by which removal of zeaxanthin and anthera- xanthin is delayed under these conditions is still unknown. Gilmore and Bj6rkman 3~ demonstrated that a low pH with- in the photosynthetic membrane can be maintained in low light or complete darkness, at low temperatures, allowing zeaxanthin and antheraxanthin to remain engaged for energy dissipation. However, there have also been reports that the synthesis of proteins is required for the return to a high photon efficiency of photosystem II after winter stress 4°. Several studies4~, 42 now suggest that maintenance of a highly de-epoxidized xanthophyll cycle is related to changes in the protein composition of photosystem II, maxi- mizing energy dissipation by virtue of a major reorganiz- ation of the light-harvesting complexes 4~ (see also Ref. 15).
Ubiquity of xanthophyll cycle-dependent energy dissipation among higher plant species, and its implications
All higher plant species examined to date possess the xanthophyll cycle. Without the built-in ability for photo- protective energy dissipation, photosynthesis could prob- ably not proceed in nature in the presence of O 2. Genetic differences in the ability of plants to increase the capacity for xanthophyll cycle-dependent energy dissipation have not yet been identified. Rather, a surprising degree of uniform- ity in the response of a wide diversity of higher-plant species to excess light has been demonstrated 22. The ability to show high levels of xanthophyll cycle-dependent energy dissi- pation may be a highly conserved trait because (1) it arose early during evolution, (2) it probably does not involve a large metabolic cost, and (3) excess light is experienced by almost all leaves at certain times, even in the deeply shaded tmderstory of a forest when sunflecks hit the leaves30. 32. Future studies will have to determine whether or not over-
(a) 1.0
U•'0.8 < >~0.6
_0.4 c -
0.; x
Summer
O• A + Z
1 I I I I
Winter
f A + Z 0 0 ~ O ~ O 0 0 g •
I I I I
(b)l. (
o
= 0.( E
~0., 0
0.:
Summer k
I I I ! 06.00 10.00
Winter
- mmmmmmnmm w i n . - ill
I I I i 14.00 18.0006.00 10.00 14,00 18.00
Time of day
Fig. 6. (a) Diurnal changes in the levels of violaxanthin (V) and antheraxanthin and zeaxanthin (A + Z) in leaves over the course of a warm day during the summer versus a cold day during the winter season. The levels of these various xanthophylls are expressed as fractions of the total pool of V + A + Z. (b) The accompanying changes in the efficiency of energy conversion in photosystem II over the course of the same two days. The efficiency of photosystem II is expressed as the ratio of variable to maximum fluorescence, obtained predawn and during energization throughout the day. Changes in this ratio reflect changes in the efficiency with which absorbed photons are delivered to open photosystem II centers. Data from Ref. 37.
expression of the enzymes of the xanthophyll cycle can improve the stress tolerance of certain species.
Future research directions In the immediate future, research on the xanthophyll
cycle in plants is likely to focus on several important a r e a s : • The molecular mechanism of xanthophylt cycle-dependent dissipation in vivo - does dissipation in vivo occur via a direct singlet energy transfer from chlorophyll to zeaxan- thin (and antheraxanthin) or is it mediated by zeaxanthin (and antheraxanthin) in a stoichiometric and obligatory but indirect function? • The mechanism(s) through which zeaxanthin and anther- axanthin can be retained - engaged for energy dissipation - in leaves under environmental stresses. • The generation of plant lines in which expression of the enzymes of the xanthophyll cycle is suppressed or enhanced, to examine the role of the xanthophyll cycle for the survival and productivity of plants.
Acknowledgements Our work has been supported by a fellowship from the
David and Lucile Packard Foundation to B. Demmig-Adams
Janua~ 1996, Voi. 1, No. 1 2 5
reviews
a n d g r a n t s f rom the U S Dept of Agr icu l tu re , Compet i t ive Resea rch G r a n t Office (Nos. 90-37130-5422 a n d 94-37100- 0291) a n d the N a t i o n a l Science F o u n d a t i o n (g ran t No. IBN- 9207653). We also wish to t h a n k m a n y col leagues for pro- r i d i n g us w i th p r e p r i n t s of t he i r m a n u s c r i p t s . We are f u r t he rmore i ndeb t ed to A d a m Gi lmore a n d Sal ly Susnowi tz for m a n y helpful c o m m e n t s on the m a n u s c r i p t .
References 1 Krause, G.H. and Weis, E. (1991) Chlorophyll fluorescence and
2 Demmig, B. et al. (1987) Photoinhibition and zeaxanthin formation in intact leaves: a possible role of the xanthophyll cycle in the dissipation of excess light energy, Plant Physiol. 84, 218-224
3 Demmig-Adams, B. (1990) Carotenoids and photoprotection in plants. A role for the xanthophyll zeaxanthin, Biochim. Biophys. Acta 1020, 1-24
4 Yamamoto, H.Y. (1979) Biochemistry of the violaxanthin cycle, Pure Appl. Chem. 51, 639-648
5 Demmig-Adams, B. and Adams, W.W., III (1993) The xanthophyll cycle, in Carotenoids in Photosynthesis (Young, A. and Britton, G., eds), pp. 206-251, Chapman & Hall
6 Pft~ndel, E. and Bilger, W. (1994) Regulation and possible function of the violaxanthin cycle, Photosynth. Res. 42, 89-109
7 Yamamoto, H.Y. and Bassi, R. Carotenoids: localization and function, in Oxygenic Photosynthesis: The Light Reactions (Ort, D.R. and Yocum, C.F., eds), Gustav Fischer (in press)
8 Hager, A. (1980) The reversible, light-induced conversions of xanthophylls in the chloroplast, in Pigments in Plants (Czygan, F-C., ed.), pp. 57-79, Gustav Fischer
9 Gilmore, A.M. and Yamamoto, H.Y. (1993) Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin- independent quenching, Photosynth. Res. 35, 67-78
16 BjSrkman, O. and Demmig-Adams, B. (1994) Regulation of photosynthetic light energy capture, conversion, and dissipation in leaves of higher plants, in Ecophysiology of Photosynthesis (Schulze, E-D. and Caldwell, M.M., eds), pp. 17-47, Springer-Veriag
11 Siefermann-Harms, D. (1987) The light harvesting and protective functions of carotenoids in photosynthetic membranes, Physiol. Plant. 69, 561-568
12 Demmig-Adams, B., Gilmore, A.M. and Adams, W.W., III In vivo functions of higher plant carotenoids, FASEB J. (in press)
13 Gilmore, A.M. and Yamamoto, H.Y. (1993) Biochemistry of xanthophyll- dependent nonradiative energy dissipation, in Photosynthetic Responses to the Environment (Yamamoto, H.Y. and Smith, C.M., eds), pp. t62-165, American Society of Plant Physiologists
14 Horton, P., Ruban, A.V. and Waiters, R.G. (1994) Regulation of light harvesting in green plants, Plant Physiol. 106, 415-420
15 Owens, T.G. Processing of excitation energy by antenna pigments, in Photosynthesis and the Environment (Baker, N.R., ed.), Kluwer (in press)
16 Gilmore, A.M. and Yamamoto, H.Y. (1992) Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP, Proc. Natl Acad. Sci. USA 89, 1899-1903
17 Gilmore, A.M., Mohanty, N. and Yamamoto, H.Y. (1994) Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of a trans-thylakoid ApH, FEBS Lett. 350, 271-274
18 Gilmore, A.M., Hazlett, T.L. and Govindjee (1995) Xanthophyl] cycle dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime, Proc. Natl Acad. Sci. USA 92, 2273-2277
19 Frank, H.A. et al. (1994) Photophysies of the carotenoids associated with the xanthophyll cycle in photosynthesis, Photosynth. Res. 41, 389-395
$0 Crofts, A.R. and Yerkes, C.T. (1994) A molecular mechanism for qE-quenching, FEBS Lett. 352, 265-270
21 Mohanty, N. and Yamamoto, H.Y. (1995) Mechanism of non- photochemical chlorophyll fluorescence quenching. I. The role of de-epoxidised xanthophylls and sequestered thylakoid membrane protons as probed by dibucaine, Aust. J. Plant Physiol. 22, 231-238
22 Demmig-Adams, B. and Adams, W.W., III Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species, Planta (in press)
23 Walters, R.G., Ruban, A.V. and Horton, P. (1994) Higher plant light-harvesting complexes LHCIIa and LHCIIc are bound by dicyclohexylcarbodiimide during inhibition of energy dissipation, Eur. J. Biochem. 226, 1063-1069
24 Jahns, P. (1995) The xanthophyll cycle in intermittent light-grown pea plants, Plant Physiol. 108, 149-156
25 H~rtel, H. and Lokstein, H. (1995) Relationship between quenching of maximum and dark-level chlorophyll fluorescence in vivo: dependence on photosystem II antenna size, Biochim. Biophys. Acta 1228, 91-94
26 Andrews, J.R., Fryer, M.J. and Baker, N.R. (1995) Consequences of LHC II deficiency for photosynthetic regulation in chlorina mutants of barley, Photosynth. Res. 44, 81-91
27 Gilmore, A.M., Govindjee and BjSrkman, O. Xanthophyll dependent non-photochemical quenching of chlorophyll a fluorescence at low physiological temperatures, in Proceedings of the 10th International Congress on Photosynthesis (Mathis, P., ed.), Kluwer (in press)
28 Grnszecki, W.I. and Krupa, Z. (1993) LHCII, the major light-harvesting pigment-protein complex is a zeaxanthin epoxidase, Biochim. Biophys. Acta 1144, 97-101
29 Adams, W.W., III and Demmig-Adams, B. (1992) Operation of the xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight, Planta 186, 390-398
39 Demmig-Adams, B., Adams, W.W., III and Grace, S.C. Physiology of light tolerance in plants, Hortic. Rev. 18 (in press)
31 Demmig-Adams, B. and Adams, W.W., III (1994) Capacity for energy dissipation in the pigment bed in leaves with different xanthophyll cycle pools, Aust. J. Plant Physiol. 21, 575-588
32 Demmig-Adams, B. et al. (1995) Xanthophyll cycle-dependent energy dissipation and flexible PSII efficiency in plants acclimated to light stress, Aust. J. Plant Physiol. 22, 249-260
33 Thayer, S.S. and BjSrkman, O. (1990) Leafxanthophyll content and composition in sun and shade determined by HPLC, Photosynth. Res. 23, 331-343
34 Demmig-Adams, B. and Adams, W.W., III (1992) Photoprotection and other responses of plants to high light stress, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 599-626
35 Gilmore, A.M. and BjSrkman, O. Temperature-sensitive coupling and uncoupling of ATPase-mediated, nonradiative energy dissipation: similarities between chloroplasts and leaves, Planta (in press)
36 Adams, W.W., III, Hoehn, A. and Demmig-Adams, B. (1995) Chilling temperatures and the xanthophyll cycle. A comparison of warm-grown and overwintering spinach, Aust. J. Plant Physiol. 22, 75-85
37 Adams, W.W., III et al. (1995) 'Photoinhibition' during winter stress: Involvement of sustained xanthophyll cycle-dependent energy dissipation, Aust. J. Plant Physiol. 22,261-276
38 Demmig, B. et al. (1988) Zeaxanthin and the heat dissipation of excess light energy in Nerium oleander exposed to a combination of high light and water stress, Plant Physiol. 87, 17-24
39 Bj6rkman, O., Demmig, B. and Andrews, T.J. (1988) Mangrove photosynthesis: response to high-irradiance stress, Aust. J. Plant Physiol. 15, 43-61
40 tG'ause, G.H. (1994) Photoinhibition induced by low temperatures, in Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field (Baker, N.R. and Bowyer, J.R., eds), pp. 331-348, Bios
41 Ottander, C., Campbell, D. and 0quist, G. (1995) Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris, Planta 197, 176-183
42 Adamska, I. and Kloppstech, K. (1994) The role of early light-induced proteins (ELIPs) during light stress, in Photoinhibition of Photosynthesis from Molecular Mechanisms to the Field (Baker, N.R. and Bowyer, J.R., eds), pp. 205-219, Bios
Barbara Demmig-Adams and W am Adams are at the Dept of Environmenta Popu at on and Organ sm c B o[ogy Un vers ty of Colorado, Boulder, CO 80309.0334, USA.
2 6 Janua~ 1996, Vol, 1, No. 1
ANRV375-PP60-12 ARI 25 March 2009 13:47
Sensing and Respondingto Excess LightZhirong Li,1,2 Setsuko Wakao,1 Beat B. Fischer,1
and Krishna K. Niyogi1,2
1Department of Plant and Microbial Biology, University of California, Berkeley,California 947202Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley,California 94720; email: [email protected]
Annu. Rev. Plant Biol. 2009. 60:239–60
First published online as a Review in Advance onDecember 9, 2008
The Annual Review of Plant Biology is online atplant.annualreviews.org
This article’s doi:10.1146/annurev.arplant.58.032806.103844
AbstractPlants and algae often absorb too much light—more than they can ac-tually use in photosynthesis. To prevent photo-oxidative damage and toacclimate to changes in their environment, photosynthetic organismshave evolved direct and indirect mechanisms for sensing and respond-ing to excess light. Photoreceptors such as phototropin, neochrome,and cryptochrome can sense excess light directly and relay signals forchloroplast movement and gene expression responses. Indirect sensingof excess light through biochemical and metabolic signals can be trans-duced into local responses within chloroplasts, into changes in nucleargene expression via retrograde signaling pathways, or even into systemicresponses, all of which are associated with photoacclimation.
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Excess light (EL):a relative term thatdescribes theabsorption of light thatexceeds photosyntheticcapacity
Excess light (EL) is the light absorbed by plantsand algae that exceeds their photosynthetic ca-pacity (Figure 1). Although light is needed forphotosynthesis, absorption of EL can lead toincreased production of highly reactive inter-
mediates and by-products that can potentiallycause photo-oxidative damage and inhibit pho-tosynthesis (117). Exactly what constitutes ex-cess light for a plant or an alga depends on itsenvironmental conditions and can vary over awide range of irradiance levels (123), but mostphotosynthetic organisms must deal with ELon a seasonal as well as daily basis. Environ-mental stresses generally decrease the maxi-mum photosynthetic capacity of plants and al-gae (Figure 1). Thus, natural conditions suchas drought, high salinity, nutrient deprivation,or temperature stress can influence and exacer-bate EL stress (30).
Photosynthetic organisms have evolveda variety of direct and indirect mechanismsfor sensing EL, as diagramed in Figure 2.Upon exposure to EL, plants and green algaecan directly sense a high incident photonflux using several classes of photoreceptors,including phototropins, neochromes, phy-tochromes, rhodopsins, and cryptochromes.The phototropins and neochromes playimportant roles in chloroplast avoidancemovement. The rhodopsins are of majorimportance in phototaxis and photophobicmovement on the basis of studies with the greenalga Chlamydomonas reinhardtii. In the modelplant Arabidopsis thaliana, cryptochromes havebeen shown to control the expression of a largenumber of EL-responsive genes. EL can alsobe sensed indirectly through biochemical andmetabolic signals that are transduced into pho-toprotective and photoacclimatory responses.
Excess light
Photon flux density (pfd)
Rate
of photo
n abso
rptio
n
Rat
e o
f ph
oto
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With additional stress
Figure 1Light response curves for photosynthesis comparedwith the rate of light absorption.
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For example, when the rate of photosynthesisreaches saturation, absorption of EL causes adecrease in thylakoid lumen pH, an increasein the reduction state of the plastoquinonepool and thiols in the chloroplast, productionof various reactive oxygen species (ROS)[including mainly hydrogen peroxide (H2O2)and singlet oxygen (1O2
∗)], and perturbationof chlorophyll biosynthesis. Some aspects ofthese topics have been covered in several recentreviews (10, 12, 16, 36, 111, 112, 118, 120,125, 127, 167, 170). We focus on advances inunderstanding how photosynthetic organismssense and respond to EL, specifically on thecellular and molecular responses in plantsand green algae that help to protect againstphotodamage and photoinhibition.
EXCESS LIGHT RESPONSESMEDIATED BYPHOTORECEPTORS
Chloroplast Avoidance Movement
Plants are generally rooted in place, but they ex-hibit several well-known movement responsesto light, such as phototropism. At a cellularlevel, chloroplasts move to the sides of a plantcell so that they are positioned parallel to the di-rection of incident light to avoid absorption ofEL and thereby minimize photodamage (70).This response is called chloroplast avoidancemovement, and it is mediated by blue light anda blue light photoreceptor, phototropin, in mostplants (165).
Phototropin was first identified as a ∼120-kilodalton (kD) plasma membrane protein thathas blue light–dependent autophosphorylationactivity (reviewed in Reference 23). A. thalianacontains two phototropins, PHOT1 (originallycalled NPH1) and PHOT2 (originally calledNPL1), each of which consists of two tandemLOV (light-, oxygen-, and voltage-sensing) do-mains at the N terminus and a Ser/Thr kinasedomain at the C terminus (56, 62). LOV do-mains bind flavin mononucleotide (FMN) andare responsible for blue/UV-A light perception.In two independent molecular genetic studies,
Change in redox state(PQ, thioredoxin, glutathione)
Production of ROS(singlet oxygen, H2O2)
Accumulation of metabolites(e.g., Chl intermediates)
Chloroplast avoidance movement
Nonphotochemical quenching (qE)
Changes in gene expression
Systemic acquired acclimation
Figure 2Schematic depiction of strategies for sensing and responding to excess light byphotosynthetic organisms. PQ, plastoquinone; ROS, reactive oxygen species;qE, pH-dependent regulation of photosynthetic light harvesting.
ROS: reactive oxygenspecies
PHOT2, but not PHOT1, was demonstratedto function as a photoreceptor that regulatesthe chloroplast avoidance movement in strongblue light (62, 66, 70). The chloroplasts in phot2mutants accumulate along periclinal walls, per-pendicular to the incident light, in response tothe EL conditions that induce the chloroplastavoidance response in wild type and phot1 mu-tants (66, 70). In the fern Adiantum capillus-veneris, PHOT2 is also required for the bluelight–induced chloroplast avoidance responseas shown by mutant analysis and rescue experi-ments using Acphot2 mutant plants (65).
Although blue light is the most effectivetype of light in inducing chloroplast avoidancemovement in angiosperms, red light has alsobeen implicated in the fern A. capillus-veneris,the green alga Mougeotia scalaris, and the mossPhyscomitrella patens (reviewed in Reference152). These red light–induced responsescan be reversed by subsequent far-red lightirradiance, indicating the involvement ofphytochrome (64). Interestingly, the pho-toreceptor controlling the red light–induced
chloroplast avoidance response of A. capillus-veneris is a novel chimeric photoreceptorcalled neochrome (AcNEO, originally namedAcPHY3), which consists of an N-terminalphytochrome-like chromophore-binding do-main fused to a full-length phototropin (119,151). Similar PHY-PHOT chimeric photore-ceptors, MsNEO1 and MsNEO2, were foundin M. scalaris. As in the case of AcNEO1, bothMsNEO1 and MsNEO2 show phytochrome-typical red/far-red reversibility and areinvolved in red light–induced chloroplastavoidance movement (72, 151, 152). An-other exceptional case of red light–inducedchloroplast avoidance is found in the mossP. patens. Four phototropin genes from P. patenswere isolated and classified into two groups(PHOTA and PHOTB ). Analysis of P. patensmutants indicates that these phototropins areinvolved in both blue and red light–inducedchloroplast avoidance movements (71). Giventhat phototropins do not absorb red lightand that no neochrome-type protein has beenisolated in this moss (104, 151), phototropinsmay function as downstream components ofsignal transduction pathways for phytochrome-dependent chloroplast avoidance in P. patens(71).
The isolation of additional chloroplastavoidance mutants of A. thaliana has shed lighton the components that act downstream of thephototropin receptor. The first described mu-tant in this class was chloroplast unusual position-ing 1 (chup1), which contains an altered F-actin-binding protein (70, 121). The chup1 mutantsexhibit aberrant chloroplast positioning regard-less of the light conditions (70), indicating thatCHUP1 most likely represents an essentialcomponent of the motility system. Similarly,the plastid movement–impaired mutant pmi1is severely impaired in chloroplast movementsunder both low and high intensities of bluelight (29). Sequence analysis of the PMI1 pro-tein suggests that it may be involved in Ca2+-mediated signal transduction to the actin cy-toskeleton (29). In contrast, the pmi2 and pmi5mutations cause attenuated chloroplast avoid-ance movement under EL while maintaining a
normal movement response under low light, in-dicating that PMI2 and PMI5 are specificallyinvolved in the downstream signaling eventsof chloroplast avoidance movement (96). PMI2and PMI5 are unknown plant-specific proteinswhose functions have yet to be determined.
Photophobic Movementin Chlamydomonas
In contrast to plants, C. reinhardtii and manyother unicellular algae exhibit flagellar motil-ity, and an abrupt increase in light intensitycauses a transient stop or reorientation of cellmovement known as the photophobic response.The photophobic response involves an eye-spot apparatus and is induced by a cascadeof electrical events that includes the genera-tion of photoreceptor currents, membrane de-polarization, activation of voltage-gated Ca2+
channels in the flagellar membrane, and finallya brief alteration of the flagella beating mode.In C. reinhardtii, retinal reconstitution stud-ies in blind mutants strongly suggest that thisphotophobic response is mediated by retinal-containing receptors (43). Two rhodopsins,Chlamydomonas sensory rhodopsin A and B(CSRA and CSRB), were discovered almost si-multaneously by three independent researchgroups (116, 147, 154). CSRA and CSRBare photoactive, seven-transmembrane-helixreceptors that use retinal as a chromophore.CSRA and CSRB were originally demonstratedto mediate the light-generated currents respon-sible for phototaxis signaling by photoelectro-physiological analysis of RNAi strains (147).CSRA absorbs maximally near 510 nm and trig-gers a rapid current that saturates at high lightintensities, whereas CSRB has an absorbancemaximum at 470 nm and induces a slow cur-rent that saturates at low light intensities.Researchers later directly showed by quantita-tive comparison of the photophobic responsein cells enriched in either CSRA or CSRB,using a motion analysis/tracking system andvideo recording of cell behavior, that CSRAand CSRB do indeed mediate the photopho-bic response in C. reinhardtii (49). Because of
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the different cellular abundance and light sat-uration characteristics of the two rhodopsins,CSRA is the dominant receptor for photopho-bic responses (49).
Cryptochrome and NuclearGene Expression
Microarray experiments have shown that ex-posure to EL results in dramatic changes ingene expression in plants (3, 15, 75, 77, 114,137, 162) and green algae (42, 59, 85). InA. thaliana, a subset of these genes appears tobe regulated in EL by the blue/UV-A light-absorbing cryptochrome photoreceptor, CRY1(77). CRY1 and its paralog CRY2 are nuclearflavoproteins that share a similar chromophore-binding domain at their N termini but differ inthe variable extensions at their C termini. CRY1and CRY2 are known to mediate specific bluelight–dependent responses such as inhibition ofhypocotyl elongation in response to relativelyhigh and low fluences of blue light, respectively(4, 95). Genome-wide gene expression analy-sis showed that the EL-dependent regulationof 77 genes was altered in a cry1 mutant, and26 of these genes were also misregulated in ahy5 transcription factor mutant (77). For ex-ample, induction of ELIP1 and ELIP2, whichencode light stress-related relatives of the light-harvesting chlorophyll a/b-binding (LHC) pro-tein family, by either EL or high-intensity bluelight was strongly attenuated in cry1 mutants(Figure 3). However, their induction was unaf-fected in cry2, phyA, phyB, or hyh mutants, indi-cating that the induction of ELIP1 and ELIP2is mediated specifically by CRY1 in a bluelight–dependent manner (77). The induction ofseveral phenylpropanoid metabolism genes, in-cluding the anthocyanin transcriptional regula-tors PAP1 and PAP2, was also inhibited in cry1,and the normal accumulation of anthocyanindid not occur in EL. Induction/repression ofother known EL-responsive genes, such as anascorbate peroxidase–encoding gene (APX2)and a light-harvesting protein–encoding gene(LHCB2.4 ) was unaffected in photoreceptormutants (77), suggesting that these genes are
regulated by other pathways (see below). Thecry1 mutant is sensitive to short-term EL (77,139), confirming a function of the CRY1 pho-toreceptor in mediating the response to EL,although acclimation of photosynthetic param-eters in cry1 does occur during long-term accli-mation to EL (168).
qE, A pH-DEPENDENT RESPONSETO EXCESS LIGHT
When light absorption exceeds the capacityfor light utilization in assimilatory reactions, adecrease in the proton conductance of thechloroplast ATPase results in a rapid decreasein thylakoid lumen pH (67, 156) that triggersa type of regulation of photosynthetic lightharvesting. This regulatory process, called qE(reviewed in References 55 and 110), dissipatesexcess absorbed light energy as heat (Figure 3)and protects the photosynthetic apparatus dur-ing short-term fluctuations in light intensity(50, 82, 93). The induction of qE occurs on atimescale of seconds to minutes, independent ofany changes in gene expression. Instead, qE isdependent on a pH-dependent, intrathylakoidsignal transduction pathway (Figure 3), whichinvolves activation of a xanthophyll cycle andsensing of lumen pH by a photosystem (PS) IIprotein, PsbS (92).
The decrease in thylakoid lumen pHin EL activates violaxanthin de-epoxidase(VDE), an enzyme that converts violaxanthinto antheraxanthin and zeaxanthin as part ofa xanthophyll cycle (172). Zeaxanthin andantheraxanthin, along with lutein, share aspecific carotenoid ring structure, and theyare necessary for qE in vivo (110). VDE is amember of the lipocalin family of proteins,and its central lipocalin domain is flankedby a cysteine-rich N-terminal domain and aglutamate-rich C-terminal domain (21). ThepH optimum of purified VDE is 5.2 (52),and estimation of the Hill coefficient (nH)for de-epoxidation in isolated chloroplasts(129) and leaves (155) yielded values of ∼5.3and ∼4.3, respectively. Low pH induces aconformational change in the enzyme (73) and
Figure 3Schematic diagram of a plant/algal cell showing the locations and interactions of some of the sensing andsignaling molecules that are involved in responses to excess light. Dashed arrows indicate diffusion ortransport of a molecule. cyt b6/f, cytochrome b6/f complex; GSH, glutathione; Mg-ProtoIX,Mg-protoporphyrin IX; qE, pH-dependent regulation of photosynthetic light harvesting; PS, photosystem;PQH2, plastoquinol; PChlide, protochlorophyllide; Trx, thioredoxin; VDE, violaxanthin de-epoxidase.
also promotes its binding to the membrane(173), where its substrate is located. Analysis ofC-terminal deletions suggests that theglutamate-rich domain is important forthis pH-dependent binding of VDE to thethylakoid membrane (53). Site-directed muta-genesis has also implicated a role for histidineresidues in membrane binding (48).
In addition to activating VDE, the low thy-lakoid pH in EL also drives protonation oflumen-exposed carboxylate side chains in spe-cific PS II proteins (55). This protonation is hy-pothesized to induce a conformational changethat allows for de-excitation of chlorophylls inthe antenna system of PS II. Mutant analysis inA. thaliana has identified PsbS (50, 91, 126) as
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a critical component that determines the levelof qE (93). N, N ′-dicyclohexylcarbodiimide(DCCD), which reacts with carboxyl groupsin hydrophobic environments, inhibits qE andbinds to PsbS in vitro (32). By site-directed mu-tant analysis in vivo, a conserved (9, 79, 142)pair of lumen-facing acidic residues (E122 andE226) was shown to be required for qE and thebinding of DCCD (92, 94), suggesting that pro-tonation of these residues in EL is involved inqE induction. A Hill coefficient of ∼1.0 wascalculated for PsbS activation (155), consistentwith the idea that each glutamate residue actsindependently rather than cooperatively (92).On the basis of its reported pigment binding(46), it was initially hypothesized that PsbSmight be the site of qE (91), but it now seemsclear that PsbS serves as a sensor of lumen pHthat turns qE on and off in neighboring an-tenna proteins where quenching of 1Chl∗ ac-tually occurs (6, 20, 32, 54, 92). This role insignal transduction has been suggested to in-volve monomerization of a PsbS dimer at lowpH and association with the antenna proteins,whereas the dimer is preferentially associatedwith the PSII reaction center (18).
REDOX-REGULATED RESPONSESTO EXCESS LIGHT
In EL, the plastoquinone (PQ) pool and thiolssuch as thioredoxin and glutathione can exhibitsignificant changes in their reduction/oxidation(redox) state and are thus possible signalingmolecules for EL responses (Figure 3). Theredox state of the PQ pool is known to reg-ulate chloroplast and nuclear gene expression(37, 128) and photosynthetic state transitions,which adjust excitation of the two photosystemsduring light-quality acclimation (reviewed inReference 135). The availability of PS I electronacceptors also impacts chloroplast and nucleargene regulation (144). Signaling roles of thiore-doxin have been appreciated for decades (143),and evidence for similar roles of glutathione andglutaredoxins is beginning to emerge (113). Asjust one example, EL alters the synthesis rateof some chloroplast-encoded proteins, such as
PQ: plastoquinone
LHCB: light-harvesting chlorophylla/b-binding proteinassociated withphotosystem II
the D1 protein of PS II (146), which must berepaired after inactivation by EL. The rate ofD1 synthesis increases in C. reinhardtii cells thatare exposed to EL, whereas synthesis of theRubisco large subunit (RbcL) decreases within15 min but recovers after ∼6 h in EL (146).This transient repression is associated with ox-idation of the glutathione pool (60). Subse-quent oxidation of thiols in the RbcL protein byglutathione (24) exposes a conserved RNArecognition motif, which binds to and inhibitstranslation of its own (rbcL) mRNA (25). Otherexamples of thiol regulation, specifically involv-ing chloroplast transcription and translation,have been documented, and many of these havebeen covered extensively in recent reviews (12,120). Here, in the context of EL, we focus onstudies of PQ redox regulation.
The oxidation of plastoquinol (PQH2) is therate-limiting step in photosynthetic electrontransport between PS II and PS I (51). UnderEL conditions or when light quality favors ab-sorption of light by PS II, the rate of electrontransport by PS II can exceed the rate of oxida-tion of PQH2, thus resulting in overreductionof the PQ pool. Among the thylakoid electroncarriers, the PQ pool is generally considered tobe an important redox sensor for signaling im-balances between the relative excitation of thetwo photosystems (Figure 3).
Many plants and green algae compensate forchanges in incident light quantity and qualityby altering the sizes of the antennae associatedwith PS II and PS I during long-term photoac-climation. For example, the PS II antenna sizeusually decreases during growth in EL com-pared with growth in low light (reviewed inReference 100). The PS II antenna is composedof multiple subunits of homologous proteinsbelonging to the light-harvesting chlorophylla/b-binding (LHCB) protein family, which areencoded by nuclear LHCB genes (61), and gen-erally the accumulation of the most peripherallylocated antenna subunits (with respect to the
reaction center) is regulated by light intensity(14).
Regulation of LHCB gene expression by theredox state of the PQ pool has been exten-sively studied in unicellular green algae, par-ticularly Dunaliella and Chlorella species, whichexhibit dramatic EL-dependent decreases inLHCB protein abundance. For example inDunaliella tertiolecta, LHCB mRNA levels de-crease in EL mainly because of lower ratesof transcription, and an effect similar to ELcan be induced in low light by inhibitingthe oxidation of PQH2 with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)(35). In contrast, preventing PQ reductionby PS II using 2-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) mimics the effect of ashift to lower light intensity (35). These exper-iments led to the proposal that a high PQ re-dox state acts via a chloroplast-to-nucleus signaltransduction pathway to repress LHCB tran-scription (33, 35). Similar LHCB gene regu-lation occurs in Dunaliella salina and Chlorellavulgaris cells (98, 169), in which effects of highlight intensity can be mimicked under relativelylow light intensity at low temperature, leadingto the concept of PS II excitation pressure asa signal for LHCB gene regulation (57). How-ever, subsequent studies with Dunaliella specieshave shown that DCMU does not block theEL-induced decrease in LHCB mRNA levelsand that the PQ redox regulation pathway actson a relatively long timescale of hours, suggest-ing that more than one pathway is involved (22,97). In C. reinhardtii, downregulation of LHCBmRNA levels by EL occurs transiently and in-volves multiple pathways (34, 158). Transla-tional regulation, possibly involving H2O2, alsoappears to play an important role in photoaccli-mation of C. reinhardtii cells (99), and a possi-ble posttranscriptional regulator of LHCB pro-tein levels has been identified by mutant analysis(115).
Investigations of photoacclimation andLHCB gene expression in plants have pro-vided mixed support for the hypothesis ofPQ redox regulation. For example, no dif-ferences in chloroplast photoacclimation re-
sponses were observed in antisense tobaccoplants with decreased levels of functional cy-tochrome b6f complex, despite a constitutivelyhigh PQ reduction state (7). In contrast, analysisof a Lemna perpusilla cytochrome b6f complex–deficient mutant revealed that the PQ pool isoverreduced even under very low illumination,and the LHCB protein level is always lower inthe mutant than in the wild type (174). Thisphenotype can be partially reversed by blockingPQ reduction with DCMU (174). In barley, nocorrelation between PQ redox state and LHCBmRNA or protein levels was found when PQ re-dox state was modulated by changing light andgas composition (107). However, analysis of thebarley PS I–less viridis zb63 mutant showed anoverreduced PQ pool and a minimal antennasize (108). The decrease in PS II antenna sizein barley viridis zb63 occurs without changesin LHCB mRNA levels, suggesting that regu-lation by chronic PQ reduction is at the levelof protein accumulation (44). Consistent withthis, microarray analysis of EL-regulated geneexpression in A. thaliana found that altered ex-pression of a subset of 50 genes (out of hun-dreds of genes that were induced or repressed byEL) is blocked by DCMU, suggesting PQ re-dox regulation, but this subset of genes includesELIP but no LHCB genes (3). Transcriptionalinduction of the ELIP2 promoter by EL hasbeen demonstrated using a luciferase reporterfusion (76), and experiments with DCMU andDBMIB confirmed that this induction dependson PQ redox state (74).
The redox state of the PQ pool has also beenimplicated in the EL-dependent regulation ofnuclear genes that encode cytosolic antioxi-dant enzymes. One example is the stimulationof cytosolic ascorbate peroxidase (APX1 andAPX2) gene expression by PQ pool reduction inA. thaliana (68). Both genes are induced within15 min after exposure to EL, and this effectcould be abolished by the application of exoge-nous glutathione. DBMIB treatment stimulatesthe increase of both APX1 and APX2 mRNAlevels, in either subsaturating or EL conditions,whereas treatment with DCMU results in con-stant or even reduced mRNA levels, suggesting
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that the redox state of the PQ pool controls nu-clear expression of APX1 and APX2 (68).
These and other studies provide evidencefor a role of the PQ pool as a sensor of ELand a regulator affecting nuclear gene expres-sion at the transcriptional and/or posttranscrip-tional levels (Figure 3). However, the mecha-nism by which the PQ redox signal is relayedfrom the chloroplast remains unclear, althoughinhibitor studies have suggested the involve-ment of a protein phosphatase type 2A in theinduction of the ELIP2 promoter by EL (76).
Possible Roles ofThylakoid-Associated Kinases
The redox state of the PQ pool can be sensedvia the cytochrome b6/f complex by thylakoidprotein kinases that are involved in phosphory-lation of PS II antenna and reaction center pro-teins, and these kinases might also have a role inmediating PQ redox-state-dependent gene ex-pression. Phosphorylation of LHCB proteinsis necessary for a mechanism called state tran-sition, which redistributes antenna proteins be-tween PS II and PS I to maintain PQ redoxhomeostasis during short-term light-quality ac-climation (reviewed in Reference 135). STT7was identified as a protein Ser-Thr kinase re-quired for LHCB phosphorylation and for statetransition in C. reinhardtii (31), and two ho-mologs, STN7 and STN8, were subsequentlyfound in A. thaliana (17, 19). As in the case ofSTT7, STN7 is required for the phosphory-lation of several LHCB proteins and for statetransition (17), whereas STN8 is not involvedin state transition (17) but is required for thephosphorylation of PS II core proteins (19).Long-term responses of photosynthetic param-eters and protein levels to changes in light qual-ity and quantity are impaired in stn7 mutants(19, 159), consistent with the idea that STN7might be involved in signaling changes in nu-clear gene expression for photosynthetic ac-climation. However, no differences or only afew differences in RNA levels were detectedby microarray analyses of wild-type and stn7plants that were grown in various light condi-
tions (19, 159), including EL, suggesting thatSTN7 is involved in PQ-regulated long-termacclimation at the level of protein abundance(Figure 3). The stn7 mutant, which is unableto perform state transition, has a more reducedPQ pool than the wild type (17), and this mighthave an effect similar to that described abovefor the L. perpusilla cytochrome b6/f mutant(174) and barley PS I mutant (44). In contrast tothe situation in long-term growth, short-termexposure to EL results in a higher inductionof stress responsive genes (such as those en-coding heat shock proteins) in the stn7 mutantthan in the wild type (159). The roles of STN7and STN8 in the signaling network of nuclear(and chloroplast) gene expression merit furtherinvestigation.
EXCESS LIGHT RESPONSESMEDIATED BY REACTIVEOXYGEN SPECIES
EL is associated with generation of ROS suchas H2O2, superoxide (O2
−), hydroxyl radi-cal, and 1O2
∗ in chloroplasts (11). H2O2 isalso produced in plant peroxisomes duringdetoxification of glycolate in photorespiration.Although these species arise from distinctphotochemical and biochemical processes, theproduction of one ROS can lead to that ofanother; thus, the examination of cellular re-sponses to a specific ROS in the context ofEL is inherently complicated. To what ex-tent does each of these ROS contribute tochanges in gene expression and acclimationto EL? To dissect the specific gene expres-sion changes caused by different ROS, variousexperimental approaches have been developedand applied in combination with EL expo-sure. These include the use of ROS-specificphotosynthetic inhibitors such as DCMU andother PS II herbicides (38, 145), methyl vi-ologen (MV) (generates O2
− mostly in thechloroplast) (122, 141), the conditional flu-orescent ( flu) mutant (generates 1O2
∗ in thechloroplast) (122), and transgenic plants withreduced ROS-detoxifying activities, includ-ing knockdown plants of Cu/Zn superoxide
dismutase (SOD) (134) and catalase (161, 162)and knockout (KO) plants of cytosolic APX1(130). Comparison of transcriptomic data frommultiple treatments and genetic modificationshas provided insight into the distinct and over-lapping transcriptional responses conferred byvarious ROS of different origin (47). By catego-rizing genes on the basis of temporal regulationby specific ROS, one can begin to infer the or-der of ROS-sensing and signaling events thatoccur in EL.
Singlet Oxygen Responses
EL conditions promote formation of the tripletexcited state chlorophyll and thus lead to thegeneration of 1O2
∗, predominantly at the reac-tion center of PS II (80). Because of its shorthalf-life, 1O2
∗ has been thought to react in theimmediate proximity of the PS II reaction cen-ter, but its lifetime in vivo appears to be longerthan expected (148). A possible signaling roleof 1O2
∗ has been proposed in C. reinhardtiicells, where 1O2
∗ was detected in the cytosol(Figure 3) after its generation in the chloroplast(41).
The existence of a specific 1O2∗ signal-
ing pathway (Figure 3) has been shownin A. thaliana through analysis of theprotochlorophyllide-accumulating flu mutant(122) and isolation of the executer (ex) mutant(166), a suppressor of flu. The flu mutant, whichis defective in feedback regulation of chloro-phyll biosynthesis, accumulates high levels ofprotochlorophyllide in the dark (101), and sub-sequent illumination results in photosensitized1O2
∗ formation (122). Microarray experimentsusing the flu mutant and plants treated with MVhave demonstrated that 1O2
∗ and O2−/H2O2
elicit distinct transcriptional responses (122); alarger number of nuclear genes are induced by1O2
∗ (including several genes encoding WRKYtranscription factors) compared with O2
− (122)or other ROS (47). The transcriptional re-sponse to 1O2
∗ in the flu mutant is impairedin ex1 flu and abolished in the ex1 ex2 flu triplemutant (87). EX1 and EX2 encode two homol-ogous proteins whose biochemical functions
are unknown but are suggested to be associ-ated with the thylakoid membrane, where theymight participate in sensing or transduction ofa 1O2
∗ signal to the nucleus (87, 166).The molecular basis of 1O2
∗-specific geneexpression has been studied in detail in C. rein-hardtii. A glutathione peroxidase homologousgene (GPXH ) is induced with high specificityby 1O2
∗ (Figure 3) generated either by photo-sensitizers (39, 89) or in EL (38). Analysis ofthe GPXH promoter narrowed the region re-sponsible for 1O2
∗-specific induction down toa region that contains sequences similar to acAMP response element and an activator pro-tein 1 binding site (89). The transcriptionalresponse to H2O2 and 1O2
∗ can be clearly sep-arated at the level of cis-acting elements usingthe HSP70A promoter in C. reinhardtii (145). InA. thaliana, researchers found an antagonizingeffect of O2
−/H2O2 on 1O2∗-induced gene ex-
pression using MV-treated flu mutants overex-pressing a thylakoid-bound APX (84), suggest-ing that interactions between different ROSsignaling pathways might be involved in the or-chestration of the response to EL in vivo.
C. reinhardtii was recently found to exhibita specific acclimation response to 1O2
∗ gen-erated by a photosensitizer, rose bengal (RB).Exposure to a sublethal dose of 1O2
∗ by RBin the light (pretreatment) leads to increasedresistance to subsequent doses of 1O2
∗ thatwould otherwise have been lethal (86). The ac-climation process is highly specific to 1O2
∗, be-cause no cross-acclimation is observed to otherROS generators (86). Pretreatment is suffi-cient to cause changes in gene expression, in-cluding the induction of GPXH and GSTS1(86), which were previously identified as 1O2
∗-induced genes (40, 89). Moreover, cells that arepretreated with EL are more resistant to chal-lenge with RB, indicating the physiological sig-nificance of 1O2
∗ signaling in response to EL(86). With the aim of identifying regulators ofthe 1O2
∗ signaling pathway in C. reinhardtii,several mutants defective in the 1O2
∗ acclima-tion response have been isolated (S. Wakao,H.K. Ledford, B.L. Chin, & K.K. Niyogi,unpublished data). Identification of the genes
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affected in such mutants is expected to shedlight on the signaling events that occur in re-sponse to 1O2
∗.
Responses to ExcessLight–Induced H2O2
Many studies have demonstrated effects ofH2O2 on gene expression in plants. For ex-ample, the effects of photorespiratory H2O2
in eliciting genetic responses were addressedby transcriptomic analysis comparing wild-typeand catalase-deficient plants during exposureto EL (161, 162). Cluster analysis of tempo-ral expression profiles suggested that consti-tutively elevated H2O2 causes many genes tobe enhanced (hypersensitive) or attenuated (de-sensitized) in their response (161). Many ofthe genes upregulated by elevated H2O2 over-lapped with those of MV-, heat-, cold-, anddrought-treated plants, indicating the centralrole of photorespiratory H2O2 in abiotic stressresponses (162). Additionally, the fact that somegenes upregulated in catalase-deficient plantsoverlap only with those genes upregulated ina single abiotic stress treatment suggests that,despite the apparent general role of H2O2 instress signaling, additional regulatory eventsoccur that distinguish different abiotic stressresponses.
Cytosolic ascorbate peroxidase genes APX1and APX2 were identified as early EL responsegenes that are induced by the redox state of PQ(68), as described above. The loss of function inAPX1-KO plants leads to stunted growth andhypersensitivity to light (130), as well as acceler-ated degradation of chloroplast proteins such asthe small subunit of RubisCO and thylakoidalAPX (tAPX) (27, 28). These results indicatethe importance of cytosolic H2O2 scavengingby APX1 and its protective role for chloro-plast proteins in EL. The induction of APX2mRNA levels by EL involves H2O2 and abscisicacid (ABA) in addition to PQ redox state (15,45, 69, 136). APX2 induction by EL is abol-ished by infiltration of leaves with catalase (69),and overexpression of catalase in chloroplastsor tAPX suppresses the EL-dependent induc-
tion of a cytosolic APX gene in tobacco (171).A screen for mutants with altered regulation ofthe APX2 promoter uncovered a glutathione-deficient mutant, rax1, which exhibits higherbasal and induced APX2 mRNA levels (13).The alx8 mutant, which was isolated in a sim-ilar screen, also overexpresses APX2 mRNA.alx8 has constitutively elevated levels of ABA,and EL increases ABA levels in wild-type leaves(136). Thus, multiple pathways appear to reg-ulate APX2 expression in EL, and further workis needed to unravel this complexity.
Transcription Factors Involvedin Responses to Excess Lightand Reactive Oxygen Species
The zinc-finger transcription factorZAT12/RHL41 was identified as one ofthe genes that are rapidly induced during ELacclimation in A. thaliana (58). Overexpressionof ZAT12 confers MV resistance (133) and aconstitutively EL-acclimated state, as shownby delayed loss of PS II activity under ELcompared with wild type (58). ZAT12 isinduced not only under EL, but also by manyother abiotic stresses such as salinity, cold, heat,salt, and wounding (28, 133). ZAT12 togetherwith ZAT10 is strongly induced in responseto seven out of nine ROS treatments (47),in agreement with previous and independentobservations that these might code for generalabiotic stress transcription factors (27, 28,103, 133, 140). Interestingly, whereas ZAT12is closely associated with the induction ofAPX1, ZAT10 appears to be involved in theinduction of APX2 (Figure 3), and photosyn-thetic parameters of ZAT10-overexpressingplants display an EL-acclimated state (138) aswell as tolerance to multiple abiotic stresses(103).
Two genes encoding heat shock transcrip-tion factors (HSFA4a/HSF21, HSFA8/HSF5 )were identified among rapidly induced genesin APX1-KO plants transferred to EL (27, 28).There are hints that other HSFs are involvedin stress- and species-specific ROS signaling(102). In mammalian cells and in Drosophila,
H2O2 induces DNA binding of a similar A-type HSF (5, 175), and in yeast, O2
− inducesthe DNA-binding conformation of the HSFtrimer (88). In A. thaliana, the APX1 promoterregion contains an HSF binding site (149), andincreased APX activity is observed in plantsoverexpressing one of the HSF genes (124).Overexpression of a dominant-negative formof HSFA4a incapable of binding DNA resultsin the loss of ZAT12 and APX1 induction,strongly suggesting that HSFA4a functions up-stream of ZAT12 in the EL response pathway(27).
Comparative transcriptomic analysis foundHSFA4A among the genes induced by 1O2
∗,O3, and MV and in APX1-KO and SOD-knockdown plants transferred to EL (47).HSFA8 and HSFB2b were found as genes thatare induced predominantly by 1O2
∗ and H2O2,respectively. Interestingly, HSFA4a and HSFA8are induced in APX1-KO but not in catalase-deficient plants, suggesting that the site ofH2O2 generation plays an important role insignaling (47). Similarly, extracellular but notphotorespiratory H2O2 has a role in inductionof APX2 (15, 45).
Systemic Acquired Acclimationto Excess Light
Plants exhibit systemic acclimation to EL, inwhich exposure of part of the plant to EL re-sults in acclimation of distal, nonexposed leavesto EL (69, 138). The acclimated state in the sec-ondary site is associated with H2O2 production,induced expression of EL-responsive genesAPX2 and ZAT10, and a slower decline in PS IIefficiency when exposed to EL (45, 69, 138).Transcriptome analysis of EL-exposed, dis-tal leaves and of ZAT10-overexpressing plantsidentified many new systemic acquired accli-mation target genes, which partly overlap withthe ZAT10 abiotic stress regulon (69, 138).Hierarchical clustering comparisons with var-ious hormone responses indicate similaritiesto ABA and jasmonic acid responses (138).However, known hormone signaling mutantsare not defective in systemic acquired acclima-
tion; thus, the systemic signal for EL remainselusive.
POSSIBLE REGULATIONOF EXCESS LIGHT RESPONSESBY CHLOROPHYLLINTERMEDIATES
EL can also potentially regulate nuclear geneexpression by affecting tetrapyrrole biosynthe-sis. The biosynthesis of chlorophyll and heme istightly controlled by multiple regulation mech-anisms to avoid the accumulation of phototoxicintermediates in the light. For example, theformation of 5-aminolevulinic acid (ALA) froml-glutamate is subject to negative feed-back regulation by various intermediates oftetrapyrrole synthesis (reviewed in Reference157). ALA formation is decreased by ELin cucumber (1) and by overexpression ofELIP2, an EL-induced gene (see above), inA. thaliana (160), suggesting that mechanismsexist to prevent accumulation of tetrapyr-role intermediates in EL. Nevertheless,increased levels of chlorophyll intermediatessuch as Mg-protoporphyrin IX (Mg-Proto)and Mg-protoporphyrin IX monomethylester (Mg-ProtoMe) have been measured inalgal cells shifted from dark to light (81). Simi-larly, increased Mg-Proto levels were detectedin plants exposed to photooxidative stress in thechloroplast caused by norflurazon or amitroletreatment, which inhibits carotenoid biosyn-thesis (8, 83, 150), although these findingshave been questioned by two recent studiesshowing that there is no rise in any chlorophyllintermediate in norflurazon-treated A. thalianaseedlings (106, 109). Increased ROS produc-tion in cucumber exposed to MV results in theaccumulation of Mg-ProtoMe, whereas thesame treatment under EL increases levels ofprotoporphyrin IX (Proto) (2). Conversely, ELdecreases the enhanced porphyrin accumula-tion in transgenic tobacco plants with loweractivity of protoporphyrinogen oxidase, whichcatalyzes the conversion of protoporphyrino-gen to Proto (90). This effect of EL protectsthe plants from photooxidative damage and
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has been attributed to stimulated degradationof the chlorophyll intermediates and higherantioxidant levels. Altogether, these resultssuggest that EL might have different effectson the accumulation of chlorophyll precursorsdepending on the specific conditions.
Accumulation of chlorophyll precursors,such as Mg-Proto (Figure 3) and Mg-ProtoMe,as well as heme, stimulates the expression of thenuclear HSP70 and HEMA genes in C. rein-hardtii (81, 163, 164). Chlorophyll intermedi-ates also repress the light-dependent expres-sion of LHCB genes in plants and algae (63, 83,150, 163; reviewed in References 16, 118, and170). Direct involvement of these intermedi-ates in signal transduction was demonstrated inC. reinhardtii by the exogenous addition of Mg-Proto to dark-incubated cultures, mimickingthe chloroplast signal responsible for HSP70induction in the light (81).
In A. thaliana, Mg-Proto was hypothesizedto be a retrograde signal from the chloroplast(Figure 3) on the basis of data obtained withmutants that are defective in the norflurazon-induced downregulation of LHCB expression(153). Five genomes uncoupled ( gun) mutantsare known, four of which directly influencetetrapyrrole biosynthesis by preventing accu-mulation of Mg-Proto (150). The gun2 andgun3 mutations are allelic to hy1 and hy2, andthe genes encode heme oxygenase and phy-tochromobilin synthase, respectively, which areenzymes acting downstream of heme in theheme branch of the tetrapyrrole pathway. Themutations presumably result in heme accu-mulation, which lowers tetrapyrrole biosyn-thesis by negative feedback inhibition (150).The gun5 mutation was localized in the CHLHgene, which codes for the Mg-chelatase Hsubunit (105), and GUN4 is a porphyrin-binding protein that stimulates the activity ofMg-chelatase at low Mg2+ levels (26). Thus,gun4 and gun5 directly inhibit the formationof Mg-Proto, which was suggested to act asthe signal to repress LHCB expression. Con-sistent with this hypothesis, a mutant affectingthe Mg-Proto methyltransferase gene showseven higher LHCB suppression than norflura-
zon treatment (131). However, two recent stud-ies excluded Mg-Proto as the plastid-to-nucleusretrograde signal by showing that the steady-state accumulation of chlorophyll intermediatesdoes not correlate with nuclear gene expres-sion profiles (106, 109). Thus, further work isneeded to unravel the nature of the plastid sig-nal that mediates norflurazon-induced LHCBrepression.
Relatively little is known about how a sig-nal that is somehow related to tetrapyrrolemetabolism might be transmitted to the nu-cleus. In C. reinhardtii, Mg-Proto or Mg-ProtoMe is proposed to be directly exportedout of the chloroplast (Figure 3) in a light-requiring process to activate the genetic re-sponse (81). Accumulation of Mg-Proto in thecytoplasm of norflurazon-treated A. thalianaplants was directly shown by confocal laserscanning spectroscopy, but only after feedingwith ALA (8). However, a role downstreamof Mg-Proto metabolism in regulating nucleargene expression was shown for GUN1, whichencodes a chloroplast-localized pentatricopep-tide repeat protein, and ABI4, a transcriptionfactor originally identified by its involvementin ABA signaling (78). Interestingly, gun1 andabi4 mutations partially block the repression ofLHCB and the increase in ZAT12 and ZAT10mRNA levels by EL (78), and gun1 seedlingsare more sensitive to EL than wild type (139).Thus, GUN1 and ABI4 seem to be involvedin multiple retrograde signaling pathways(Figure 3). The DNA binding activity ofGUN1 suggests that it controls plastid gene ex-pression, and a component acting downstreamof GUN1 is hypothesized to be responsiblefor transmitting the signal out of the chloro-plast (78). An abi4 mutant is also deficientin ABA-dependent LHCB repression, whereasgun1 is not affected in this response, show-ing that ABA is not the downstream signalmolecule for the GUN1-mediated retrogradesignal. The data indicate that ABI4 could bea master switch that regulates nuclear geneexpression in response to the developmen-tal and physiological state of the chloroplast(78, 132).
Although several different EL-induced signalscan clearly cause physiological and geneticresponses in photosynthetic organisms, rela-tively little is known about how the signals aretransmitted to their endpoints and how the dif-ferent signaling pathways interact. These ques-tions are difficult to address, because it is prac-tically impossible to stimulate one pathway byEL without affecting others. Indeed, a lot ofcross-talk appears to occur between EL signal-ing pathways and with abiotic stress signaling,including various ROS signaling pathways. Forexample, in microarray experiments there is astrong overlap between a set of putative PQ-regulated genes (3) and the set of genes whoseexpression in EL is affected in the cry1 mu-tant (77), most notably the ELIP and phenyl-propanoid genes. Furthermore, cry1 mutantshave been isolated by screening for new gunmutants (139). Indeed, as we learn more about
EL signaling, more examples of interactions be-tween EL signaling pathways will likely emerge.
Why did photosynthetic organisms evolveso many different ways to sense and respondto EL? One possible explanation is that differ-ent sensing mechanisms and signaling pathwaysoperate on different timescales and in responseto different ranges of light intensity (degrees ofEL). Some very rapid responses to EL, such asqE, need to occur locally (in the chloroplast),whereas slower responses can involve changesin gene expression. At the lower end of the ELscale, redox signals might have a more promi-nent role, and ROS might come into play un-der more extreme EL conditions when certainprotection mechanisms are overwhelmed. Byemploying a variety of EL sensing and signal-ing mechanisms, photosynthetic organisms areable to maintain photosynthetic efficiency andto cope with the changes in light intensity thatoccur in nature.
SUMMARY POINTS
1. Excess light (EL) can be sensed directly by photoreceptors or indirectly through bio-chemical and metabolic signals.
2. Photoreceptors mediate chloroplast avoidance movement, some changes in gene expres-sion, and photophobic responses in algae.
3. Because EL perturbs photosynthesis, responses in the chloroplast such as qE (pH-dependent regulation of photosynthetic light harvesting) and several retrograde signalingpathways from the chloroplast to the nucleus have important roles in EL acclimation.
4. Reactive oxygen species (ROS) generated as by-products of photosynthesis, especiallysinglet oxygen and H2O2, are signals involved in responses to EL.
5. Integration of multiple signals and coordination with other environmental stress signaltransduction pathways are important for acclimation to EL.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.
ACKNOWLEDGMENTS
We thank Graham Peers for comments on the manuscript. Relevant work in our lab is supportedby grants from the Office of Basic Energy Sciences, Chemical Sciences Division, U.S. Department
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of Energy (contract DE-AC03–76SF000098) and the National Institutes of Health (GM071908).We apologize to colleagues whose relevant work we were not able to cite and/or discuss extensivelybecause of space constraints.
LITERATURE CITED
1. Aarti D, Tanaka R, Ito H, Tanaka A. 2007. High light inhibits chlorophyll biosynthesis at the level of5-aminolevulinate synthesis during de-etiolation in cucumber (Cucumis sativus) cotyledons. Photochem.Photobiol. 83:171–76
2. Aarti PD, Tanaka R, Tanaka A. 2006. Effects of oxidative stress on chlorophyll biosynthesis in cucumber(Cucumis sativus) cotyledons. Physiol. Plant. 128:186–97
3. Adamiec M, Drath M, Jackowski G. 2008. Redox state of plastoquinone pool regulates expression ofArabidopsis thaliana genes in response to elevated irradiance. Acta Biochim. Pol. 55:161–73
4. Ahmad M, Cashmore AR. 1993. HY4 gene of A. thaliana encodes a protein with characteristics of ablue-light photoreceptor. Nature 366:162–66
5. Ahn SG, Thiele DJ. 2003. Redox regulation of mammalian heat shock factor 1 is essential for Hsp geneactivation and protection from stress. Genes Dev. 17:516–28
6. Ahn TK, Avenson TJ, Ballottari M, Cheng Y-C, Niyogi KK, et al. 2008. Architecture of a charge-transferstate regulating light harvesting in a plant antenna protein. Science 320:794–99
7. Anderson JM, Price GD, Chow WS, Hope AB, Badger MR. 1997. Reduced levels of cytochromebf complex in transgenic tobacco leads to marked photochemical reduction of the plastoquinone pool,without significant change in acclimation to irradiance. Photosynth. Res. 53:215–27
8. Ankele E, Kindgren P, Pesquet E, Strand A. 2007. In vivo visualization of Mg-ProtoporphyrinIX, acoordinator of photosynthetic gene expression in the nucleus and the chloroplast. Plant Cell 19:1964–79
9. Anwaruzzaman M, Chin BL, Li X-P, Lohr M, Martinez DA, Niyogi KK. 2004. Genomic analysis ofmutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in Chlamy-domonas reinhardtii. Photosynth. Res. 82:265–76
10. Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction.Annu. Rev. Plant Biol. 55:373–99
11. Asada K. 2006. Production and scavenging of reactive oxygen species in chloroplasts and their functions.Plant Physiol. 141:391–96
12. Baier M, Dietz KJ. 2005. Chloroplasts as source and target of cellular redox regulation: a discussion onchloroplast redox signals in the context of plant physiology. J. Exp. Bot. 56:1449–62
13. Ball L, Accotto G-P, Bechtold U, Creissen G, Funck D, et al. 2004. Evidence for a direct link betweenglutathione biosynthesis and stress defense gene expression in Arabidopsis. Plant Cell 16:2448–62
14. Ballottari M, Dall’Osto L, Morosinotto T, Bassi R. 2007. Contrasting behavior of higher plant photo-system I and II antenna systems during acclimation. J. Biol. Chem. 282:8747–958
15. Bechtold U, Richard O, Zamboni A, Gapper C, Geisler M, et al. 2008. Impact of chloroplastic- andextracellular-sourced ROS on high light-responsive gene expression in Arabidopsis. J. Exp. Bot. 59:121–33
16. Beck CF. 2005. Signaling pathways from the chloroplast to the nucleus. Planta 222:743–5617. Bellafiore S, Bameche F, Peltier G, Rochaix J-D. 2005. State transitions and light adaptation require
chloroplast thylakoid protein kinase STN7. Nature 433:892–9518. Bergantino E, Segalla A, Brunetta A, Teardo E, Rigoni F, et al. 2003. Light- and pH-dependent structural
changes in the PsbS subunit of photosystem II. Proc. Natl. Acad. Sci. USA 100:15265–7019. Bonardi V, Pesaresi P, Becker T, Schleiff E, Wagner R, et al. 2005. Photosystem II core phosphorylation
and photosynthetic acclimation require two different protein kinases. Nature 437:1179–8220. Bonente G, Howes BD, Caffarri S, Smulevich G, Bassi R. 2008. Interactions between the photosystem
II subunit PsbS and xanthophylls studied in vivo and in vitro. J. Biol. Chem. 283:8434–4521. Bugos RC, Yamamoto HY. 1996. Molecular cloning of violaxanthin de-epoxidase from romaine lettuce
and expression in Escherichia coli. Proc. Natl. Acad. Sci. USA 93:6320–25
23. Christie JM. 2007. Phototropin blue-light receptors. Annu. Rev. Plant Biol. 58:21–4524. Cohen I, Knopf JA, Irihimovitch V, Shapira M. 2005. A proposed mechanism for the inhibitory effects
of oxidative stress on Rubisco assembly and its subunit expression. Plant Physiol. 137:738–4625. Cohen I, Sapir Y, Shapira M. 2006. A conserved mechanism controls translation of Rubisco large subunit
in different photosynthetic organisms. Plant Physiol. 141:1089–9726. Davison PA, Schubert HL, Reid JD, Iorg CD, Heroux A, et al. 2005. Structural and biochemical charac-
terization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis. Biochemistry 44:7603–1227. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, et al. 2005. Cytosolic ascorbate peroxidase
1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17:268–8128. Davletova S, Schlauch K, Coutu J, Mittler R. 2005. The zinc-finger protein Zat12 plays a central role
in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol. 139:847–5629. DeBlasio SL, Luesse DL, Hangarter RP. 2005. A plant-specific protein essential for blue-light-induced
chloroplast movements. Plant Physiol. 139:101–1430. Demmig-Adams B, Adams WW III. 1992. Photoprotection and other responses of plants to high light
stress. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:599–62631. Depege N, Bellafiore S, Rochaix J-D. 2003. Role of chloroplast protein kinase Stt7 in LHCII phospho-
rylation and state transition in Chlamydomonas. Science 299:1572–7532. Dominici P, Caffarri S, Armenante F, Ceoldo S, Crimi M, Bassi R. 2002. Biochemical properties of
the PsbS subunit of Photosystem II either purified from chloroplast or recombinant. J. Biol. Chem.277:22750–58
34. Durnford DG, Price JA, McKim SM, Sarchfield ML. 2003. Light-harvesting complex gene expressionis controlled by both transcriptional and post-transcriptional mechanisms during photoacclimation inChlamydomonas reinhardtii. Physiol. Plant. 118:193–205
35. Escoubas J-M, Lomas M, LaRoche J, Falkowski PG. 1995. Light intensity regulation of cab gene tran-scription is signaled by the redox state of the plastoquinone pool. Proc. Natl. Acad. Sci. USA 92:10237–41
37. Fey V, Wagner R, Brautigam K, Wirtz M, Hell R, et al. 2005. Retrograde plastid redox signals in theexpression of nuclear genes for chloroplast proteins of Arabidopsis thaliana. J. Biol. Chem. 280:5318–28
38. Fischer BB, Eggen RIL, Trebst A, Krieger-Liszkay A. 2006. The glutathione peroxidase homologousgene Gpxh in Chlamydomonas reinhardtii is upregulated by singlet oxygen produced in photosystem II.Planta 223:583–90
39. Fischer BB, Krieger-Liszkay A, Eggen RIL. 2004. Photosensitizers neutral red (type I) and rose bengal(type II) cause light-dependent toxicity in Chlamydomonas reinhardtii and induce the Gpxh gene viaincreased singlet oxygen formation. Environ. Sci. Technol. 38:6307–13
40. Fischer BB, Krieger-Liszkay A, Eggen RIL. 2005. Oxidative stress induced by the photosensitizers neutralred (type I) or rose bengal (type II) in the light causes different molecular responses in Chlamydomonasreinhardtii. Plant Sci. 168:747–59
41. Fischer BB, Krieger-Liszkay A, Hideg E, Snyrychova I, Wiesendanger M, Eggen RIL. 2007. Role ofsinglet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii. FEBS Lett.581:5555–60
42. Fischer BB, Wiesendanger M, Eggen RIL. 2006. Growth condition-dependent sensitivity, photodamageand stress response of Chlamydomonas reinhardtii exposed to high light conditions. Plant Cell Physiol.47:1135–45
43. Foster KW, Saranak J, Patel N, Zarilli G, Okabe M, et al. 1984. A rhodopsin is the functional photore-ceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature 311:756–59
44. Frigerio S, Campoli C, Zorzan S, Fantoni LI, Crosatti C, et al. 2007. Photosynthetic antenna sizein higher plants is controlled by the plastoquinone redox state at the post-transcriptional rather thantranscriptional level. J. Biol. Chem. 282:29457–69
254 Li et al.
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:23
9-26
0. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-12 ARI 25 March 2009 13:47
45. Fryer MJ, Ball L, Oxborough K, Karpinski S, Mullineaux PM, Baker NR. 2003. Control of AscorbatePeroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals afunctional organisation of Arabidopsis leaves. Plant J. 33:691–705
46. Funk C, Schroder WP, Napiwotzki A, Tjus SE, Renger G, Andersson B. 1995. The PSII-S protein ofhigher plants: a new type of pigment-binding protein. Biochemistry 34:11133–41
47. Gadjev I, Vanderauwera S, Gechev TS, Laloi C, Minkov IN, et al. 2006. Transcriptomic footprintsdisclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiol. 141:436–45
48. Gisselsson A, Szilagyi A, Akerlund H-E. 2004. Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis. Physiol. Plant. 122:337–43
49. Govorunova EG, Jung KH, Sineshchekov OA, Spudich JL. 2004. Chlamydomonas sensory rhodopsins Aand B: cellular content and role in photophobic responses. Biophys. J. 86:2342–49
50. Graßes T, Pesaresi P, Schiavon F, Varotto C, Salamini F, et al. 2002. The role of �pH-dependentdissipation of excitation energy in protecting photosystem II against light-induced damage in Arabidopsisthaliana. Plant Physiol. Biochem. 40:41–49
51. Haehnel W. 1984. Photosynthetic electron transport in higher plants. Annu. Rev. Plant Physiol. 35:659–9352. Hager A. 1969. Lichtebedingte pH-Erniedrigung in einem Chloroplasten-Kompartiment als Ursache
der enzymatischen Violaxanthin-Zeaxanthin-Umwandlung: Beziehungen zur Photophosphorylierung.Planta 89:224–43
53. Hieber AD, Bugos RC, Verhoeven AS, Yamamoto HY. 2002. Overexpression of violaxanthin de-epoxidase: properties of C-terminal deletions on activity and pH-dependent lipid binding. Planta214:476–83
54. Horton P, Johnson MP, Perez-Bueno ML, Kiss AZ, Ruban AV. 2008. Photosynthetic acclimation:does the dynamic structure and macro-organisation of photosystem II in higher plant grana membranesregulate light harvesting states? FEBS J. 275:1069–79
55. Horton P, Ruban AV, Walters RG. 1996. Regulation of light harvesting in green plants. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 47:655–84
56. Huala E, Oeller PW, Liscum E, Han IS, Larsen E, Briggs WR. 1997. Arabidopsis NPH1: a protein kinasewith a putative redox-sensing domain. Science 278:2120–23
57. Huner NPA, Oquist G, Sarhan F. 1998. Energy balance and acclimation to light and cold. Trends PlantSci. 3:224–30
58. Iida A, Kazuoka T, Torikai S, Kikuchi H, Oeda K. 2000. A zinc finger protein RHL41 mediates the lightacclimatization response in Arabidopsis. Plant J. 24:191–203
59. Im C-S, Zhang Z, Shrager J, Chang C-W, Grossman AR. 2003. Analysis of light and CO2 regulation inChlamydomonas reinhardtii using genome-wide approaches. Photosynth. Res. 75:111–25
60. Irihimovitch V, Shapira M. 2000. Glutathione redox potential modulated by reactive oxygen speciesregulates translation of Rubisco large subunit in the chloroplast. J. Biol. Chem. 275:16289–95
61. Jansson S. 1999. A guide to the Lhc genes and their relatives in Arabidopsis. Trends Plant Sci. 4:236–4062. Jarillo JA, Gabrys H, Capel J, Alonso JM, Ecker JR, Cashmore AR. 2001. Phototropin-related NPL1
controls chloroplast relocation induced by blue light. Nature 410:952–5463. Johanningmeier U, Howell SH. 1984. Regulation of light-harvesting chlorophyll-binding protein mRNA
accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors.J. Biol. Chem. 259:13541–49
64. Kadota A, Sato Y, Wada M. 2000. Intracellular chloroplast photorelocation in the moss Physcomitrellapatens is mediated by phytochrome as well as by a blue-light receptor. Planta 210:932–37
65. Kagawa T, Kasahara M, Abe T, Yoshida S, Wada M. 2004. Function analysis of phototropin2 using fernmutants deficient in blue light-induced chloroplast avoidance movement. Plant Cell Physiol. 45:416–26
66. Kagawa T, Sakai T, Suetsugu N, Oikawa K, Ishiguro S, et al. 2001. Arabidopsis NPL1: a phototropinhomolog controlling the chloroplast high-light avoidance response. Science 291:2138–41
67. Kanazawa A, Kramer DM. 2002. In vivo modulation of nonphotochemical exciton quenching (NPQ) byregulation of the chloroplast ATP synthase. Proc. Natl. Acad. Sci. USA 99:12789–94
68. Karpinski S, Escobar C, Karpinska B, Creissen G, Mullineaux PM. 1997. Photosynthetic electron trans-port regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess lightstress. Plant Cell 9:627–40
69. Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P. 1999. Systemic signalingand acclimation in response to excess excitation energy in Arabidopsis. Science 284:654–57
70. Kasahara M, Kagawa T, Oikawa K, Suetsugu N, Miyao M, Wada M. 2002. Chloroplast avoidancemovement reduces photodamage in plants. Nature 420:829–32
71. Kasahara M, Kagawa T, Sato Y, Kiyosue T, Wada M. 2004. Phototropins mediate blue and red light-induced chloroplast movements in Physcomitrella patens. Plant Physiol. 135:1388–97
72. Kawai H, Kanegae T, Christensen S, Kiyosue T, Sato Y, et al. 2003. Responses of ferns to red light aremediated by an unconventional photoreceptor. Nature 421:287–90
73. Kawano M, Kuwabara T. 2000. pH-dependent reversible inhibition of violaxanthin de-epoxidase bypepstatin related to protonation-induced structural change of the enzyme. FEBS Lett. 481:101–4
74. Kimura M, Manabe K, Abe T, Yoshida S, Matsui M, Yamamoto YY. 2003. Analysis of hydrogen peroxide-independent expression of the high-light-inducible ELIP2 gene with the aid of the ELIP2 promoter-luciferase fusion. Photochem. Photobiol. 77:668–74
75. Kimura M, Yamamoto YY, Seki M, Sakurai T, Sato M, et al. 2003. Identification of Arabidopsis genesregulated by high light-stress using cDNA microarray. Photochem. Photobiol. 77:226–33
76. Kimura M, Yoshizumi T, Manabe K, Yamamoto YY, Matsui M. 2001. Arabidopsis transcriptional regula-tion by light stress via hydrogen peroxide-dependent and -independent pathways. Genes Cells 6:607–17
77. Kleine T, Kindgren P, Benedict C, Hendrickson L, Strand A. 2007. Genome-wide gene expressionanalysis reveals a critical role for CRYPTOCHROME1 in the response of Arabidopsis to high irradiance.Plant Physiol. 144:1391–406
78. Koussevitzky S, Nott A, Mockler TC, Hong F, Sachetto-Martins G, et al. 2007. Signals from chloroplastsconverge to regulate nuclear gene expression. Science 316:715–19
79. Koziol AG, Borza T, Ishida K-I, Keeling P, Lee RW, Durnford DG. 2007. Tracing the evolution of thelight-harvesting antennae in chlorophyll a/b-containing organisms. Plant Physiol. 143:1802–16
80. Krieger-Liszkay A. 2005. Singlet oxygen production in photosynthesis. J. Exp. Bot. 56:337–4681. Kropat J, Oster U, Rudiger W, Beck CF. 2000. Chloroplast signalling in the light induction of
nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility tocytoplasm/nucleus. Plant J. 24:523–31
82. Kulheim C, Agren J, Jansson S. 2002. Rapid regulation of light harvesting and plant fitness in the field.Science 297:91–93
83. La Rocca N, Rascio N, Oster U, Rudiger W. 2001. Amitrole treatment of etiolated barley seedlingsleads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes. Planta213:101–8
84. Laloi C, Stachowiak M, Pers-Kamczyc E, Warzych E, Murgia I, Apel K. 2007. Cross-talk betweensinglet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA 104:672–77
85. Ledford HK, Baroli I, Shin JW, Fischer BB, Eggen RIL, Niyogi KK. 2004. Comparative profiling oflipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas reinhardtii. Mol. Genet. Genomics 272:470–79
86. Ledford HK, Chin BL, Niyogi KK. 2007. Acclimation to singlet oxygen stress in Chlamydomonas rein-hardtii. Eukaryot. Cell 6:919–30
87. Lee KP, Kim C, Landgraf F, Apel K. 2007. EXECUTER1- and EXECUTER2-dependent transfer ofstress-related signals from the plastid to the nucleus of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA104:10270–75
88. Lee S, Carlson T, Christian N, Lea K, Kedzie J, et al. 2000. The yeast heat shock transcription factorchanges conformation in response to superoxide and temperature. Mol. Biol. Cell. 11:1753–64
89. Leisinger U, Rufenacht K, Fischer B, Pesaro M, Spengler A, et al. 2001. The glutathione peroxidasehomologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen.Plant Mol. Biol. 46:395–408
90. Lermontova I, Grimm B. 2006. Reduced activity of plastid protoporphyrinogen oxidase causes attenuatedphotodynamic damage during high-light compared to low-light exposure. Plant J. 48:499–510
91. Li X-P, Bjorkman O, Shih C, Grossman AR, Rosenquist M, et al. 2000. A pigment-binding proteinessential for regulation of photosynthetic light harvesting. Nature 403:391–95
256 Li et al.
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:23
9-26
0. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-12 ARI 25 March 2009 13:47
92. Li X-P, Gilmore AM, Caffarri S, Bassi R, Golan T, et al. 2004. Regulation of photosynthetic lightharvesting involves intrathylakoid lumen pH sensing by the PsbS protein. J. Biol. Chem. 279:22866–74
93. Li X-P, Muller-Moule P, Gilmore AM, Niyogi KK. 2002. PsbS-dependent enhancement of feedbackde-excitation protects photosystem II from photoinhibition. Proc. Natl. Acad. Sci. USA 99:15222–27
94. Li X-P, Phippard A, Pasari J, Niyogi KK. 2002. Structure-function analysis of Photosystem II subunit S(PsbS) in vivo. Funct. Plant Biol. 29:1131–39
95. Lin C, Yang H, Guo H, Mockler T, Chen J, Cashmore AR. 1998. Enhancement of blue-light sensitivityof Arabidopsis seedlings by a blue light receptor cryptochrome 2. Proc. Natl. Acad. Sci. USA 95:2686–90
96. Luesse DR, DeBlasio SL, Hangarter RP. 2006. Plastid movement impaired 2, a new gene involved innormal blue-light-induced chloroplast movements in Arabidopsis. Plant Physiol. 141:1328–37
97. Masuda T, Tanaka A, Melis A. 2003. Chlorophyll antenna size adjustments by irradiance in Dunaliellasalina involve coordinate regulation of chlorophyll a oxygenase (CAO) and Lhcb gene expression. PlantMol. Biol. 51:757–71
98. Maxwell DP, Laudenbach DE, Huner NPA. 1995. Redox regulation of light-harvesting complex II andcab mRNA abundance in Dunaliella salina. Plant Physiol. 109:787–95
99. McKim SM, Durnford DG. 2006. Translational regulation of light-harvesting complex expression duringphotoacclimation to high-light in Chlamydomonas reinhardtii. Plant Physiol. Biochem. 44:857–65
100. Melis A. 1991. Dynamics of photosynthetic membrane composition and function. Biochim. Biophys. Acta1058:87–106
101. Meskauskiene R, Nater M, Goslings D, Kessler F, op den Camp R, Apel K. 2001. FLU: a negativeregulator of chlorophyll biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 93:12026–34
102. Miller G, Mittler R. 2006. Could heat shock transcription factors function as hydrogen peroxide sensorsin plants? Ann. Bot. 98:279–88
103. Mittler R, Kim Y, Song L, Coutu J, Coutu A, et al. 2006. Gain- and loss-of-function mutations in Zat10enhance the tolerance of plants to abiotic stress. FEBS Lett. 580:6537–42
104. Mittmann F, Brucker G, Zeidler M, Repp A, Abts T, et al. 2004. Targeted knockout in Physcomitrellareveals direct actions of phytochrome in the cytoplasm. Proc. Natl. Acad. Sci. USA 101:13939–44
105. Mochizuki N, Brusslan JA, Larkin R, Nagatani A, Chory J. 2001. Arabidopsis genomes uncoupled 5 (GUN5 )mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc.Natl. Acad. Sci. USA 98:2053–58
106. Mochizuki N, Tanaka R, Tanaka A, Masuda T, Nagatani A. 2008. The steady-state level of Mg-protoporphyrin IX is not a determinant of plastid-to-nucleus signaling in Arabidopsis. Proc. Natl. Acad.Sci. USA 105:15184–89
107. Montane M-H, Tardy F, Kloppstech K, Havaux M. 1998. Differential control of xanthophylls and light-induced stress proteins, as opposed to light-harvesting chlorophyll a/b proteins, during photosyntheticacclimation of barley leaves to light irradiance. Plant Physiol. 118:227–35
108. Morosinotto T, Bassi R, Frigerio S, Finazzi G, Morris E, Barber J. 2006. Biochemical and structuralanalyses of a higher plant photosystem II supercomplex of a photosystem I-less mutant of barley. Con-sequences of a chronic over-reduction of the plastoquinone pool. FEBS J. 273:4616–30
109. Moulin M, McCormac AC, Terry MJ, Smith AG. 2008. Tetrapyrrole profiling in Arabidopsis seedlingsreveals that retrograde plastid nuclear signaling is not due to Mg-protoporphyrin IX accumulation.Proc. Natl. Acad. Sci. USA 105:15178–83
110. Muller P, Li X-P, Niyogi KK. 2001. Non-photochemical quenching. A response to excess light energy.Plant Physiol. 125:1558–66
111. Mullineaux P, Karpinski S. 2002. Signal transduction in response to excess light: getting out of thechloroplast. Curr. Opin. Plant Biol. 5:43–48
112. Mullineaux PM, Karpinski S, Baker NR. 2006. Spatial dependence for hydrogen peroxide-directedsignaling in light-stressed plants. Plant Physiol. 141:346–50
113. Mullineaux PM, Rausch T. 2005. Glutathione, photosynthesis and the redox regulation of stress-responsive gene regulation. Photosynth. Res. 86:459–74
114. Murchie EH, Hubbart S, Peng S, Horton P. 2005. Acclimation of photosynthesis to high irradiance inrice: gene expression and interactions with leaf development. J. Exp. Bot. 56:449–60
115. Mussgnug JH, Wobbe L, Elles I, Claus C, Hamilton M, et al. 2005. NAB1 is an RNA binding proteininvolved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonasreinhardtii. Plant Cell 17:3409–21
116. Nagel G, Ollig D, Fuhrmann M, Kateriya S, Musti AM, et al. 2002. Channelrhodopsin-1: a light-gatedproton channel in green algae. Science 296:2395–98
117. Niyogi KK. 1999. Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol.Plant Mol. Biol. 50:333–59
118. Nott A, Jung H-S, Koussevitzky S, Chory J. 2006. Plastid-to-nucleus retrograde signaling. Annu. Rev.Plant Biol. 57:739–59
119. Nozue K, Kanegae T, Imaizumi T, Fukuda S, Okamoto H, et al. 1998. A phytochrome from the fernAdiantum with features of the putative photoreceptor NPH1. Proc. Natl. Acad. Sci. USA 95:15826–30
120. Oelze M-L, Kandlbinder A, Dietz K-J. 2008. Redox regulation and overreduction control in the photo-synthesizing cell: complexity in redox regulatory networks. Biochim. Biophys. Acta 1780:1261–72
121. Oikawa K, Kasahara M, Kiyosue T, Kagawa T, Suetsugu N, et al. 2003. CHLOROPLAST UNUSUALPOSITIONING1 is essential for proper chloroplast positioning. Plant Cell 15:2805–15
122. op den Camp RGL, Przybyla D, Ochsenbein C, Laloi C, Kim C, et al. 2003. Rapid induction of distinctstress responses after the release of singlet oxygen in Arabidopsis. Plant Cell 15:2320–32
123. Ort DR. 2001. When there is too much light. Plant Physiol. 125:29–32124. Panchuk II, Volkov RA, Schoffl F. 2002. Heat stress- and heat shock transcription factor-dependent
expression and activity of ascorbate peroxidase in Arabidopsis. Plant Physiol. 129:838–53125. Pesaresi P, Schneider A, Kleine T, Leister D. 2007. Interorganellar communication. Curr. Op. Plant Biol.
10:600–6126. Peterson RB, Havir EA. 2001. Photosynthetic properties of an Arabidopsis thaliana mutant possessing a
defective PsbS gene. Planta 214:142–52127. Pfannschmidt T, Brautigam K, Wagner R, Dietzel L, Schroter Y, et al. 2008. Potential regulation of gene
expression in photosynthetic cells by redox and energy state: approaches towards better understanding.Ann. Bot.: doi: 10.1093/aob/mcn081
128. Pfannschmidt T, Nilsson A, Allen JF. 1999. Photosynthetic control of chloroplast gene expression.Nature 397:625–8
129. Pfundel EE, Dilley RA. 1993. The pH dependence of violaxanthin deepoxidation in isolated pea chloro-plasts. Plant Physiol. 101:65–71
130. Pnueli L, Liang H, Rozenberg M, Mittler R. 2003. Growth suppression, altered stomatal responses, andaugmented induction of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsisplants. Plant J. 34:187–203
131. Pontier D, Albrieux C, Joyard J, Lagrange T, Block MA. 2007. Knock-out of the magnesium protopor-phyrin IX methyltransferase gene in Arabidopsis: effects on chloroplast development and on chloroplast-to-nucleus signaling. J. Biol. Chem. 282:2297–304
132. Richly E, Dietzmann A, Biehl A, Kurth J, Laloi C, et al. 2003. Covariations in the nuclear chloroplasttranscriptome reveal a regulatory master-switch. EMBO Rep. 4:491–98
133. Rizhsky L, Davletova S, Liang H, Mittler R. 2004. The zinc finger protein Zat12 is required for cytosolicascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J. Biol. Chem. 279:11736–43
134. Rizhsky L, Liang H, Mittler R. 2003. The water-water cycle is essential for chloroplast protection in theabsence of stress. J. Biol. Chem. 278:38921–25
135. Rochaix J-D. 2007. Role of thylakoid protein kinases in photosynthetic acclimation. FEBS Lett. 581:2768–75
136. Rossel JB, Walter PB, Hendrickson L, Chow WS, Poole A, et al. 2006. A mutation affecting ASCORBATEPEROXIDASE 2 gene expression reveals a link between responses to high light and drought tolerance.Plant Cell Environ. 29:269–81
137. Rossel JB, Wilson IW, Pogson BJ. 2002. Global changes in gene expression in response to high light inArabidopsis. Plant Physiol. 130:1109–20
138. Rossel JB, Wilson PB, Hussain D, Woo NS, Gordon MJ, et al. 2007. Systemic and intracellular responsesto photooxidative stress in Arabidopsis. Plant Cell 19:4091–110
258 Li et al.
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:23
9-26
0. D
ownl
oade
d fr
om a
rjou
rnal
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nual
revi
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Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-12 ARI 25 March 2009 13:47
139. Ruckle ME, DeMarco SM, Larkin RM. 2007. Plastid signals remodel light signaling networks and areessential for efficient chloroplast biogenesis in Arabidopsis. Plant Cell 19:3944–60
140. Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, et al. 2004. Arabidopsis Cys2/His2-typezinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stressconditions. Plant Physiol. 136:2734–46
141. Scarpeci TE, Zanor MI, Carrillo N, Mueller-Roeber B, Valle EM. 2008. Generation of superoxide anionin chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes. PlantMol. Biol. 66:361–78
142. Schultes NP, Peterson RB. 2007. Phylogeny-directed structural analysis of the Arabidopsis PsbS protein.Biochem. Biophys. Res. Comm. 355:464–70
143. Schurmann P, Buchanan BB. 2008. The ferredoxin/thioredoxin system of oxygenic photosynthesis.Antiox. Redox Signal. 10:1235–74
144. Shaikhali J, Heiber I, Seidel T, Stroher E, Hiltscher H, et al. 2008. The redox-sensitive transcriptionfactor Rap2.4a controls nuclear expression of 2-Cys peroxiredoxin A and other chloroplast antioxidantenzymes. BMC Plant Biol. 8:48
145. Shao N, Krieger-Liszkay A, Schroda M, Beck CF. 2007. A reporter system for the individual detectionof hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced invivo. Plant J. 50:475–87
146. Shapira M, Lers A, Heifetz PB, Irihimovitz V, Osmond CB, et al. 1997. Differential regulation ofchloroplast gene expression in Chlamydomonas reinhardtii during photoacclimation: light stress transientlysuppresses synthesis of the Rubisco LSU protein while enhancing synthesis of the PS II D1 protein.Plant Mol. Biol. 33:1001–11
147. Sineshchekov OA, Jung KH, Spudich JL. 2002. Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 99:8689–94
148. Snyder JW, Skovsen E, Lambert JD, Poulsen L, Ogilby PR. 2006. Optical detection of singlet oxygenfrom single cells. Phys. Chem. Chem. Phys. 8:4280–93
149. Storozhenko S, De Pauw P, Van Montagu M, Inze D, Kushnir S. 1998. The heat-shock element is afunctional component of the Arabidopsis APX1 gene promoter. Plant Physiol. 118:1005–14
150. Strand A, Asami T, Alonso J, Ecker JR, Chory J. 2003. Chloroplast to nucleus communication triggeredby accumulation of Mg-protoporphyrinIX. Nature 421:79–83
151. Suetsugu N, Mittmann F, Wagner G, Hughes J, Wada M. 2005. A chimeric photoreceptor gene,NEOCHROME, has arisen twice during plant evolution. Proc. Natl. Acad. Sci. USA 102:13705–9
152. Suetsugu N, Wada M. 2007. Phytochrome-dependent photomovement responses mediated by pho-totropin family proteins in cryptogam plants. Photochem. Photobiol. 83:87–93
153. Susek RE, Ausubel FM, Chory J. 1993. Signal transduction mutants of Arabidopsis uncouple nuclearCAB and RBCS gene expression from chloroplast development. Cell 74:787–99
154. Suzuki T, Yamasaki K, Fujita S, Oda K, Iseki M, et al. 2003. Archaeal-type rhodopsins in Chlamydomonas:model structure and intracellular localization. Biochem. Biophys. Res. Commun. 301:711–17
155. Takizawa K, Cruz JA, Kanazawa A, Kramer DM. 2007. The thylakoid proton motive force in vivo.Quantitative, noninvasive probes, energetics, and regulatory consequences of light-induced pmf. Biochim.Biophys. Acta 1767:1233–44
156. Takizawa K, Kanazawa A, Kramer DM. 2008. Depletion of stromal Pi induces high ‘energy-dependent’antenna exciton quenching (qE) by decreasing proton conductivity at CF0-CF1 ATP synthase. Plant CellEnviron. 31:235–43
157. Tanaka R, Tanaka A. 2007. Tetrapyrrole biosynthesis in higher plants. Annu. Rev. Plant Biol. 58:321–46158. Teramoto H, Nakamori A, Minagawa J, Ono T. 2002. Light-intensity-dependent expression of the
Lhc gene family encoding light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonasreinhardtii. Plant Physiol. 130:325–33
159. Tikkanen M, Piippo M, Suorsa M, Sirpio S, Mulo P, et al. 2006. State transitions revisited—a bufferingsystem for dynamic low light acclimation of Arabidopsis. Plant Mol. Biol. 62:779–93
160. Tzvetkova-Chevolleau T, Franck F, Alawady AE, Dall’Osto L, Carriere F, et al. 2007. The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana. Plant J. 50:795–809
161. Vandenabeele S, Vanderauwera S, Vuylsteke M, Rombauts S, Langebartels C, et al. 2004. Catalasedeficiency drastically affects gene expression induced by high light in Arabidopsis thaliana. Plant J. 39:45–58
162. Vanderauwera S, Zimmermann P, Rombauts S, Vandenabeele S, Langebartels C, et al. 2005. Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-inducedtranscriptional cluster involved in anthocyanin biosynthesis. Plant Physiol. 139:806–21
163. Vasileuskaya Z, Oster U, Beck CF. 2004. Involvement of tetrapyrroles in interorganellar signaling inplants and algae. Photosynth. Res. 82:289–99
164. von Gromoff ED, Alawady A, Meinecke L, Grimm B, Beck CF. 2008. Heme, a plastid-derived regulatorof nuclear gene expression in Chlamydomonas. Plant Cell 20:552–67
165. Wada M, Kagawa T, Sato Y. 2003. Chloroplast movement. Annu. Rev. Plant Biol. 54:455–68166. Wagner D, Przybyla D, op den Camp R, Kim C, Landgraf F, et al. 2004. The genetic basis of singlet
oxygen-induced stress responses of Arabidopsis thaliana. Science 306:1183–85167. Walters RG. 2005. Towards an understanding of photosynthetic acclimation. J. Exp. Bot. 56:435–47168. Walters RG, Rogers JJM, Shephard F, Horton P. 1999. Acclimation of Arabidopsis thaliana to the light
environment: the role of photoreceptors. Planta 209:517–27169. Wilson KE, Huner NPA. 2000. The role of growth rate, redox-state of the plastoquinone pool and the
trans-thylakoid �pH in photoacclimation of Chlorella vulgaris to growth irradiance and temperature.Planta 212:93–102
170. Woodson JD, Chory J. 2008. Coordination of gene expression between organellar and nuclear genomes.Nat. Rev. Genet. 9:383–95
171. Yabuta Y, Maruta T, Yoshimura K, Ishikawa T, Shigeoka S. 2004. Two distinct redox signaling pathwaysfor cytosolic APX induction under photooxidative stress. Plant Cell Physiol. 45:1586–94
172. Yamamoto HY, Bugos RC, Hieber AD. 1999. Biochemistry and molecular biology of the xanthophyllcycle. In The Photochemistry of Carotenoids, ed. HA Frank, AJ Young, G Britton, RJ Cogdell, pp. 293–303.Dordrecht: Kluwer Academic Publishers
174. Yang D-H, Andersson B, Aro E-M, Ohad I. 2001. The redox state of the plastoquinone pool controlsthe level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoac-climation. Photosynth. Res. 68:163–74
175. Zhong M, Orosz A, Wu C. 1998. Direct sensing of heat and oxidation by Drosophila heat shock tran-scription factor. Mol. Cell 2:101–8
An online log of corrections to Annual Review of Plant Biology articles may be found athttp://plant.annualreviews.org/
vi Contents
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Light-derived energy enters the biosphere through photosyn-thesis, and ultimately sustains virtually all living organisms.The rates of reactions involved in primary photochemistry,
the subsequent transformation of the light into reducing power(NADPH) and chemical energy (ATP) and its ultimate utilizationin metabolism and growth differ by at least 15 orders of magni-tude (Fig. 1). Because the primary photochemical reactions ofphotosystem II (PSII) and PSI occur on a much faster time scalethan electron transport and metabolism, exposure of plants to lightenergy that is in excess of that required for photosynthesis re-sults in an energy imbalance that generally leads to photoinhibi-tion1. Whenever the absorbed light energy exceeds the capacity ofthe organism either to use the trapped energy through photosyn-thesis (photochemical quenching) or to dissipate it as heat (non-photochemical quenching), damage to PSII may occur (Fig. 1).Furthermore, exposure to photoinhibitory conditions in oxygenicorganisms can result in the formation of damaging reactive oxy-gen species either through the reduction of O2 to yield the super-oxide anion radical (O2
2.) or through energy transfer from excitedtriplet chlorophyll to ground-state O2 to form singlet oxygen (1O2)(Ref. 2). If unchecked, irreversible photoinhibition can lead tosignificant decreases in plant productivity1. Thus, it is imperativethat plants maintain a balance between the energy supplied throughphotochemistry and the energy consumed through photosyntheticCO2 assimilation and contiguous metabolic pathways3–6.
Cold acclimation: a potential ‘energy crisis’Plants can experience and must adjust to wide daily and seasonalfluctuations in temperature. Plant responses to these changes canbe divided into two principal components. Adaptation is a geno-typic response to long-term environmental changes. The alterationsin the genome are stable and remain in the population over gener-ations. By contrast, acclimation is a response induced by an environ-mental change that causes a phenotypic alteration with no changein genetic complement. However, acclimation is usually initiated bya stress response to an abrupt change in the environment that is char-acterized by transient, physiological, biochemical and molecularperturbations. The stress response subsequently leads to stable,long-term adjustments that reflect a developmental response to thenew environmental condition. Thus, cold acclimation is a long-termdevelopmental response to low temperature that results in the at-tainment of maximum freezing tolerance. This is estimated as thefreezing temperature required to kill 50% of a plant population(LT50). The energy required to attain the cold-acclimated state isderived from photosynthesis.
The potential for an energy imbalance between photochem-istry, electron transport and metabolism is exacerbated under con-ditions of either high light or cold temperatures, which lead toincreased PSII excitation pressure (Box 1). On a time scale ofminutes, organisms can acclimate in an attempt to compensate forexposure to high PSII excitation pressure by reducing energytransfer efficiency to PSII either by diverting energy from PSII toPSI through state transitions or by dissipating excess energy asheat by non-photochemical quenching (Fig. 1). On a longer timescale, photosynthetic acclimation to high PSII excitation pres-sure may occur as a consequence of a reduction in PSII antennasize. These mechanisms result in adjustments in the functionalabsorption cross-sectional area of PSII (sPSII), which would re-duce photosynthetic efficiency measured as either the quantumyield of CO2 assimilation (FCO2) or the quantum yield of O2 evo-lution (FO2). In addition, some plants reduce leaf angle relative to the incident radiation, alter leaf optical properties8 or changetheir position in the water column in the case of algae9 to reducethe incident photon flux. Alternatively, photoautotrophs couldacclimate by increasing the number of components acting as elec-tron-consuming sinks by elevating the levels of Calvin cycleenzymes, which would increase the capacity for CO2 assimilationor photorespiration relative to electron transport. Clearly, innature, photoautotrophs may exploit any one or a combination ofthese mechanisms to offset the potential ‘energy crisis’ duringexposure to fluctuations in environmental conditions such as tem-perature and irradiance (Fig. 1). However, modulation of energybalance is not restricted to light and temperature. CO2 limitationsassociated with drought and deprivation of macronutrients such asnitrate might reduce sink capacity, which also leads to the over-reduction of PSII.
Changes in PSII excitation pressure are reflected in alterationsin the redox state of PSII, which can be monitored in vivo byexploiting chlorophyll a fluorescence as a non-invasive probe10.Photochemical fluorescence quenching can be used to estimatethe proportion of PSII reaction centres that are ‘open’ (reflectingthe relative oxidation state of QA, the first stable quinone electronacceptor of PSII reaction centres) or ‘closed’ (thought to reflectthe relative reduction state of PSII) (Fig. 2). Thus, an estimate canbe made of the relative PSII excitation pressure to which photo-autotrophs are exposed1,6,11. Although not providing an exact esti-mate of the reduction state of PSII, this fluorescence parameterdoes provide a useful estimate of relative changes in PSII exci-tation pressure of organisms exposed to changing environmentalconditions12.
Energy balance and acclimation tolight and coldNorman P.A. Huner, Gunnar Öquist and Fathey Sarhan
Changes in environmental conditions such as light intensity or temperature result in an im-balance between the light energy absorbed through photochemistry versus the energy utilizedthrough metabolism. Such an energy imbalance is sensed through alterations in photo-system II excitation pressure, which reflects the relative reduction state of the photosystem.Modulation of this novel, chloroplastic redox signal either by excess light or by low temperatureinitiates a signal transduction pathway. This appears to coordinate photosynthesis-relatedgene expression and to influence the nuclear expression of a specific cold-acclimation gene,plant morphology and differentiation in cyanobacteria. Thus, in addition to its traditional rolein energy transduction, the photosynthetic apparatus might also be an environmental sensor.
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Fig. 1. Photosynthetic transformation of light energy and mechanisms to maintain an energy balance. Primary photochemistry involves theabsorption of sunlight by the pigments of the light-harvesting protein complex (LHC) and the transfer of this energy to the reaction centres ofphotosystem II (PSII) and photosystem I (PSI) to induce charge separation in 10215 to 1026 s. Coupled biochemical redox reactions transfer elec-trons from PSII through the intersystem electron-transport chain, consisting of plastoquinone (PQ), the cytchrome f/b6 complex (CYT f/b6) andplastocyanin (PC), to PSI in about 1023 s. Photosynthetic electron transport converts light into reducing power (NADPH) and, concomitantly,chemical potential energy (ATP), through chemiosmosis. These products are consumed through the reduction of C, N and S in the chloroplast,all of which occurs in seconds to minutes. These metabolic processes, in turn, are required for normal growth and development. Several mecha-nisms have evolved to ensure a balance between light energy absorbed versus energy utilized through electron transport and metabolism toprotect PSII from over-excitation. Energy transfer from PSII to PSI (state transitions) and dissipation of excess absorbed energy as heat by non-photochemical quenching can occur in minutes. Over a longer time (hours to days), the following mechanisms occur: reduction of LHCIIantenna size; alteration of PSII–PSI stoichiometry; stimulation of the rate of repair of the damaged D1 polypeptide of the PSII reaction centre;and stimulation of the capacity to utilize ATP and NADPH through metabolism to maintain high photochemical quenching of PSII excitation.Minimal levels ( 3%) of the absorbed light energy are always lost through chlorophyll a fluorescence. The detection of modulated chlorophylla fluorescence emission is exploited to estimate non-photochemical and photochemical quenching.
ADP +Pi
LHC
I
ATP NADPH NADP+
LHC
II
LHC
II
LHC
II
Chlorophyll afluorescence
O2
O2 PQ CYT f/b6 PC
Reduction of C, N and S
Growth and developmentReproduction
PSII PSIHeat
Sun
Non-photochemical
Photochemical
quenching
quenching
Decrease in size of LHCII
Increase in size of LHCII
Damage Repair
Box 1. Energy balance and photosystem II excitation pressure during acclimation to light and temperature
The rate of excitation of photosystem II (PSII) under light-limiting conditions can be estimated as:sPSII
• Iwhere sPSII is the functional absorption cross-sectional area of PSII and I is the incident photon flux (units mmol photons m22 s21) (Ref. 7). Underlight-saturating conditions, the rate of utilization of this excitation energy through electron transport and metabolism can be estimated as:
n • 1/twhere n is the number of components acting as sinks that consume electrons, t is the life-time and, hence, 1/t is the turnover rate of these sinks.A balance between energy absorbed and energy utilized is attained when:
sPSII• I = n • 1/t
Consequently, any environmental condition that satisfies the following inequality would result in over-excitation of PSII (i.e. PSII excitationpressure):
sPSII• I > n • 1/t
Thus, an energy imbalance can result from exposure to excessive irradiance, I , at constant temperature. Alternatively, an energy imbalance canoccur as a consequence of exposure to low temperature at constant irradiance. Low temperature causes a reduction in 1/t. Thus, PSII excitationpressure can be created either by modulating irradiance, which affects temperature-insensitive photochemical processes (Q10 = 1), or by modu-lating temperature, which affects temperature-dependent, biochemical processes (Q10 = 2).
Photosynthetic adjustment during coldacclimationIt appears that nature has provided severalalternative solutions to the common prob-lem of stress-induced energy imbalance(Fig. 1). The mechanisms employed areintegrally related to the time scale ofexposure and to the developmental strat-egy of the species in question.
Evergreen plantsIn the Northern hemisphere, the leaves ofevergreen plants typically develop and ex-pand during the warm spring and summermonths and are subsequently retained andmaintained during the winter monthswhen all growth ceases. The reversibleinterconversion of the light-harvestingxanthophyll, violaxanthin, to the energyquencher, zeaxanthin, occurs over periodsof minutes upon exposure to excessivelight whereas an increase in the total xan-thophyll pool size and an accumulation ofzeaxanthin can occur over a period of daysor months in response to extensive periodsof low, winter temperatures, when light-saturated photosynthetic rates as well asphotosynthetic efficiency are depressed.This results in a sustained capacity to dis-sipate excess light as heat by non-photo-chemical quenching mechanisms12 (Fig.1). However, in Pinus sylvestris, LHCII isreorganized into large pigment–proteinaggregates containing both chlorophyll
and zeaxanthin that appear to dissipate excess energy as heat13.Because non-photochemical quenching appears to play a majorrole in the protection of the photosynthetic apparatus from severephotodamage, evergreens appear to adjust sPSII in response to theimbalance in energy budget that occurs during the winter months.
Other temperate conifers such as P. banksiana exhibit ‘purpling’caused by the accumulation of anthocyanin in epidermal cells(Fig. 3) in response to a complex interaction of photoperiod, lowtemperature and irradiance14. Accumulation of anthocyanins ap-pears to protect the needles against photoinhibition of PSII whenexposed to light and cold temperatures through a simple screeningeffect that reduces the absorbed photon flux. This is consistentwith the suggestion that anthocyanins are a natural sun screenagainst both UV-B radiation and high visible irradiance15.
CerealsIn contrast to evergreens, cold-tolerant winter rye and wheat mustgrow and develop at low temperatures for maximum cold toleranceand successful winter survival in temperate climates. Thus, theseplant species must maintain the capacity for active photosynthesisduring prolonged exposure to low, non-freezing temperatures dur-ing the cold acclimation period with minimal changes in pigmentcomposition. Somersalo and Krause16 were the first to report thatcold acclimation results in an increased tolerance to photoinhibition,a result that was subsequently confirmed for winter rye and wheat17.This was not because of changes in leaf optical properties, but wasassociated with minimal changes in thylakoid membrane micro-viscosity based on differential scanning calorimetry and electronspin resonance measurements17 . In contrast to the alpine arcticspecies Oxyria digyna18, the increased tolerance to photoinhibition
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Fig. 2. Regulation of the redox state of photosystem II (PSII). The PSII reaction centrepolypeptides, D1 and D2, bind the following redox components: Z, a tyrosine residue of D1; P680, the reaction centre chlorophyll a; Pheo, pheophytin; QA, quinone A (the first, stableelectron acceptor of PSII reaction centres); and QB, quinone B (the secondary quinone elec-tron acceptor). The transfer of absorbed light energy to ‘open’ PSII reaction centres causesthe photo-oxidation of P680 to P680
+ and subsequent electron transfer to Pheo to reduce QA
to QA2. This results in ‘closed’ PSII reaction centres10. Excitation of closed reaction cen-
tres results in damage to PSII. Conversion of closed to open PSII reaction centres requires thereduction of P680
+ to P680 through the oxidation of water and electron transfer through Z. Concomitantly, QA
2 is oxidized by the CYT f/b6 complex via the PQH2 pool, which is the rate-limiting step of photosynthetic electron transport. The proportion of closed reactioncentres is estimated in vivo by the relative reduction state of QA. Thus, 1 2 qP, where qP isthe photochemical quenching parameter, is approximately equal to [QA
2] / [QA] + [QA2] and
is an estimate of the relative PSII excitation pressure to which an organism is exposed.
‘Open’ PSII
‘Closed’ PSII
[ Z P680 Pheo QA QB ]
[ Z P680+ Pheo QA
− QB ]
(qP)
(I – qP) H2O
1/2O2 + 2e− + 2H+
PQ(oxidized)
PQ(reduced)
CYT f/b6(oxidized)
CYT f/b6(reduced)
Absorbedlight energy
Fig. 3. Cross-section of a needle of cold-acclimated Pinus bank-siana. The arrow indicates a layer of epidermal cells that containsanthocyanin.
in wheat and rye is not caused by an increased capacity to repairdamaged PSII reaction centres or increased non-photochemicalquenching, but rather an increased capacity to keep the quinoneQA oxidized (i.e. high photochemical quenching) due to an el-evated photosynthetic capacity with no change in photosyntheticefficiency. The potential to keep QA oxidized and the potential toincrease the photosynthetic capacity as a consequence of cold ac-climation are correlated with both tolerance to photoinhibition andthe capacity of winter rye and wheat cultivars to develop maxi-mum freezing tolerance17.
The increased capacity of rye and wheat to keep QA oxidizedappears to be a consequence of a cold acclimation-induced stimu-lation of mRNA and protein levels associated with the major regu-latory enzymes of photosynthetic carbon metabolism: Rubisco,stromal and cytosolic fructose bisphosphatase and sucrose phos-phate synthase19. This is reflected in an increased enzyme activityas well as an increased activation state of these important regula-tory enzymes. This is translated into increased growth rates underpotentially photoinhibitory conditions17. Thus, the elevated sucroselevels normally associated with cold acclimation do not result inthe repression of photosynthetic gene expression in Arabidopsis20,rye or wheat6,17,19, contrary to current models related to sucroseregulation of plant gene expression. It appears that increased coldtolerance and tolerance to photoinhibition may be the result of a‘reprogramming’ of carbon metabolism in these species20. Thus,winter wheat and rye appear to adjust the number of electron-consuming sinks in response to growth at low temperatures. How-ever, this response appears to be species specific.
Green algaeSimilar to the evergreens, low-temperature acclimation of the uni-cellular green algae, Chlorella vulgaris and Dunaliella salina,results in a depression of the capacity for CO2 assimilation andphotosynthetic efficiency, calculated on a per cell basis, concomi-tant with an increase in the total xanthophyll pool size as well as alower epoxidation state of the xanthophyll cycle pigments due tothe conversion of violaxanthin to zeaxanthin6. However, in contrastto evergreens and both winter rye and wheat, cold acclimation ofthese green algae is associated with: a six-fold lower chlorophyllcontent per cell; a lower abundance of Lhcb mRNA as well as Lhcbpolypeptides; and an increased level of the carotenoid-binding pro-tein, Cbr (Fig. 4a: compare 27/150 and 5/150). However, thesealgae appear unable to up-regulate carbon metabolism and thus areunable to adjust the number of electron-consuming sinks duringgrowth and development at low temperature6. As a consequence,algal cultures grown at low temperature exhibited a distinctive yel-low colour (Fig. 4a). The repression in the accumulation of LhcbmRNA and Lhcb polypeptides is not due to sucrose suppression,since the capacity for sucrose accumulation is depressed uponcold acclimation of Chlorella vugaris.
Cultures grown at low temperature exhibited a three- to four-fold increased tolerance to photoinhibition. Thus, in contrast toeither evergreens or wheat and rye, these green algae alter theirpigmentation significantly in response to growth at low growthtemperature or high growth irradiance. This reflects a reduction inlight-harvesting capacity coupled with an increased capacity todissipate excess light non-photochemically as heat through zeax-anthin and possibly lutein21, which results in a decrease in sPSII.
CyanobacteriaIn contrast to chloroplasts, the cyanobacterium Synechococcus sp.PCC 7942 possesses three homologous genes for the D1 proteinof PSII reaction centres22, designated psbAI, psbAII and psbAIII.The psbAI gene encodes form 1, designated D1:1; psbAII and
psbAIII encode form 2, designated D1:2. Mutants expressing D1:2exhibit greater tolerance to photoinhibition than wild-type cells thatexpress D1:1. Recently, it has been shown that exposure of wild-type Synechococcus sp. PCC 7942 to low temperature induces atransient exchange of the D1:1 form for the D1:2 form of the PSIIreaction centre polypeptide, resulting in an increased tolerance tophotoinhibition22.
There is a consensus that a rapid cycle of damage and repair ofthe D1 polypeptide during photoinhibition is an intrinsic feature ofPSII (Ref. 1). A decrease in membrane lipid unsaturation inhibitssubsequent recovery from photoinhibition through an impairmentof the D1 repair process in cyanobacteria23. This appears to be dueto an inability to process the newly synthesized D1 protein, result-ing in the accumulation of inactive PSII reaction centres. More-over, exposure to cold enhances thylakoid fatty acid unsaturation,and increases the tolerance of cyanobacteria to low-temperaturephotoinhibition and chilling injury23. Thus, cyanobacteria appearto respond to photoinhibition by adjusting the capacity to repairPSII (Fig. 1). It may be that changes in membrane fluidity act as aprimary signal for changes in temperature in cyanobacteria24.
Acclimation to cold and high light intensityPhotosynthetic adjustment of Chlorella to growth at low tempera-ture and moderate irradiance is comparable to cells grown at highlight with respect to pigmentation, gas exhange and sensitivity to
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Fig. 4. Effects of growth regime on Chlorella vulgaris cultures:(a) pigmentation; (b) immunoblot showing the accumulation oflight-harvesting polypeptides (Lhcb polypeptides); (c) RNA-gel blotshowing Lhcb mRNA accumulation; and (d) immunoblot showingthe accumulation of the carotenoid-binding protein, Cbr. The growthregimes were x°C and y mmol m22 s21, indicated by x/y, with controlcells cultured at 27/150 or 27/2200. Cells grown at either 27/2200or 5/150 were exposed to comparable high photosystem II (PSII) ex-citation pressure whereas cells grown at either 27/150 or 5/20 wereexposed to comparable low PSII excitation pressure. The differenteffects of high or low PSII excitation pressure are very distinct.
27/150 5/20 5/150 27/2200
(a)
(b)
(c)
(d)
photoinhibition, as well as the accumulation of Lhcb polypeptidesand cbr, the carotenoid-binding protein (Fig. 4). This cannot beexplained as either a simple growth temperature effect or a simpleirradiance response. Cultures grown at either 5°C and low lightintensity or 27°C and high light intensity are photosyntheticallyadjusted to growth at high PSII excitation pressures; cultures grownat either 27°C and low light or 5°C and very low light are photo-synthetically adjusted to growth at low PSII excitation pressures21.Similar conclusions regarding the role of PSII excitation pressurehave been reported for thermal and photoacclimation of Laminaria
saccharina25 and the expression of earlylight-inducible proteins (ELIPs) in barley26.
It may be that exposure to high excitationpressure initiates a signal transduction path-way from the chloroplast to the nucleus thatrepresses Lhcb and concomitantly dere-presses Cbr gene expression. This leads tolow levels of Lhcb, but high levels of Cbrprecursor polypeptides synthesized in thecytoplasm – these are subsequently pro-cessed during transport into the chloroplastand inserted into the thylakoid membrane.
Thus, photoautotrophs sense imbalancesin the energy budget through changes in therelative reduction state of PSII (i.e. changesin PSII excitation pressure). However, PSIIcannot be the primary redox sensor thatregulates nuclear Lhcb and cbr expressionin Chlorella and Dunaliella6. Rather, thechanges in the relative reduction state ofPSII probably reflect the redox status of acomponent further downstream of PSII,which is consistent with the proposal thateither the thylakoid plastoquinone pool27,28
or the CYT b6/f complex acts as the chloro-plast redox sensor for the regulation ofphotosynthesis-related genes29.
Cold, light and freezing toleranceThe photosynthetic response and toleranceto photoinhibition of cold-acclimated wheatand rye are also due to growth at elevatedPSII excitation pressure30. However, doesthe modulation of PSII excitation pressureby either irradiance or temperature influ-ence events beyond photosynthesis andsensitivity to photoinhibition? In contrastto the Wcs120 family of cold-acclimationgenes31, expression of the nuclear-encodedcold acclimation gene Wcs19 as well as theinduction of the short, compact growthhabit associated with cold hardening arenot direct responses to cold acclimation,but are rather responses to changes in PSIIexcitation pressure30 (Fig. 5). The sensingand signalling events in photomorpho-genic development are typically associatedwith photoreceptors such as phytochromeas well as blue light and UV-light photo-receptors32. However, the results for thecompact growth morphology cannot bedue to phytochrome, because the compactgrowth morphology is induced simply bylowering the temperature with no change
in either light quality or photoperiod (Fig. 5). Thus, processes asdiverse as photosynthesis, morphogenesis and cold acclimation arenot only regulated by photoreceptors, but also appear to be verysensitive to the overall metabolic energy balance3–6,33.
Photosynthesis provides the energy necessary for the attain-ment of a cold-acclimated state and, hence, maximum freezingtolerance. Thus, any environmental factor that has a negativeimpact on photosynthesis may ultimately influence the inductionof freezing tolerance. Since maximum LT50 is dependent uponboth light and temperature, the physiology of the plant may, in
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Fig. 5. (a) Effect of growth regime on photosystem II (PSII) excitation pressure in winter ryeleaves. PSII excitation pressure was estimated as 1 2 qP, where qP is the photochemicalquenching parameter. Rye plants were grown at x°C and y mmol m22 s21, indicated by x/y. The‘normal’ growth temperature was 20°C, with cold-acclimated plants grown at 5/250 or 5/50.Rye grown at either 20/800 or 5/250 exhibits comparable elevated PSII excitation pressure,even though the plants are grown under very different light and temperature regimes; simi-larly, rye grown at either 20/250 or 5/50 also exhibits comparable low PSII excitation pres-sure. (b) Effect of growth regime on Wcs19 mRNA accumulation. Leaves exposed to highexcitation pressure accumulated high levels of Wcs19 mRNA whereas leaves exposed to lowexcitation pressure accumulated lower levels. (c) Effects of growth regime on plant mor-phology. The compact growth morphology of cold-acclimated plants (5/250) was mimickedby rye plants exposed to comparable PSII excitation pressures induced by growth at moder-ate temperatures but high light (20/800). Similarly, the elongated growth morphology of non-cold-acclimated rye plants (20/250) was mimicked by rye plants exposed to comparable lowexcitation pressures induced by growth at low temperature and low light (5/50). (d) Effect ofgrowth regime on rye freezing tolerance. The maximum freezing tolerance (LT50) of rye isdependent on growth at low temperature and moderate irradiance (5/250). However, growthat low temperature and low irradiance (5/50) results in an LT50 comparable to non-cold-acclimated plants. Furthermore, increasing irradiance at a growth temperature of 20°Cincreases LT50, although not to the same extent as growth at low temperature. Clearly, LT50is dependent on both the temperature and the light for growth. However, maximum LT50 isnot dependent on PSII excitation pressure, but rather appears to be dependent upon the addi-tive effects of temperature and irradiance during growth.
0.0
0.1
0.2
0.3
0.4
1] q
P
216
220
212
28
24
0
LT50 (
°C)
20/8
00
20/2
50
20/5
0
5/2
50
5/5
0
20/8
00
20/2
50
5/2
50
5/5
0
20/8
00
20/2
50
20/5
0
5/2
50
5/5
0
(a)
(b)
(c)
(d)
Growth regime Growth regime
fact, supersede the genetic potential of the organism to exhibit itsmaximum freezing tolerance. This has important implications notonly for genetic engineering of cold tolerant and freezing-resistantplants but also for research focused on the elucidation of stress-tolerance mechanisms.
Although maximum freezing tolerance (LT50) is altered bychanging either the growth temperature or the growth light (Fig.5d), LT50 is not modulated by PSII excitation pressure. It is de-pendent on both light and temperature in an independent but ad-ditive manner30 (Fig. 5d). Thus, cold acclimation and freezingtolerance may well be the result of complex interactions of lowtemperature, light and chloroplastic redox poise, as reflected bythe sensitivity to PSII excitation pressure. The transduction path-ways associated with each of these signals probably interact notonly with each other but also synergistically with other importantsignal transduction pathways – involving phytochrome32, sugarsensing34, protein phosphorylation and dephosphorylation, Ca2+
and plant growth regulators31 – to elicit the appropriate physiologi-cal response.
Recent results indicate that chromosome 5A of wheat carriesthe regulatory gene or genes that not only control the expressionof the gene families correlated with freezing tolerance but alsogenes controlling plant morphology31. Perhaps a locus or loci onchromosome 5A represents a master regulator that co-ordinatesthe expression of genes associated with cold acclimation, freezingtolerance and plant morphology. If this is the case, clearly thisnuclear-encoded, master regulator must be sensitive to PSII exci-tation pressure and hence chloroplastic redox poise.
Photosynthesis: a dual role? Any change in environmental conditionssuch as temperature, light, nutrient status,CO2 concentrations or water availabilitycan potentially cause a metabolic energyimbalance which, through metabolic feed-back loops, will affect chloroplast metab-olism and hence modulate PSII excitationpressure (Fig. 6). However, plants do notsense environmental changes only throughmodulation of PSII excitation pressure.Clearly, all organisms must be able tosense specific changes in temperature24 aswell as perturbations of other environ-mental conditions. Nevertheless, in na-ture, changes in environmental parameterssuch as temperature rarely occur indepen-dently of other factors such as irradiance.Thus, caution must be exercised in inter-preting data obtained from an experimen-tal design that does not include controls toallow one to distinguish between re-sponses caused by the specific environ-mental perturbation of interest from thosedue to changes in PSII excitation pressure.
In addition to the traditional role ofphotosynthesis in energy transduction, theredox state of the photosynthetic apparatusmight also act as an environmental sensorby detecting energy imbalances betweenphotochemistry and biochemistry (seeBox 1). As initially proposed by Fuijita andco-workers29 and subsequently supportedby others6,7,27,28, a putative photosyntheticredox signal might initiate a transduction
pathway that coordinates photosynthesis-related gene expressionand, hence, photosynthetic acclimation. In addition, this photo-synthetic redox sensing and signalling mechanism might alsoinfluence such diverse processes as cold acclimation, plant mor-phology (Fig. 6) and cyanobacterial differentiation35. Evidence foran intrachloroplastic redox signalling pathway has recently beenprovided with the identification of the ferredoxin–thioredoxinsystem as a critical component of redox regulation of chloroplasttranslation36. Although signalling between the chloroplast and thenucleus has been investigated for some time37, the nature of thechloroplastic signal (the elusive ‘plastid factor’) and the signaltransduction pathway have yet to be elucidated. However, it hasrecently been shown that Mg-protoporphyrin IX acts as a plastidfactor in the signal transduction pathway involved in the light in-duction of nuclear heat-shock genes38. Thus, the process of photo-synthesis appears to exert a broad influence on diverse molecular,physiological and developmental processes, consistent with thenotion of a ‘grand design of photosynthesis’5,39.
DedicationThis article is dedicated to Dr Fergus D.H. Macdowall, mentorand friend.
AcknowledgementsMuch of the work described in this review is taken from the doc-toral theses of Denis P. Maxwell and Gordon R. Gray. We aregrateful to Drs Leo Savitch, Alex Ivanov and Marianna Krol fortheir help in the preparation of this article. Research in the labs of
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Fig. 6. The chloroplast as an environmental sensor that initiates intracellular as well as inter-cellular signal transduction pathways. It is proposed that photoautotrophs sense changes inenvironmental conditions through imbalances in the energy absorbed versus energy utilizedthrough electron transport and metabolism [i.e. through modulation of photosystem II (PSII)excitation pressure]. Modulation of chloroplastic redox poise initiates a signal transductionpathway whereby the chloroplast affects nuclear gene expression (Lhcb, Cbr and Wsc19).However, in addition, this redox signal transduction pathway may act synergistically withother known signal transduction pathways. This appears to extend the influence of chloro-plastic energy imbalance beyond leaf mesophyll cells to the meristematic regions of thecrown to affect plant morphology and cold acclimation.
Environmental stress
TemperatureIrradiance
Water availabilityNutrient status
Absorbed lightenergy
Protein
DNA mRNA
Nucleus
Gene expression
Acclimation
Low excitationpressure
High excitationpressure
Growth habit
Metabolitesor sucrose?
Growth-regulatorycompounds?
Ca2+?
ExcitationpressurePSII PSI
Chloroplasticredox poise
Chloroplastsignal?
N.P.A.H. and F.S. was supported by the Natural Sciences andEngineering Research Council of Canada and Fonds pour la Formation de Chercheurs et l’Aide à la Recherche. Research inG.Ö.’s lab was supported by the Swedish Natural Science ResearchCouncil. G.Ö. and N.P.A.H. acknowledge the financial support ofthe Swedish Foundation for International Cooperation in Researchand Higher Education.
References01 Osmond, C.B. (1994) What is photoinhibition? Some insights from the
comparison of shade and sun plants, in Photoinhibition of Photosynthesis –from Molecular Mechanisms to the Field (Baker, N.R. and Bowyer, J.R., eds),pp 1–24, Bios
02 Asada, K. (1996) Radical production and scavenging in the chloroplasts, inAdvances in Photosynthesis. Photosynthesis and the Environment (Vol. 5)(Baker, N.R., ed.), pp. 123–150, Kluwer
03 Melis, A. et al. (1985) The mechanism of photosynthetic membraneadaptation to environmental stress conditions: a hypothesis on the role ofelectron-transport capacity and of the ATP/NADPH pool in the regulation ofthylakoid membrane organization and function, Physiol. Vég. 23, 757–765
04 Horton, P. et al. (1988) Regulation of the photochemical efficiency ofphotosystem II: consequences for the light response of field photosynthesis,Plant Physiol. Biochem. 26, 453–460
05 Anderson, J.M., Chow, W.S. and Park, Y-I. (1995) The grand design ofphotosynthesis: acclimation of the photosynthetic apparatus to environmentalcues, Photosynth. Res. 46, 129–139
06 Huner, N.P.A. et al. (1996) Sensing environmental temperature changethrough imbalances between energy supply and energy consumption: redoxstate of photosystem II, Physiol. Plant. 98, 358–364
07 Durnford, D.G. and Falkowski, P.G. (1997) Chloroplast redox regulation ofnuclear gene transcription during photoacclimation, Photosynth. Res. 53,229–241
08 Vogelman, T.C., Nishio, J.N. and Smith, W.K. (1996) Leaves and lightcapture: light propagation and gradients of carbon fixation within leaves,Trends Plant Sci. 1, 65–70
09 Falkowski, P.G.(1983) Light–shade adaptation and vertical mixing of marinephytoplankton: a comparative field study, J. Mar. Res. 41, 215–237
10 Krause, G.H. and Weis, E. (1991) Chlorophyll fluorescence and photo-synthesis: the basics, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 313–349
11 Adams, W.W., III et al.(1995) ‘Photoinhibition’ during winter stress:involvement of sustained xanthophyll cycle-dependent energy dissipation,Aust. J. Plant Physiol. 22, 261–276
12 Adams, W.W., III, Hoehn, A. and Demmig-Adams, B. (1995) Chillingtemperatures and the xanthophyll cycle. A comparison of warm-grown andoverwintering spinach, Aust. J. Plant Physiol. 22, 75–85
13 Ottander, C., Campbell, D. and Öquist, G. (1995) Seasonal changes inphotosystem II organization and pigment composition in Pinus sylvestris,Planta 197, 176–183
14 Krol, M. et al. (1995) Low temperature stress and photoperiod affect anincreased tolerance to photoinhibition in Pinus banksiana seedlings, Can. J.Bot. 73, 1119–1127
15 Gould, K.S. et al. (1996) Why leaves are sometimes red, Nature 378, 241–24216 Somersalo, S. and Krause, G.H. (1989) Photoinhibition at chilling
temperatures. Fluorescence characteristics of unhardened and cold-hardenedspinach leaves, Planta 177, 409–416
17 Huner, N.P.A. et al. (1993) Photosynthesis, photoinhibition and lowtemperature acclimation in cold tolerant plants, Photosynth. Res. 37, 19–39
18 Koroleva, O.Y., Bruggemann, W. and Krause, G.H. (1994) Photoinhibition,xanthophyll cycle and in vivo chlorophyll fluorescence quenching of chilling-tolerant Oxyria digyna and chilling-sensitive Zea mays, Physiol. Plant. 92,577–584
19 Hurry, V.M. et al. (1998) Photosynthesis at low growth temperatures, inPhotosynthesis: a Comprehensive Treatise (Raghavendra, A.S., ed.) pp. 238–249, Cambridge University Press
20 Strand, A. et al. (1997) Development of Arabidopsis thaliana leaves at lowtemperature releases the suppression of photosynthesis and photosyntheticexpression despite the accumulation of soluble carbohydrates, Plant J. 12,605–614
21 Maxwell, D.P., Falk, S. and Huner, N.P.A. (1995) Photosystem II excitationpressure and development of resistance to photoinhibition. I. Light harvestingcomplex II abundance and zeaxanthin content in Chlorella vulgaris, PlantPhysiol. 107, 687–694
22 Campbell, D. et al. (1995) Electron transport regulates exchange of two formsof photosystem II D1 protein in the cyanobacterium Synechococcus, EMBO J.14, 5457–5466
23 Nishida, I. and Murata, N. (1996) Chilling sensitivity in plants andcyanobacteria: the crucial contribution of membrane lipids, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 47, 541–568
24 Murata, N. and Los, D.A. (1997) Membrane fluidity and temperatureperception. Plant Physiol. 115, 875–879
25 Machalek, K.M., Davison, I.R. and Falkowski, P.G. (1996) Thermalacclimation and photoacclimation of photosynthesis in the brown algaLaminaria saccharina, Plant Cell Environ. 19, 1005–1016
26 Montane, M.H. et al. (1997) Early light-inducible proteins during long-termacclimation of barley to photooxidative stress caused by light and cold – highlevel of accumulation by posttranscriptional regulation, Planta 202, 293–302
27 Allen, J.F., Alexciev, K. and Hakansson, G. (1995) Regulation by redoxsignalling, Curr. Biol. 5, 869–872
28 Escoubas, J-M. et al. (1995) Light intensity regulates cab gene transcriptionvia the redox state of the plastoquinone pool in the green alga, Dunaliellatertiolecta, Proc. Natl. Acad. Sci. U. S. A. 92, 10237–10241
29 Fujita, Y. et al. (1994) Short-term and long-term adaptation of thephotosynthetic apparatus: homeostatic properties of thylakoids, in Advances inPhotosynthesis. The Molecular Biology of Cyanobacteria (Vol. 1) (Bryant,D.A., ed.), pp. 677–692, Kluwer
30 Gray, G.R. et al. (1997) Cold acclimation and freezing tolerance. A complexinteraction of light and temperature, Plant Physiol. 114, 467–474
31 Sarhan, F., Ouellet, F. and Vazquez-Tello, A. (1997) The wheat wcs120 genefamily. A useful model to understand the molecular genetics of freezingtolerance in cereals, Physiol. Plant. 101, 439–445
32 Chory, J. (1997) Light modulation of vegetative development, Plant Cell 9,1225–1234
33 Walters, R.G. and Horton, P. (1995) Acclimation of Arabidopsis thaliana tolight environment: regulation of chloroplast composition, Planta 197, 475–481
34 Jang, J-C., Léon, P. and Sheen, J. (1997) Hexokinase as a sugar sensor inhigher plants, Plant Cell 9, 5–19
35 Campbell, D., Houmard, J. and Tandeau de Marsac, N. (1993) Electrontransport regulates cellular differentiation in the filamentous cyanobacteriumCalothrix, Plant Cell 5, 451–463
36 Kim, J. and Mayfield, S.P. (1997) Protein disulfide isomerase as a regulator ofchloroplast translational activation, Science 278, 1954–1957
37 Taylor, W.C. (1989) Regulatory interactions between nuclear and plastidgenomes, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 211–233
38 Kropat, J. et al. (1997) Chlorophyll precursors are signals of chloroplast origininvolved in light induction of nuclear heat-shock genes, Proc. Natl. Acad. Sci.U. S. A. 94, 14168–14172
39 Arnon, D.I. (1982) Sunlight, earth, life: the grand design of photosynthesis,The Sciences 22, 22–27
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Norman Huner* is at the Dept of Plant Sciences, University ofWestern Ontario, London, Canada N6A 5B7; Gunnar Öquist is atthe Dept of Plant Physiology, Umeå University, Umeå, S-901 87 Sweden; Fathey Sarhan is at the Dépt des SciencesBiologiques, Université du Québec à Montréal, C.P. 8888, Succ. Centre-ville, Montréal, Canada H3C 3P8
Life: Comparative Analysis of Water Relationships at theOrganismic, Cellular and Molecular Level, pp. 338–362.Berlin: Springer-Verlag.
Thomashov MF (1999) Plant cold acclimation: freezingtolerance genes and regulatory mechanisms. AnnualReview of Plant Physiology and Plant Molecular Biology50: 571–591.
Free Radicals, Oxidative Stressand AntioxidantsK S Gould, University of Auckland, Auckland, NewZealand
Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction
Plants are continuously exposed to free radicals.These unstable and often highly reactive moleculespresent a formidable challenge to all plants, evenunder optimal growing conditions. If left unchecked,free radicals can cause oxidative injury by initiatingchain reactions that disrupt membranes, denatureproteins, fragment DNA, and ultimately precipitatecell death. The problems are exacerbated in plantsthat face additional stressors such as high light,temperature extremes, drought, or fungal infections.In these situations, protection from the effects of freeradicals is not a luxury – it may be critical forsurvival! Fortunately, plants have evolved a sophis-ticated armory of antioxidant defense, a diverseassortment of enzymes, pigments, and secondarymetabolites that serve to scavenge or quench thereactive molecules before they inflict injury. Effortsto enhance the antioxidant levels in plants have far-reaching implications both for crop productivity andhuman nutrition.
Free Radicals
A free radical is defined as any atom or moleculecapable of independent existence that contains oneor more unpaired electrons. An unpaired electron isone that occupies an atomic or molecular orbital byitself. Free radicals are usually denoted by a super-script dot after the chemical formula, such as OH�
for the hydroxyl radical. A related group ofcompounds, including hydrogen peroxide, singletoxygen, and ozone, lack unpaired electrons, but, likethe radicals, can participate in cellular redox reac-tions; these together with the oxygen containing
radicals are collectively known as reactive (or active)oxygen species (ROS or AOS).
Free radicals can be generated in plants when anonradical gains or loses an electron, or receivesexcitation energy from a photoactivated pigment.Most radicals are unstable and are therefore short-lived; half-lives in the order of a microsecond or ananosecond are common. Stability is restored whentwo radicals meet and share their unpaired electronsin a covalent bond. However, when a free radicalreacts with an organic molecule such as an unsatu-rated fatty acid in a membrane bilayer, a new radicalresults, and chain reactions are established. Suchreactions can eventually lead to oxidative injury.
Free radicals and ROS are generated in thechloroplasts, mitochondria, endoplasmic reticulum,peroxisomes, glyoxysomes, plasma membrane, andapoplasm of plant cells. They are present in the rootsand shoots of both vegetative and reproductiveindividuals. Many different types of radicals havebeen identified (Table 1). The oxygen-centeredradicals have been most extensively characterized,possibly because of the pivotal roles of oxygen in theprocesses of respiration and photosynthesis. Indeed,diatomic oxygen is itself a free radical, which,although relatively stable, is toxic to plants at higherconcentrations. However, it is becoming increasinglyevident that the nitrogen radicals, primarily nitricoxide and the reactive nitrogen species peroxynitrite,also contribute significantly to a plant’s oxidativeload.
Sources of Reactive Oxygen and Nitrogen
Routine processes such as photosynthesis, respira-tion, and nitrogen metabolism generate free radicalsand ROS. These are quickly scavenged by anti-oxidants, and are not usually harmful to plants.Indeed, some oxidants are beneficial at low concen-trations; H2O2, for example, is required in theapoplast for lignin biosynthesis, and has been
Table 1 Examples of free radicals and reactive oxygen species
in plant cells
Name Formula
Diatomic oxygen O2
Singlet oxygen 1O2
Superoxide O��2
Hydroxyl OH�
Hydrogen peroxide H2O2
Transition metal atoms/ions Fe, Cu
Thiyl RS�
Peroxyl, alkoxyl RO�2; RO
�
Nitric oxide NO�
Peroxynitrite ONOO–
ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants 9
implicated as a signaling molecule in defense res-ponses to fungal elicitors. Environmental and bioticstressors further increase the numbers of radicalsproduced, and can cause permanent oxidative injury.
Photosynthesis
Chloroplasts in leaves, stems, and other green tissuesare the primary source of ROS in plants under light.In photosynthesis, chloroplasts harvest light energy,transform it into chemical energy and reducingequivalents, and generate diatomic oxygen from theoxidation of water. Electrons travel along theelectron transport chains of photosystems II (PSII)and I (PSI), eventually to reduce the terminalaccepter NADPþ to NADPH. However, even undermoderate light conditions, a proportion of theelectron flux through PSI does not reach NADPþ.Instead, those electrons are diverted from thepenultimate acceptor, ferredoxin (Fd) in its reducedstate, to molecular oxygen. This results in thereduction of O2 to superoxide radicals ðO��
2 Þ: Theprocess is known as the Mehler reaction after itsdiscoverer:
Fdred þO2-O��2 þ Fdox
Superoxide itself is a relatively unreactive freeradical. It is capable of both oxidation and reductionand is known to react with some organic molecules,yet it diffuses only slowly and cannot penetrate cellmembranes. However, chloroplasts attempt to re-move all superoxide almost as soon as it is formed.An extremely efficient enzyme, superoxide dismutase(SOD), is used to disproportionate O��
2 into H2O2
and O2:
2O��2 þ 2Hþ -
SODO2 þH2O2
Thus, in the process of removing O��2 ; SOD generates
another ROS, hydrogen peroxide. H2O2 is a weakoxidizing agent that is toxic to most cells in the10–100mmol l� 1 range. It diffuses quickly withinand between cells, and crosses the lipid bilayer ofcell membranes faster than can be accounted for bydiffusion alone. Described as only mildly reactive,H2O2 can nevertheless inactivate essential enzymesin the Calvin cycle and, by oxidizing thiol groups,disrupts the mechanism that couples carbon fixationin the stroma to photosynthetic electron flow. Itmust, therefore, be kept below micromolar concen-trations in the chloroplasts. This is normallyachieved by enzymatic antioxidants.
There is another very good reason why chloro-plasts and other cellular components need to removeO��
2 and H2O2. The two species can react together, inthe presence of transition metal ions Cu2þ or Fe2þ ,
to form hydroxyl radicals (OH�). The reaction takesplace in two steps:
ð1Þ H2O2 þ Fe2þ-OH� þOH� þ Fe3þ
ðthe Fenton reactionÞð2Þ O��
2 þ Fe3þ-O2 þ Fe2þ
ðferrous ion recyclingÞThe net sum of reactions (1) and (2) is called theHaber–Weiss reaction:
H2O2 þO��2 -O2 þOH� þOH�
Hydroxyl radicals are potentially the most danger-ous molecules in biological systems. In plants,animals, and humans, OH� reacts nonspecificallywith almost every type of molecule found in livingcells: sugars, amino acids, phospholipids, nucleo-tides, and organic acids. Indeed, it is among the mostreactive chemicals known. OH� radicals diffuserapidly, and react with any molecule in their vicinityto produce secondary radicals. No specific scaven-gers of OH� are known. Because OH� radicals aretoo reactive to be controlled directly, plants eliminateinstead the less reactive precursors, H2O2 and O��
2 :
Respiration
The problems with ROS faced by mitochondriaduring respiration are essentially similar to those ofthe chloroplasts. In the process of oxidative phos-phorylation, electrons from NADH are transferred todiatomic oxygen via an electron transport chain onthe inner mitochondrial membrane. This transferreleases free energy, much of which is conservedthrough the synthesis of ATP from ADP and Pi.Although electron flow through the chain is tightlyregulated, it has been estimated that 1–3% of the O2
reduced in mitochondria may form O��2 : Electrons
are believed to leak directly onto O2 from varioussites (e.g., the cyanide insensitive alternative oxidase)early in the mitochondrial electron transport chain.Damage to mitochondrial membranes favors elec-tron leakage, and thus increases O��
2 production. Asoccurs in the chloroplasts, O��
2 in the mitochondria israpidly dismutated into H2O2 by SOD. In addition tothe risk of producing OH� radicals via Haber–Weissreactions, H2O2 can also degrade proteins essentialto mitochondrial function, such as cytochrome c.Mitochondrial H2O2 is scavenged predominantly bythe enzyme catalase.
Nitrogen Metabolism
Shoots release into the atmosphere significantamounts of nitric oxide (NO�), a gaseous, unstable,and reactive free radical. NO� emissions as high as5� 10� 14 mol cm�2 s�1 have been reported for
10 ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants
various species under strong light, and even greaterspurts occur when the plants are returned todarkness.
The biochemical source of NO� in plants iscontentious, but there is growing evidence forenzymatic control. In mammalian cells, nitric oxidesynthase generates NO� in the conversions of theamino acid L-arginine to L-citrulline, a reaction thatconsumes oxygen and NADPH. Several, indirectlines of enquiry have indicated that a similar processoccurs in plants.
An alternative explanation, recently supported bydirect, in vivo evidence, holds that NO� is formedduring the metabolism of nitrate. Normally, nitrate isconverted in the cytosol to nitrite by nitratereductase. The nitrite is then reduced in thechloroplast to ammonium by nitrite reductase. Thereducing power for this second step, the productionof ammonium is by reduced nitrite reductase.However, at times when the supplies of Fdred arelow (at nightfall, or under environmental stress),nitrite is instead converted to NO� by nitratereductase (Figure 1). This mechanism accounts forthe burst in NO� evolution evident when plants aremoved from the light to the dark, but it does notexplain why NO� can also be generated in the light,when Fdred is plentiful. It is highly likely, thereforethat two or more pathways operate for the biosyn-thesis of NO� in plants.
NO� has both cytotoxic and cytoprotective proper-ties. NO� applications at concentrations above10� 6mol l� 1 inhibit photosynthesis and retardshoot growth. In plant cell cultures, high NO� levels
have been associated with irreversible DNA frag-mentation and cell death. In contrast, NO� at lowerconcentrations promotes normal growth and devel-opment. It stimulates seed germination, root elonga-tion, leaf expansion and phytoalexin production,inhibits etiolation, and retards the onset of senes-cence. NO� is also involved as a signaling molecule inplant defense responses to fungal pathogens, and hasbeen implicated as a promoter of tolerance toherbicides and drought.
Protective properties of NO� have been tentativelyascribed to an antioxidant function of this radical inplant cells. NO� diffuses freely, and can readilypermeate cell and organelle membranes. It has a highaffinity both for the transition metals and O��
2 ; andmay, therefore, prevent the formation of OH�
radicals from Haber–Weiss reactions. However, thereaction between NO� and O��
2 (Figure 1) generatesthe peroxynitrite ion (ONOO–), which itself isconsidered to be a major cytotoxic agent of reactivenitrogen. Moreover, ONOO– can react with H2O2 togive singlet oxygen (1O2), which has the potential todamage photosynthetic membranes.
Stress Responses
Laboratories and controlled greenhouses are prob-ably the only places where plants can experienceconstant environments. In more natural conditions,abrupt daily and seasonal changes are inevitable.Anthropogenic factors, such as the emission ofpollutants, the deposition of heavy metals, and theimpact of human activities on the climate, compoundthe stresses faced by plants. Stress has been defined as
NO3−
NO2−
NO2−
NH4+
FdoxFdred
NO.
NO.
ONOO−
O2O2−.
Thylakoid
Chloroplast
Nitratereductase
Nitritereductase
Cytosol
Amino acids
Nitratereductase Light
Figure 1 Tentative scheme for the biosynthesis of nitric oxide (NO�) and peroxynitrite (ONOO� ) in plant cells. Reproduced with
permission from Yamasaki H (2000) Nitrite-dependent nitric oxide production pathway: implications for involvement of active nitrogen
species in photoinhibition in vivo. Philosophical Transactions of the Royal Society, London, Series B 355: 1477–1488.
ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants 11
a state in which increasing demands made upon aplant lead to a destabilization of functions. Plantsoften recover from the effects of stress, and, indeed,subsequently show improved resistance. However,when the limits of tolerance are exceeded and theadaptive capacity is overworked, the result may bepermanent damage or even death.
Almost all environmental and biotic stressors canlead to the overproduction of reactive oxygen and/ornitrogen in plants (Table 2). The experimentalevidence is mostly indirect, based on observationsof changes in levels of different antioxidants uponthe application of a stressor. Recently, however,fluorescent probes have become available thatare specific for a given reactive species. Theseprobes have facilitated direct observations ofO��
2 ; H2O2, and NO� inside plant cells under themicroscope, and spectacular surges have been wit-nessed in response to fungal elicitors and mechanicalinjury (Figure 2).
Stressors can induce ROS formation both byupregulating the normal mechanisms for their bio-synthesis, and by creating new routes (Table 2).Under hot, sunny conditions, for example, the Mehlerreaction assumes increasing importance, accountingfor up to 20% of the electron flux through PSI.These conditions can also trigger photorespiration(the C-2 oxidative photosynthetic cycle), which,in the process of converting 2-phosphoglycolate to3-phosphoglycerate, generates H2O2 in the peroxi-somes. Significant oxidative loads are generated evenby those plants that are considered to be well adaptedto arid conditions, such as the CAM (crassulacean
acid metabolism) species of the desert. CAM plantsclose their stomata during the day to conserve water,and photosynthesize using CO2 obtained via thedecarboxylation of malic acid from vacuolar reserves.O2 is formed, but because the stomata are closed, itcannot readily escape from the leaves. As a con-sequence, internal O2 concentrations soar to around30% (41.5% in the exceptional case of Kalanchoegastonis-bonnieri), far in excess of the requirementsfor respiration. The excess oxygen leads to O��
2 andH2O2 by photooxidation.
Plants rarely experience a single stressor inisolation. Combinations of stressors can be synergis-tically effective in promoting ROS formation. Forexample, the combination of high light and lowtemperatures can impair photosynthesis through theformation of singlet oxygen (1O2). Low temperaturesabate the activities of Calvin cycle enzymes, but donot affect light capture. A sunny, cold climate thusleads to conditions where the absorbed light energy isin excess of the capacity to utilize ATP and NADPHin photosynthesis. The quanta excite ground-statechlorophyll first to its singlet state (1Chl*), and thento its overexcited triplet state (3Chl*). Tripletchlorophyll has a lifetime of around 1ms, andreleases its energy as heat, as fluorescence, or byreacting with O2 to form 1O2.
1O2 is highlydiffusible, but it is not a radical, and therefore doesnot initiate chain reactions directly. It can, however,oxidize the amino acids methionine, tryptophan,histidine, and cysteine, and 1O2 leads to peroxidationof lipid membranes in the chloroplast envelope,thylakoids, tonoplast, and plasmalemma.
Table 2 Examples of primary reactive species, and putative mechanisms for their overproduction in response to environmental,
UV-B radiation OH�, O��2 ; H2O2 Inhibition of PSII reaction center enzymes; possibly fission of H2O2
Drought O��2 ; H2O2, NO
� Inhibition of rubisco; uncoupling of electron transport from ATP synthesis;
enhanced Mehler activity; photoinhibition; inhibition of mitochondrial
antioxidants; enhanced root respiration
Mechanical injury O��2 ; H2O2, NO
� Elicitation by cell wall fragments; interference with redox systems on plasma
membrane
Salinity O��2 ; H2O2, NO
� Stomatal closure, causing NADPþ deficit and O2 reduction in mitochondria
Pathogens O��2 ; H2O2, NO
� Activation of membrane bound NADPH oxidase or cell wall peroxidase
Pollutant gases OH�, O��2 ; H2O2, NO
�,ONOO–
Metabolism of O3, SO2, NOx entering through stomata
Herbicides O��2 ; H2O2,
1O2 Interference with photosynthetic electron transport; photoactivated herbicide
interactions with O2; inhibition of antioxidants
Heavy metals Fe2þ , Cu2þ , OH�, 1O2,
O��2 ; H2O2
Direct uptake from contaminated soils; Haber–Weiss reactions; Fe
dependent photosensitization
12 ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants
Reactive species of oxygen and nitrogen arecertainly not the only causes of cellular injury inplants under stress, yet they are evidently a sig-nificant contribution. Crops that have been genetic-ally engineered to overproduce antioxidant enzymesoften show enhanced cross-tolerance to severaldifferent stressors. Conversely, the underproductionof antioxidant enzymes is associated with anincreased sensitivity to stress, indicating that ade-quate protection from the effects of ROS is essential.
Oxidative Injury
Lipid membranes, DNA, and proteins are all directtargets for attack by free radicals. Radicals also acton cell metabolism indirectly, by initiating signalingcascades involving the release of secondary messen-gers such as free Ca2þ . Oxidative injury manifestsitself as chlorotic and/or necrotic lesions, reducedproductivity, abnormal development, and ultimatelythe death of the plant.
Chlorophyll bleaching is often the first visible signof oxidative injury. Lipids in the chloroplast envelopeand thylakoid membranes contain a high percentageof polyunsaturated fatty acids, and are thereforeparticularly susceptible to peroxidation. Radicalinduced lipid peroxidation involves the initiation,propagation, and termination of a chain reaction.Initiation occurs when a radical (e.g., OH�) extractsa hydrogen atom from a methylene group in the fattyacid, creating a lipid alkyl radical (R�). During thepropagation phase, the alkyl radical reacts withoxygen to form a lipid peroxyl radical (RO2
�), whichcan then remove a hydrogen from an adjacent fatty-acid side chain to form a new alkyl radical. Lipidperoxides are formed in ever-increasing numbers asthe reaction propagates along the fatty-acid chains.Termination of the chain reaction may be achievedby dimerization of adjacent radicals. By disrupting
lipid structure, peroxidation increases the leakinessof membrane bilayers, inactivates membrane boundenzymes, and generates cytotoxic aldehydes andketones.
Oxidative stress greatly accelerates DNA damage.OH�, in particular, causes single- and double-strandbreakages, fragmentation of the deoxyribose sugar,chemical alterations to bases, and cross-links be-tween DNA and protein, which impair chromatinunfolding, DNA repair, and transcription. Proteinsexposed to OH� show damage to amino acidresidues, polypeptide fragmentation, denaturation,and aggregation.
Antioxidants
An antioxidant has been defined as ‘‘any substancethat, when present at low concentrations comparedto those of an oxidizable substrate, significantlydelays or inhibits oxidation of that substrate.’’ Thecaveat that antioxidants be present at low concen-trations is critical; because many antioxidantspromote redox reactions, they can act as pro-oxidants if present in large amounts. Antioxidantsfunction by: (1) preventing the production of freeradicals; (2) scavenging the unpaired electron; (3)quenching the energy of excited molecules such as1O2; or (4) terminating chain reactions.
Plant cells normally contain a suite of antioxidantslocalized in different compartments (Figure 3). Theantioxidant complement often changes as organsgrow and develop, and is markedly affected byenvironmental stresses. Antioxidants are broadlycategorized into two groups: the enzymatic antiox-idants and the low molecular weight (nonenzymatic)antioxidants. Both groups are apparently requiredfor the effective removal of reactive species. Theenzymatic antioxidants are further subdivided intotwo types: those that deal directly with toxic
1 min 5 min 10 min 15 min 20 min
0 256
Figure 2 Oxidative burst in the epidermal cells of the leaves of Pseudowintera colorata at various times after puncturing with a
needle. Hydrogen peroxide (red color), produced immediately after wounding, is gradually scavenged by antioxidants (increasing blue
coloration). Scale bar¼200mm. Reproduced with permission from Gould KS, McKelvie J, and Markham KR (2002) Do anthocyanins
function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury. Plant, Cell & Environment 25:
1261–1269.
ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants 13
oxidants and those that help to restore the spentantioxidants to their reduced state.
Superoxide dismutase (SOD) is probably the bestresearched of the enzymatic antioxidants. It isextraordinarily efficient – one of the fastest enzymesknown – scavenging O��
2 produced both by thechloroplasts and the mitochondria. Three types ofSOD isozymes occur in plants, distinguished by thetype of metal cofactor required to catalyze thedismutation reaction: MnSOD (in mitochondria);FeSOD (in chloroplasts); and Cu/ZnSOD (in cytosol,peroxisomes, and chloroplasts). SOD activities in-crease appreciably in harsh environments, implicat-ing a role in stress tolerance.
H2O2 can be scavenged either by catalase (CAT) orthe peroxidases (PX). CAT occurs predominantly inthe peroxisomes, where it scavenges H2O2 producedby photorespiration, but it may also be found in themitochondria:
2H2O2 -CAT
2H2OþO2
In the chloroplasts, ascorbate peroxidase (APX) isthe key enzyme involved in H2O2 scavenging. APXuses ascorbate (vitamin C) as a substrate in a redoxreaction, producing monodehydroascorbate (MDA):
2 ascorbateþH2O2 -APX
2MDAþ 2H2O
Although H2O2 has been eliminated, chloroplastsnow need to recover ascorbate, a particularly useful
molecule, from MDA. This can be achieved byvarious routes. In the thylakoids, MDA is reducednonenzymatically by ferredoxin:
MDAþ Fdred-ascorbateþ Fdox
In the stroma, the enzyme MDA reductase (MDAR)is used:
MDAþNADPH -MDAR
2 ascorbateþNADPþ
Further enzymes are needed because MDA can alsospontaneously dissociate into ascorbate and dehydro-ascorbate (DHA). DHA is reduced to ascorbate bydehydroascorbate reductase (DHAR), using gluta-thione (GSH) as the reducing substrate:
DHAþ 2 GSH -DHAR
ascorbateþGSSG
Glutathione reductase (GR) completes the ‘‘ascor-bate–glutathione’’ pathway, regenerating GSH usingNADPH from the light reaction:
GSSGþNADPH-GR
2GSHþNADPþ
In addition to the enzymatic antioxidants, there hasbeen increasing recognition of the role of low-molecular-weight antioxidants (LMWAs) as protec-tants from oxidative stress. This has, in part,stemmed from research that shows that such com-pounds are important components of human nutri-tion, potentially protecting us from the developmentof diseases such as cancer. LMWAs are a diverse
Figure 3 Localization of enzymatic and low-molecular-weight antioxidants in plant cells.
14 ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants
assortment of water and lipid soluble compounds,including various vitamins, glutathione, and severalplant pigments. They occur in almost all compart-ments of the plant cell (Figure 3).
Vitamin E (a-tocopherol) is probably the mostimportant of the mobile lipid soluble LMWAs.Found in thylakoid membranes, a-tocopherol effec-tively scavenges O��
2 ; serves as a chain breakingagent to stop the propagation of lipid peroxides, andprotects the membrane proteins from oxidation ofS-H groups. In performing these tasks, a-tocopherolis itself oxidized into a radical, and is regenerated toits reduced state by ascorbate.
Ascorbate (vitamin C) is a particularly versatileantioxidant. Located in the cytosol, apoplast, and thechloroplasts, ascorbate can both quench 1O2 andscavenge O��
2 : It also regenerates various antioxi-dants, such as a-tocopherol and the flavonoid andxanthophyll pigments, and serves as a substrate inenzyme catalyzed reactions for the detoxificationof H2O2.
Plant pigments have unique, multiple roles in theprotection from oxidative stress. First, they effi-ciently eliminate reactive oxygen species. b-carotene,an orange pigment that is a precursor of vitamin A,quenches 1O2 produced by chloroplasts. The flavo-noid pigments are particularly potent scavengers ofmost reactive species, including H2O2,
1O2, O��2 ;
OH�, RO�2; and ONOO–. Indeed, cyanidin-3-gluco-
side (an anthocyanin) and quercetin (a flavonol) haveantioxidant capacities more than four times greaterthan those of vitamins C and E, as estimated usingautomated ‘‘oxygen radical absorbance capacity’’(ORAC) assays. Anthocyanins, the pigments thatcause red, purple, and blue colors in various plantorgans, are predominantly located in the cell vacuole.Recent studies have shown that red leaves often havehigher capacities to combat oxidative stress than dogreen leaves, and that cells with anthocyanicvacuoles remove H2O2 more rapidly than do color-less cells.
A second, important role of the plant pigments isto prevent the generation of ROS in the first place.Xanthophyll pigments (comprising three carotenoidpigments) achieve this by absorbing the excess energyin triplet chlorophyll before it can be transferred tomolecular oxygen. Under strong light, violaxanthinis converted to antheraxanthin, and then to zeax-anthin, which dissipates the excess energy as heat.The anthocyanins in leaves function as a light filter,intercepting the energetic green wavelengths beforethey have an opportunity to cause free radicals in thechloroplasts. Similarly, the colorless flavonoids andhydroxycinnamates, which are especially abundantin cell walls of the upper epidermis, attenuate
ultraviolet radiation before it can reach chloroplastsin the underlying cells.
Concluding Remarks
The potential for oxidative injury is a seriousconcern, particularly for crop plants, which are oftengrown in environments far-removed from those oftheir ancestral origins. Current changes to the globalclimate, the elevated temperatures and UV-B levels,and increasing incidence of severe droughts andfloods are expected to increase the likelihood ofoxidative stress even further. It is not surprising,therefore, that there has been a concerted effort bychemists, plant scientists, and medical researchers tounderstand and improve upon the natural anti-oxidant systems in plants. The emerging data areextremely encouraging. Cultivars of wheat (Triticumspp.), for example, have been identified that producesupernumerary levels of antioxidant enzymes inresponse to abiotic stressors; these cultivars have anenhanced tolerance to water stress, salinity, and heat.Similarly, in several species, plants that have beengenetically engineered to overexpress the genes forSOD, APX, and/or CAT are demonstrably moretolerant to drought, salinity, chilling, freezing, andherbicides. Thus, the problems stemming fromoxidative stress in cultivated plants, though signifi-cant, might not be insurmountable.
See also: Abiotic Stresses: Cold Stress; MechanicalStress and Wind Damage. Genetic Modification, Appli-cations: Oxidative Stress. Photosynthesis and Parti-tioning: Photoinhibition; C3 Plants; C4 Plants; CAMPlants. Plants and the Environment: Global WarmingEffects; Ozone Depletion; Plants and Atmospheric Pollu-tion. Regulators of Growth: Photoperiodism. WaterRelations of Plants: Drought Stress; Salt Stress.
Further Reading
Foyer CH, Lelandais M, and Kunert KH (1994) Photo-oxidative stress in plants. Physiologia Plantarum 92:696–717.
Gould KS and Lee DW (eds) (2002) Anthocyanins inLeaves: Advances in Botanical Research, vol. 37.London: Academic Press.
Inze D, and Van Montagu M (eds) (2002) Oxidative Stressin Plants. London: Taylor & Francis.
Halliwell B and Gutteridge JM (1999) Free Radicals inBiology and Medicine, 3rd edn. Oxford: OxfordUniversity Press.
Larson RA (1997)Naturally Occurring Antioxidants. BocaRaton: Lewis Publishers.
Leshem YY (2000) Nitric Oxide in Plants: Occurrence,Function and Use. Dordrecht: Kluwer AcademicPublishers.
ABIOTIC STRESSES /Free Radicals, Oxidative Stress and Antioxidants 15
Polle A (1997) Defense against photooxidative damage inplants. In: Scandalios JG (ed.) Oxidative Stress andthe Molecular Biology of Antioxidant Defenses, pp.623–666. New York: Cold Spring Harbor Laboratory.
Wendehenne D, Pugin A, Klessig DF, and Durner J (2001)Nitric oxide: comparative synthesis and signalingin animal and plant cells. Trends in Plant Science 6:177–183.
Mechanical Stress and WindDamageJ Grace, University of Edinburgh, Edinburgh, UK
Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction
Erasmus Darwin wrote about the sensitivity of plantsto touch in 1794. Later, his grandson Charles Darwincarried out experiments on the touch-sensitivity ofinsectivorous and climbing plants. One of the mostspectacularly sensitive plants, Mimosa pudica, foldsits leaves completely within a few seconds of beingtouched. Many climbing plants have tendrils thatrespond to touch by curling, a process that requiresdifferential growth on different sides of the tendril.Less obvious touch-sensitivity occurs in most otherplants. They respond to mechanical stimulation bydeveloping more structural tissue, enabling them towithstand the forces of gravity and wind. Thesestructural tissues take various forms. They are mostevident in the stems and branches of trees, whichoften develop mechanical tissues eccentrically inorder to compensate for the tendency for directionaldisplacement by the prevailing wind or by slope.However, similar growth responses are more or lessubiquitous in green plants, affecting leaves and rootsas well as stems and branches, and can affect annualplants, grasses, and shrubs as well as trees.
Historically, the effects of wind were noted in theecological and forestry literature, usually in thecontext of stress; but the observations almost alwaysrelate to situations where effects of other factors areinvolved. For example, the wind shaping and‘‘flagging’’ of trees in coastal regions may be causedby the deposition of salt on the windward leavesrather than to the direct effect of wind. Likewise, thewind shaping of trees on mountains may be theconsequence of small differences in the temperatureof tissues on the windward versus leeward side of thecrown, rather than the direct effect of mechanicalstimulation on the tissues. Thus, to understand the
effect of wind and mechanical stress on plants, it isusually necessary to carry out controlled experimentsusing wind tunnels, shaking machines, and mechan-ical rigs, which enable forces to be applied to plants.When plants are exposed to wind or to almost anymechanical challenge, their rate of growth declinesand the allocation of new biomass shifts to achievemore mechanical tissue and less photosynthesizingtissue.
In trees, this mechanical tissue takes the form ofthickened cells in the woody tissues: these tissues arefound in particular parts of the tree where mechanicalstrengthening is required. The material formed ismuch denser than normal wood and is called reactionwood or compression wood, depending on whether ithas been formed under tension or compression. Suchstructural changes have long been known to anato-mists and wood technologists, but it took physiol-ogists rather a long time to realize that there reallywas a direct response to mechanical stimulation by allplants. It was not until 1973 that the term thigmo-morphogenesis was coined by Jaffe to describe thegeneral phenomenon whereby plants perceive mech-anical stimuli and undergo a growth response.
In this article, it is convenient to discuss the effectsof wind and mechanical stresses under two headings:direct and indirect effects.
Direct Effects
Response to Movement of Plants in the Wind
In nature, terrestrial plants are more often in motionthan not, as a result of air movement. There havebeen some notable experiments in which trees wereprevented from moving. The result is very clear:when they are restrained, trees grow taller, but theirstems are thinner (Figure 1). A lesser proportion ofthe water conducting cells, the tracheids, developthick walls to confer mechanical strength. Whensupported at 451 and rotated, the thickeningresponse was especially well developed (Figure 1).Somewhat similar results have been obtained fromexperiments on herbaceous plants such as corn (Zeamays; maize) and sunflower (Helianthus spp.). Manysuch observations point toward a self-regulatingcapacity whereby the plant builds its own supportingstructure, enabling it to withstand the forces actingupon it. Thus, when external support is provided, thestems of the plant do not need to be as thick, andassimilates are directed to an increase in heightgrowth at the expense of growth in diameter.
There are some interesting arboricultural implica-tions of this phenomenon. When trees are planted inurban environments they are often provided with a
16 ABIOTIC STRESSES /Mechanical Stress and Wind Damage
Ozone and Reactive OxygenSpeciesAndrew C Allan, HortResearch, Auckland, New Zealand
Robert Fluhr, Plant Sciences, Weizmann Institute of Science, Rehovot, Israel
Increasing levels of the pollutant ozone are likely to have a dramatic effect on future plant
productivity. Ozone’s damaging effect on the plant is mediated primarily via an induction
of cellular reactive oxygen species (ROS).
Introduction
Ozone (O3) is an ever-present pollutant gas, formed by theinteraction of nitrogen oxides with hydrocarbons and UVradiation. Ozone has useful UV-B screening effects; itsdepletion from the stratosphere is therefore the cause ofsome alarm. However, despite these reductions in totalatmospheric ozone, it is predicted that over the next50 years, the tropospheric (from the surface to about 10 kmabove sea level) levels of ozone will increase by 1% peryear. The source of this ozone is anthropogenic emissionsof nitrogen oxides that after photolysis react with carboncompounds to yield ozone. Ambient ozone concentrationsgenerally range from 20 to 60 nanolitres (nL) per litre of air(20–60 ppb), but local increases of up to 250 ppb have beenobserved. Problematic levels of ozone are not restricted totheir source in cities and industrial sites; because of dis-persal patterns it is often rural areas that suffer the highestincreases in ozone. Western Europe, mid-western andeastern USA, and eastern China are currently beingexposed to some of the highest background levels of ozone(Morgan et al., 2006). Current ozone levels can have dam-aging effects on plants at levels above 40 ppb, depending onduration of exposure and the sensitivity of the plant spe-cies. A reported increase of 13 ppb in mean daytime ozonecauses a 20% decrease in soybean seed yield. This increasein ozone concentration could be reached as soon as 2020in south Asia. Thus, the effect of ozone on the health ofour major crops must be of major concern. See also: AcidRain; Biogeochemical Cycles
Effects of Ozone on Plant Performance
Ozone is 12 timesmore soluble inwater than is oxygen. It isunstable and decomposes to highly oxidizing free radicals.The type of damage that ozone exerts on the plant can bedivided into two categories: (1) acute effects, involvingelicitation of plant defence responses and switching on ofeither hypersensitive responses or necrosis, resulting innecrotic areas of brown or white colour appearing in
punctate areas on leaves and fruit; (2) chronic effects,including reduction in growth and fitness, with resultingpigmentation change and chlorosis, the outcome of accel-erated senescence. It is difficult to distinguish chronic fromacute effects. Some parts of the plant may be undergoingacute effects while the plant in general is experiencing asystemic chronic effect. See also: Enzymatic Free RadicalReactions; Plant Stress PhysiologyCertain plant species are ozone tolerant, while others are
hypersensitive. It has been proposed that damage resultingfrom a 4-h exposure to ozone at 50ppb indicates a sensitivespecies; damage due to exposure to 100ppb indicates anintermediate plant; and 150ppb indicates a tolerant species.The so-called ‘sentinels’ are sensitive plants that are intro-duced into an area to serve as early warning devices or aschecks on the efficiency of abatement practices. Examples ofsentinels include sensitive cultivars of tobacco. Importantly,sensitivity to ozone in tobacco and other species is clearlyunder complex genetic control, implying that tolerance isthe result of a combination of traits. In practice, under con-trolled conditions, yield reductions of up to 30% areobserved in such common crops as potato, bean and wheatwhen exposed to local ambient ozone concentrations.Recently, controlled laboratory experiments have beenextrapolated to field conditions. In a free-air gas concen-tration enrichment (FACE)field experiment for the effect ofozone fumigation, a 23% increase in ozone concentrationdecreased seed yield by 20% (Morgan et al., 2006).Being gaseous, water soluble and highly reactive, ozone
will have many effects on the plant’s physiology. For sim-plicity only, wewill divide the probable causes of decreasedplant performance into discrete areas.
Ozone effects on components ofphotosynthesis
Both light and dark photosynthetic reactions are affectedby ozone. Ozone decreases carbon dioxide assimilation in
Article Contents
Advanced article
. Introduction
. Effects of Ozone on Plant Performance
. Induction by Ozone of Plant Stress Responses
. Ozone, ROS Generation and Programmed Cell Death
. ROS during Photosynthesis
. ROS during Senescence
. Ozone and ROS during Pathogenesis
. Ozone and ROS Detoxification in Normal and in
Transgenic Plants
doi: 10.1002/9780470015902.a0001299.pub2
1ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net
Vicia faba by inhibiting guard cellK+ channels, which thendrives the closing of stomates (Torsethaugen et al., 1999).However, the resultant stomatal closure is eventually ben-eficial as it limits further ozone entry.
Exposure to ozone has been shown to decrease the levelsof mRNA for Rubisco small subunit, chlorophyll a/b pro-tein, and glyceraldehyde-3-phosphate dehydrogenase(Glick et al., 1995) either by specifically switching offtranscription or by increased messenger ribonucleic acid(mRNA) instability. Rubisco protein, the major solubleprotein in leaf extracts, was shown to degrademore rapidlywhen exposed to ozone. See also: Photosynthesis; Rubisco
Ozone effects on primary metabolism andhormones
Anumberofmetabolic processes are switchedonor alteredfollowing exposure to ozone. At the whole-plant level,carbohydrate assimilation and carbon allocation to theroots of large trees (mostly studied in conifer species) aredramatically reduced following ozone exposure. The directincrease in stomate resistance and desensitization to ABAnoted above is one mode of action of ozone that will effectmetabolism by limiting carbon dioxide. However, theinduction by ozone of the plant hormone ethylene plays anadditional critical role in this process. Inappropriateinduction of ethylene by ozone appears to trigger featuresof senescence damaging to plant metabolism. Ethyleneproduction in the plant occurs via the enzyme 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, whichcatalyses the conversion ofS-adenosylmethionine toACC.ACC is then converted into ethylene by ACC oxidase.Increases in the conversion of ACC into ethylene, due tothe presence of ozone, appear to result from damagedmembranes, perhaps allowing normally sequestered subst-rate and enzyme to mix. Moreover, ozone also increasesACC levels by altering ACCmetabolism; levels of mRNAfor ACC synthase and ACC oxidase increase rapidly uponexposure to ozone (Tuomainen et al., 1997). In addition,ozone and ethylene can interact directly, producing super-oxide (O22) and reactive aldehydes,which drivemembranedamage. See also: Plant Growth Factors and Receptors;Stress-induced Premeture Senescence(SIPS)
Induction by Ozone of Plant StressResponses
Microarray experiments have revealed a fuller extent ofplant transcriptional response to ozone; when Arabidopsisis treatedwith 350 ppbozone (3 and 6 h), 2385 genes showadifferential expression equal or greater than 2-fold change(Ludwikow et al., 2004).Many of these genes are part of anethylene and jasmonic acid (JA)-induced stress-response
pathway indicating that damage has occurred and thetranscriptomehas adjusted to induce aprotective response.Indeed, increased ozone-induced foliar lesions wereobserved in jar1 mutant plants that are insensitive to JAproduction (Tamaoki et al., 2003). These responses are inturn antagonizedby the elicited production of salicylic acid(SA) as ozone promotes SA synthesis via ethylene. In npr1mutants of Arabidopsis that cannot promote the SAresponse the upregulation of protective genes is furtherenhanced (Tamaoki et al., 2003). A complex scenario hasemerged in which ozone-induced spreading of cell death isstimulated by early, rapid accumulation of ethylene, whichthen suppresses the protective actions of JA. Further, celldeath induces late accumulation of JA, which inhibits thefurther propagation of cell death via inhibition of the eth-ylene pathway.The fluorescent (flu) mutant of Arabidopsis accumulates
protochlorophyllide in the dark. This protochlorophyllidethen generates singlet oxygen when the plants are returnedto the light. Therefore, transfer of dark grown plants intothe light causes a massive, and specific increase, in singletoxygen within the chloroplast (op den Camp et al., 2003).Microarray analysis of transcript expression inArabidopsisfollowing exogenous application of oxidative stress-caus-ing agents including ozone treatment, and the flu mutant,shows which gene transcripts are specifically altered intheir expression by a particular type of ROS and which aremore generally responsive to ROS (Gadjev et al., 2006).For example, a group of 66 transcripts are commonbetween ozone and methyl viologen treatments and the flumutant singlet oxygen response. Five transcription factorsappear to be ozone specific, including AtWRKY70, whichshows a 37-fold increase in transcript levels after ozonetreatment. This transcription factor has been implicated inSA-mediated suppression of JA-induciblePDF1. 2 expres-sion (Li et al., 2004), lending a molecular basis for thecomplex scenario of cell death and subsequent suppressionof the protective actions of JA described above.Enzymes that function to dissipate ROS are induced by
ozone. Among these are glutathione S-transferase, whichaids in the restoration of molecules oxidized by free rad-icals; superoxide dismutase (SOD), which converts O2
2
into H2O2; and catalase isoform 2, which dissipates H2O2.Secondary effects associated with cell death are the elici-tation of pathogenesis-related proteins (PR proteins) andincreases in SA, phytoalexins, flavonoids, lignin and cal-lose. These responses may aid in plant defence against thepollutant (e.g. production of flavonoids acting as antioxi-dants) or be part of a more general response to stress. ROSare used by the plant as secondary messengers for plantpathogen response (see later sections), so it is not surprisingthat ozone inadvertently switches on pathogenesisresponse pathways. For example, ozone exposure oftobacco results in enhanced resistance to tobacco mosaicvirus infection (Yalpani et al., 1994). In contrast, the dam-age caused by ozone can allow a second abiotic or biotic
Ozone and Reactive Oxygen Species
2
stress to become more damaging. For example, pine treessuffering nutrient deficiency (Mg2+, Ca2+orK+) aremoreaffected by the presence of ozone than those not deficient(Siefermann-Harms et al., 2005). See also: Ecophysiologi-cal Responses of Plants to Air Pollution
Ozone, ROS Generation andProgrammed Cell Death
Ozone enters the plant through open stomata and diffusesthrough the apoplast. The low permeability and strongreactivity of ozone mean that the cytosol and other organ-elles of the plant probably do not experience free ozone.Instead, ozone appears to degrade rapidly into other ROSwithin the apoplast or plasma membrane (Figure 1). ROSare the primarymediators of the oxidative damage in plantcells. They include superoxide radicals (O2
2), .OH, singletoxygen (1O2) and H2O2. ROS, and in particular O2
2, arestrong oxidizing species that can rapidly react with othermolecules, including deoxyribonucleic acid (DNA), lead-ing to metabolic changes and hypersensitive response(HR). Ozone also reacts directly with the cell membrane toproduce aldehyde and organic radicals. Ozone elicitationof ROS has characteristic bimodal kinetics; an early
apoplastic peak in ROS has been observed in response toacute ozone exposure (in the first few hours), with a secondlonger lasting burst (after 15–72 h) observed in ozone-sen-sitive tobacco cultivars in association, both temporally andspatially, with HR. It has been shown, using electron spinresonance, that hydroxyl radicals (.OH) appear in plantcells during exposure to ozone. This conversion is aided bythe presence of free iron. See also: Oxidative StressProbably, the earliest cellular response to ozone is an
elevation in cytosolic-free calcium, which occurs withinseconds of exposure (Evans et al., 2005). The presence of aparticular calcium ‘signature’ and ROS activates a mito-gen-activated protein kinase (MAPK) kinase (MAPKK)signalling pathway.A tobaccoMAPKkinase,NtMPK4, isexpressed in the epidermis. Plants with less NtMPK4 showenhanced sensitivity to ozone and an abnormal regulationof stomatal closure in anABA-independentmanner (Gomiet al., 2005).Ozone-induced ROS is not restricted to the cytosol;
when tobacco cv Bel W3 plants are fumigated with acuteozone levels there is an accumulation of H2O2 inmitochondria and chloroplasts, as well as an early accu-mulation of nitric oxides (NO) in leaf tissues (Ederli et al.,2006). During ozone exposure, H2O2 continues to accu-mulate in the apoplast, apparently because of the activa-tion of both the plasma membrane-bound NAD(P)H
RbohO2
−
O2
H2O2
G-proteins amplification
O3
Ascorbic acid scavenging
Low concentrationchronic exposure
High concentrationshort exposure (acute)
Apoplast
Membrane
Scavengers of ROS andantioxidants
Induction of PR genesAntioxidant pathwaysPAL, flavonoids
Figure 1 A model for plant response to ozone. Elicitation of different plant responses ranging from senescence to rapid necrosis can be generallyexplainedby the relative dose of ozone that theplant receives.Ozone is dissipated in the apoplast to other free radicals andROS. These act asmore diffusiveagents, entering the cytosolwhere they are either scavengedor act as secondarymessengers for a number of plant responses(seeboxes). Cytosolic calciumsignalling is elicited, via influx of Ca2+ from the apoplast. The balance of ROS concentration versus antioxidant scavenging potential helps decide the cellfate. Good evidence exists for an amplification of ozone-elicited ROS, in susceptible plant varieties, by triggering of the plasmamembrane enzymeNADPHoxidase tomake superoxide. This candrive programmed cell death (e.g. hypersensitive response). PR, pathogen response; PAL, phenylalanine lyase; Rboh,respiratory burst oxidase homologues.
Ozone and Reactive Oxygen Species
3
oxidase complex and cell-wall located NAD(P)Hperoxidases. Using Arabidopsis mutants in the a and bsubunits of heterotrimeric G proteins, Joo et al. (2005)dissected out the pathwayproducing the bimodal oxidativeburst elicited by ozone. The early component of the oxi-dative burst, arising primarily from chloroplasts, requiressignalling through the G b–g complex to the membrane-bound nicotinamide-adenine-dinucleotide phosphatereduced (NADPH) oxidase. The late, tissue damage-asso-ciated component of the oxidative burst requires only theG a protein and arises from multiple cellular sources.Necrotic lesions have been seen to appear in tobacco after asingle acute dose of ozone (5 h of 150 ppb). In this case, thelocal concentration of ozone could be high enough for anunregulated cell necrosis to occur as a result of ozone-induced oxidation of membrane fatty acids, leading tomembrane rupture. Alternatively, ROS build-up in theapoplast and the cytosol can be below acute levels andmayelicit HR. HR is a highly regulated process of cell death.The cell undergoes characteristic developmental steps, in-cluding chromosome condensation, release of cytochromeC from the mitochondria, regulated DNA cleavage andvacuolar degradation. These characteristic markers of HR(or programmed cell death, PCD) are seen in ozone-treatedtobacco cv Bel W3 plants (Pasqualini et al., 2003). Tran-sient application of ozone causes the formation of celldeath lesions on the leaves of the ozone-sensitive radical-induced cell death1 (rcd1)mutant ofArabidopsis. The dyingcells exhibited several of the typical morphological char-acteristics of the hypersensitive response and PCD. Dou-ble-mutant analyses indicated a requirement for SA andthe function of the cyclic nucleotide-gated ion channelAtCNGC2 in cell death (Overmyer et al., 2005). See also:Plant Programmed Cell Death
ROS during Photosynthesis
The photosynthetic electron transport chain and the chlo-rophyll pigments themselves can, under certain conditions,produce singlet oxygen and O2
2; this is termed photo-oxi-dative stress. During photosynthesis, any ROS inadvert-ently generated is rapidly removed by antioxidativemechanisms. However, this removal can be compromised(e.g. by exposure to ozone). The balance between produc-tion and removal of ROS in the chloroplast may be per-turbed by abiotic stresses such as ozone or extremetemperature (e.g. low temperatures and sunny conditions).As a result, intracellular ROSmay rapidly rise. One sourceof ROS during photosynthesis is the direct photoreductionof oxygen to superoxide by reduced electron transportcomponents associated photosystems I or II. Furthermore,during photoinhibition, which occurs when plants areexposed to high light intensities, singlet oxygen can becontinuously produced by PSII. Thus, in the presence of
ozone and during excess light the damage wrought bysinglet oxygen and superoxide production drasticallyincreases due to acute overloading of the scavengingcapacity of the cell. See also: Photosynthesis: DarkReactions; Photosynthesis: Light Reactions
ROS during Senescence
Senescence involves a directed general degradation of thecellular components such as proteins and the mobilizationof the products of degradation to other parts of the plant.During both naturally induced senescence and ozone-pro-moted senescence there is a cessation of photosynthesis,disintegration of organelle structures, loss of chlorophylland certain proteins, and increases in lipid peroxidation,membrane leakiness, ethylene and jasmonic acid.Adrivingforce behind senescence is due to the enhanced release ofO2
2 from the peroxisome. The relationship to ozone is notclear. For example, yellowing of Arabidopsis leaf is accel-erated by ozone application that is accompanied by theappearance ofmanybut not all of the naturally senescence-associated genes (Miller et al., 1999). See also: PlantPeroxisomes and Glyoxysomes
Ozone and ROS during Pathogenesis
An oxidative burst is often induced when the plant firstdetects the presence of a pathogen. Pathogen-induced oxi-dative bursts are harnessed by the plant cell both as ameans of ‘poisoning’ an invading pathogen (a massiveburst that drives cell death) and as an intra- and extracel-lular message of this invasion. Therefore, ROS play a cen-tral role in the plant defence against pathogens. H2O2 andO2
2 appear to be agents of this burst and can be generatedby activation of a number of enzymatic sources: a plasmamembrane NAD(P)H oxidase, cell wall-located peroxi-dases, or amine, xanthine or aldehyde oxidase activities. Inaddition, both the activity and level of theROSdetoxifyingenzymes such as ascorbate peroxidase (APX) and catalaseare suppressed. The simultaneous production of ROS anddownregulation of ROS scavenging mechanisms can thentriggerPCD.Asone effect of ozone,mentioned above, is animmediate production of ROS via activation of NADPHoxidase (Joo et al., 2005) its influence on plant resistance todisease may be to short-circuit and jumble the disease-response signal. Necrotroph-style pathogens may be at anadvantage in this state as they utilize the plants investmentin ROS metabolites for their own benefit. An ozone-induced oxidative burst results in a cell death processsimilar to pathogen-induced PCD. However, the ROSgenerated by ozone can later serve to elevate ROS scav-enging enzymes that may compromise an appropriatedefence to biotic attack. This would explain why, in many
Ozone and Reactive Oxygen Species
4
instances, plants exposed to ozone suffer from a secondbiotic infection.
Ozone and ROS Detoxification inNormal and in Transgenic Plants
The elevation of cytoplasmic levels of ROS triggers stresssignalling pathways. However, the plant possesses efficientantioxidative defence systems, including SODs, catalases,peroxidases, ascorbate (vitamin C), glutathione, tocophe-rol (vitamin E), carotenoids and flavonoids.Under normalconditions, these antioxidative enzymes and small mole-cules provide protection against ROS, but when the mag-nitude of the oxidative stress is large, or when defences arepurposely downregulated (e.g. duringHR), large increasesof ROS in the cell can cause acute damage. Thus, attemptshave been made to engineer increased resistance to oxida-tive stresses, such as from ozone, through overexpressionof enzymes that dissipate ROS. Tolerance to ozone hasbeen achieved by targeting these enzymes to the cytoplasm.For example, overexpression of pea cytosolic Cu,ZnSODin tobacco conferred partial protection against ozonedamage, while overexpression of petunia Cu,Zn-SOD inthe chloroplast had no effect (Pitcher and Zilinskas, 1996).Reduced peroxisomal catalase increases the sensitivityof Arabidopsis to both ozone and photorespiratory H2O2-induced cell death. See also: Arabidopsis thaliana as anExperimental Organism; Transgenic Plants
Apparently, apoplastic ascorbic acid levels are a keydeterminant to sensitivity or resistance to ozone; distinctdifferences in apoplastic ascorbate are found in specieswith differences in ozone sensitivity. When expression lev-els of APX are modified in transgenic plants, a 10-foldincrease of APX detected in the chloroplast led to noincrease in ozone tolerance (Torsethaugen et al., 1997).Conversely, plants expressing antisense RNA of cytosolicAPX have an increase in sensitivity to ozone (Orvar andEllis, 1997). Both spinach and beech responded to ozonetreatment by exporting ascorbate into the apoplast in anapparent attempt to detoxify radicals formed before theirentry into the cytoplasm. The role of ascorbate in thedefence against ozone-induced damage is further high-lighted by analysis of Arabidopsis mutants. Arabidopsisplants containing the soz1mutation accumulate only 30%of normal ascorbate levels and are hypersensitive to ozonewhileVitamin c-1 (vtc1) is anozone-sensitivemutantwhichis deficient in ascorbic acid. The mutated locus was clonedand found to be GDP-Man pyrophosphorylase (Conklinet al., 1999), an enzyme which converts D-mannose-1-PintoGDP-mannose, a step in the ascorbic acid biosyntheticpathway.
Increasing the endogenous level of ascorbic acid hasbeen achieved by overexpressing dehydroascorbate red-uctase (DHAR). DHAR-overexpressing plants have a
lower oxidative stress, a lower level of oxidative-relatedenzyme activities, a higher level of chlorophyll and pho-tosynthetic activity following acute (2 h of 200 ppb) andchronic ozone exposure (30 d–100 ppb). Suppression ofDHAR expression had the opposite effect (Chen andGallie, 2005). There are threeDHARgenes inArabidopsis,but it is only cytosolic DHAR that increases with ozoneexposure. A mutant with no cytosolic DHAR activity ishighly ozone sensitive. Although total amounts of ascor-bate are not reduced in this mutant, the apoplastic ascor-bate is 60% lower. It therefore appears that apoplasticascorbate, generated through the reduction of dihydro-ascorbate by cytDHAR, is important for ozone tolerance(Yoshida et al., 2006). However, by elevating the ascorbatelevel, the guard cell is rendered less responsive to ABA andozone. Nevertheless, the harmful effects of ozone on theplant is reduced, mainly due to enhanced protectionagainst ROS.These findings show that engineering ozone tolerance
through a transgenic approach will have to take intoaccount the complex physiology of the ozone effect.Perhaps, the goal of removing the ozone problem wouldbe the discovery of an ‘ozonase activity’ that, like super-oxide dismutase, would dissipate the oxidant. Alterna-tively, breeding of crops with natural tolerance will also beaided through understanding genetic variation in the keymajor genes (e.g. cytDHAR). Indeed, naturally tolerantsoybean cultivars have been shown to have elevatedascorbate levels (Chernikova et al., 2000). Given the mul-titiered nature of the plant response, stacking of multipletransgenes or themanipulationof hierarchical gene controlelements are also reasonable genetic approaches.
References
Chen Z andGallie DR (2005) Increasing tolerance to ozone by elevating
foliar ascorbic acid confers greater protection against ozone than
Pitcher LH and Zilinskas BA (1996) Overexpression of copper/zinc
superoxide dismutase in the cytosol of transgenic tobacco confers
partial resistance to ozone-induced foliar necrosis. Plant Physiology
110: 583–588.
Siefermann-Harms D, Payer HD, Schramel P and Lutz C (2005) The
effect of ozone on the yellowing process of magnesium-deficient clonal
Norway spruce grown under defined conditions. Journal of Plant
Physiology 162: 195–206.
Tamaoki M, Nakajima N, Kubo A et al. (2003) Transcriptome analysis
of O3-exposed Arabidopsis reveals that multiple signal pathways act
mutually antagonistically to induce gene expression. Plant Molecular
Biology 53: 443–456.
Torsethaugen G, Pell EJ and Assmann SM (1999) Ozone inhibits guard
cell K+channels implicated in stomatal opening. Proceedings of the
National Academy of Sciences of the USA 96: 13577–13582.
Torsethaugen G, Pitcher LH, Zilinskas BA and Pell EJ (1997) Overpro-
duction of ascorbate peroxidase in the tobacco chloroplast does not
provide protection against ozone. Plant Physiology 114: 529–537.
Tuomainen J, Betz C, Kangasjarvi J et al. (1997) Ozone induction of
ethylene emission in tomato plants: regulation by differential accu-
mulation of transcripts for the biosynthetic enzymes. The Plant Jour-
nal 12: 1151–1162.
Yalpani N, Enyedi AJ, Leon J and Raskin I (1994) Ultraviolet light and
ozone stimulate accumulation of salicylic acid, pathogenesis-related
proteins and virus resistance in tobacco. Planta 193: 372–376.
Yoshida S, TamaokiM, ShikanoT et al. (2006) Cytosolic dehydroascor-
bate reductase is important for ozone tolerance in Arabidopsis
thaliana. Plant Cell Physiology 47: 304–308.
Further Reading
Foyer CH, Lelandais M and Kunert KJ (1994) Photooxidative stress in
plants. Physiologia Plantarum 92: 696–717.
Heagle AS (1989) Ozone and crop yield. Annual Review of Phytopathol-
ogy 27: 397–423.
Kangasjarvi J, Talvinen J, Utriainen M and Karjalainen R (1994) Plant
defence systems induced by ozone. Plant Cell and the Environment 17:
783–794.
Langebartels C, Kerner K, Leonardi S et al. (1991) Biochemical plant
responses to ozone. Plant Physiology 95: 882–889.
Mahalingam R, Shah N, Scrymgeour A and Fedoroff N (2005) Tem-
poral evolution of the Arabidopsis oxidative stress response. Plant
Molecular Biology 57: 709–730.
Overmyer K, Brosche M and Kangasjarvi J (2003) Reactive oxygen
species and hormonal control of cell death. Trends in Plant Science 8:
335–342.
Pell EJ, Schlagnhaufer CD and Arteca RN (1997) Ozone-induced
oxidative stress: mechanisms of action and reaction. Physiologia
Plantarum 100: 264–273.
Sandermann H (1996) Ozone and plant health.Annual Review of Phyto-
pathology 34: 347–366.
SandermannH, Ernst D,HellerW andLangebartels C (1998) Ozone: an
abiotic elicitor of plant defence reactions. Trends in Plant Science
3: 47–50.
Schmieden U and Wild A (1995) The contribution of ozone to forest
decline. Physiologia Plantarum 94: 371–378.
Schraudner M, Langebartels C and Sandermann H (1997) Changes in
the biochemical status of plant cells induced by the environmental
pollutant ozone. Physiologia Plantarum 100: 274–280.
Ozone and Reactive Oxygen Species
6
List of Technical Nomenclature
Carbon assimi-
lation
The process of converting carbon fromits gaseous carbon dioxide form toorganic constituents of plants.
Photoinhibition Reduction of photosynthesis at highlight levels resulting from photo-oxida-tive damage.
Respiration Process in which organic compounds arebroken down to produce energy usuallythrough an oxygen-dependent process toproduce carbon dioxide.
See also: Photosynthesis and Partitioning: C3 Plants;C4 Plants; CAM Plants.
Further Reading
Leegood RC (1999) Photosynthesis in C3 plants: thebenson–calvin cycle and photorespiration. In: Lea PJand Leegood RC (eds) Plant Biochemistry and MolecularBiology, pp. 29–50. Chichester: John Wiley.
Leegood RC, Lea PJ, Adcock MD, and Hausler RE(1995) The regulation and control of photorespiration.Journal of Experimental Botany (Special Edition) 46:1397–1414.
Portis AR (1992) Regulation of ribulose 1,5-bisphosphatecarboxylase/oxygenase activity. Annual Review of PlantPhysiology and Plant Molecular Biology 43: 415–437.
PhotoinhibitionB Demmig-Adams and W W Adams III, University ofColorado, Boulder, CO, USA
Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction and Definitions
Photosynthesis provides the energy and building-blocks for life on this planet as well as being a majorcarbon sink. Therefore changes in photosynthesisrates have the potential to affect global ecosystemsand climate. In response to environmental stress, theefficiency with which solar energy is collected andutilized by plants can be diminished for prolongedperiods of time.
What Is Photoinhibition?
Photoinhibition of photosynthesis is defined as apersistent decrease in the efficiency of solar energy
conversion into photosynthesis in combination witha decreased overall capacity for photosynthesis.Environmental stresses that trigger photoinhibitioninclude, for example, adverse temperatures, limitednutrient or water availability, and salinity. Evergreenconiferous forests in temperate climates, for exam-ple, can show a complete shutdown of photosynth-esis during the winter, and the efficiency of solarenergy conversion in photosystem II can drop tonegligible levels in needles that remain green andcontinue to absorb considerable amounts of solarenergy. Furthermore, high light stress can result fromsudden increases in growth irradiance that may occurnaturally when a canopy gap opens in a forest.
What is causing this loss in energy conversionefficiency and photosynthetic capacity under envir-onmental stresses? And what does it mean for theproductivity of plants? Does photoinhibition ofphotosynthesis limit the productivity of plants inenvironments with intermittent stress periods? Ordoes it reflect a photoprotective process that down-regulates photosynthesis during stressful times whenthe growth of plants is arrested? While manyresearchers in this field tacitly assume thatphotoinhibition of photosynthesis results fromphotodamage, others have suggested that this phe-nomenon is a regulatory and protective adjustmentto environmental stress and change. While manyassume that photoinhibition lowers plant productiv-ity – and consequently expect that more photosynth-esis and carbon uptake would occur in the absence ofphotoinhibition – others argue that photoinhibitiondoes not lower the productivity of plants and that itonly occurs when the opportunity for growth and theactivity of the plant’s sink tissues are low or absent.
It is quite surprising that a phenomenon asimportant as photoinhibition, which affects photo-synthesis of many species in a profound way, is notbetter understood.
Features of Photoinhibition
What features of this phenomenon of photoinhibi-tion are generally accepted? ‘‘Photoinhibition’’ ofphotosynthesis may include some or all of thefollowing characteristics:
1. Lasting decreases in the efficiency with whichabsorbed solar radiation is converted into photo-synthesis are detected as sustained decreases in thelevel of CO2 fixed or O2 evolved per unit ofphotons absorbed at light intensities limiting to therate of photosynthesis. In particular, the level ofphotosystem II photochemistry per unit of photonsabsorbed in the photosystem II light-collecting
PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition 707
antennae is decreased persistently. Photosystem IIphotochemical efficiency is quantified from chlor-ophyll fluorescence signals as the ratio of variableto maximal fluorescence (Fv/Fm), with maximalvalues of 0.85 equaling an 85% efficiency of theconversion of absorbed light into photochemistry.
In sun-exposed leaves, solar energy conversionefficiency undergoes pronounced changes over thecourse of a clear day (Figure 1; summer day). Thisoccurs under favorable environmental conditionswhen growth rates and carbon exchange rates aremaximal. Even maximal rates of utilization ofabsorbed light for photosynthesis in the fastest-growing species do not consume all of the energyabsorbed at peak irradiance, and the excess energyis dissipated as heat in the light-harvestingantennae of photosystem II before it reaches thereaction center. This phenomenon is sometimesdescribed as ‘‘dynamic photoinhibition’’ (as op-posed to ‘‘chronic photoinhibition’’ for sustainedchanges). However, there is no reason to addressthis phenomenon as photoinhibition, as long asthese decreases in solar energy conversion effi-ciency relax quickly upon return to low light levels(cf. Figure 1, summer day).
2. Decreases in the maximal, light-saturated, andCO2-saturated capacity of photosynthetic electronflow and photosynthesis are commonly observedfeatures in response to longer exposure to condi-tions that induce photoinhibition.
3. Inactivation and degradation of the photosystemII reaction center in which high-energy electronsfor electron transport are generated. The photo-system II reaction center core consists of twoproteins, the D1 and D2 proteins. During photo-
inhibition, inactivation of the D1 protein isobserved, followed by degradation of photosys-tem II reaction center cores (Figure 2). Whileshort-term exposures can result mainly in aninactivation of D1 function, naturally growingplants exhibiting photoinhibition commonly showdecreased levels of D1 and photosystem II coresduring periods of stress. Much work has focusedon the turnover of the D1 protein that is extremelyrapid in plants growing under favorable condi-tions. D1 protein turnover is frequently addressedas the damage/repair cycle of the D1 protein.
4. Maximization of photoprotection through con-version of excess absorbed solar radiation intoharmless thermal energy. This thermal dissipationof excess absorbed energy is catalyzed by thexanthophylls zeaxanthin and antheraxanthin ofthe xanthophyll cycle (Figure 3) and a specializedprotein of the family of light-harvesting proteins.In nonphotoinhibited leaves, the rate of thermalenergy dissipation increases and decreases rapidlyin response to the level of excess light (Figure 1;summer day), and these rapid fluctuations aretriggered by changes in the magnitude of the
Solar energy conversion efficiency (Fv/Fm) Solar energy conversion efficiency (Fv/Fm)
dawn noon dusk dawn noon dusk
Time of day
Figure 1 Diurnal patterns of formation of zeax-
anthinþantheraxanthin in the xanthophyll cycle as well as
changes in thermal energy dissipation rate and in solar energy
conversion efficiency of photosystem II (Fv/Fm) under favorable
conditions versus environmental stress.
Photoinhibition
PSIIreactioncenter
(singletoxygen)
D1
H2Oe
e
Chl*
Chl
Z+A1O2*
O2− (superoxide)
Heat
PSII light-harvesting antenna
NADPH
Figure 2 Schematic depiction of changes in the photosystem II
(PSII) reaction center (with the D1 protein) and light-harvesting
antenna in photoinhibited leaves. Photoinhibition causes an
inactivation of D1 (or removal of PSII cores), and sustained
(ZþA)-dependent dissipation of energy as heat. ZþA, zeax-
anthinþ antheraxanthin; Chl*, singlet excited state of chlorophyll.
The cross marks stand for D1 protein inactivation/removal
(leading to a suppression of the formation of high-energy
electrons and of superoxide), a decrease in energy delivery from
light-collecting antennae to PSII reaction centers, and a
suppression of singlet oxygen formation by enhanced thermal
dissipation.
708 PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition
proton gradient across the photosynthetic mem-brane (presumably sensed by the specializedprotein). In photoinhibited leaves, these rapidfluctuations no longer occur and maximal thermalenergy dissipation is ‘‘locked in’’ (Figures 1 and 3).Zeaxanthin and antheraxanthin no longer becomeremoved at the end of the day and can be retainedat high levels for the entire duration of the stressperiod. Likewise, photosystem II remains in ahighly dissipative state (Figures 1 and 3).
Relationship between Inactivation ofPhotochemistry and Dissipation of AbsorbedEnergy as Heat
In theory, decreases in the efficiency of the conversionof absorbed energy into photochemistry in photo-inhibited leaves can result either from a loss ofphotochemical competency or from an increase inthermal energy dissipation, or from a combination ofboth. Any decrease in photochemical competency willlower the energy conversion efficiency – and so willremoval of excess absorbed energy in the chlorophyllpigment bed as heat before this energy reaches thephotochemical reaction centers (Figures 1 and 2).Consequently, an observation of sustained decreasesin photosynthetic energy conversion cannot tacitly betaken to indicate either the presence of photochemicaldisabling, photodamage, or the pres-ence of sustained high levels of thermal energydissipation. Further characteristics of these photo-inhibited plants need to be established to make suchan assessment. Recent studies do, however, indicatethat these two phenomena co-occur under a range ofenvironmental stresses, particularly in evergreenspecies. This may not be unexpected. Under environ-mental stress there is typically an imbalance between(greater) absorption of solar energy and (decreased)utilization of this energy in carbon fixation. Underthese conditions of excess light, reactive oxygenspecies can be formed during light-harvesting and
photochemistry (Figure 2). These reactive oxygenspecies have the potential to destroy cellular compo-nents directly and lead to cell death. They can alsotrigger signal transduction pathways leading to arrestof protein turnover, net degradation of proteins, and,ultimately, programmed cell death. Two main sites ofreactive oxygen formation in chloroplasts are (1) thelight-absorbing chlorophyll pigments that can cata-lyze singlet oxygen formation and (2) photochemicaland electron transport reactions that can lead tosuperoxide formation (Figure 2). Dissipation of excessexcitation energy in the chlorophyll pigment bed viathe xanthophyll cycle counteracts singlet oxygenformation, and a lowering of the rate of photochemi-cal charge separation (via D1 inactivation or removal)should decrease the transfer of electrons to oxygen toform superoxide (Figure 2). A lasting combination ofthe two processes may be a prerequisite for themaintenance of evergreen leaves through seasons withextreme environmental stress by some species, such asoverwintering conifers or desert evergreens.
Photoinhibition: Friend or Foe?
Based on periods during which plants experiencephotoinhibition (frequently quantified as decreases insolar energy conversion from Fv/Fm), extrapolationshave been made in the literature of how much morecarbon uptake would be expected in the absence ofthese decreases in solar energy conversion intophotochemistry. In such extrapolations, it is tacitlyassumed that photochemistry is limiting carbon gainin these situations.
As will be discussed in the following section, thehighest levels of photoinhibition are exhibited byevergreen species with a lifespan of many years. Inclimates with seasonal environmental stress, thesespecies typically exhibit growth during the favorableseason(s) and arrest growth during the stressfulseason(s). This growth arrest is associated withphotosynthetic downregulation.
Zeaxanthin (Z)
Antheraxanthin (A)
Violaxanthin (V)
OH
HO
OHO
HO
OH
OH
Excesslight
Excesslight
Lowlight
Lowlight
O
O
Xanthophyll cycle operation Photosystem II state
Photoinhibition
Dissipative state
High solarenergy conversion
efficiency
Figure 3 Changes in xanthophyll cycle operation and photosystem II state during photoinhibition. Photoinhibition inhibits (shown by
the cross mark) the reconversion of zeaxanthin and antheraxanthin to violaxanthin as well as the return of photosystem II to a state of
high solar energy conversion efficiency in low light.
PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition 709
The demand for products of photosynthesis at thewhole-plant level regulates the level of photosyntheticproteins, resulting in increases in the capacity forphotosynthesis when growth rates are high anddecreases in photosynthetic capacities when growthrates are low (Figure 4). These changes in the capacityfor photosynthesis are achieved via changes in thelevels of photosynthetic proteins and result frommodulation of gene expression at various levels. Inevergreens it is during the times when downregula-tion of photosynthesis occurs that photoinhibition isobserved. It thus appears that photoinhibition isunlikely to limit productivity in these species.
More studies are needed to establish a cause-and-effect relationship of photoinhibition and growtharrest in a large variety of plant species. For anydecreases in solar energy conversion efficiency inresponse to limitations in the demand for photo-synthate it would be highly inappropriate to extra-polate to ‘‘potential plant productivity in the absenceof photoinhibition.’’
Does Photoinhibition Reflect Damage orRegulation?
Even if photoinhibition were not limiting to plantproductivity, the argument has been made that itcould still be the consequence of either photodamageor photoregulation.
When rapid D1 turnover was first discovered, itwas assumed to reflect a regulatory process. How-ever, soon thereafter the focus shifted to consideringthis process as a damage and repair cycle, and to invitro characterization of sites of inactivation, theeffect of blocking D1 synthesis, observing accumula-
tion of inactivated D1 and PSII reaction centers, andaltered structure and levels of the D1 protein.
Current research is uncovering a remarkable levelof regulation of D1 synthesis and degradation. Thisinvolves environmental control of D1 synthesis atseveral levels as well as environmental control of theexpression and activity of D1-degrading proteases.The D1 protein also possesses a motif characteristicof key regulatory proteins that turn over rapidly toallow rapid adjustments in metabolism. For suchproteins, the investment in a high turnover ratewould thereby allow a high level of metaboliccontrol.
Key components in the regulation of D1 turnoverare reactive oxygen species and other redox pro-cesses. For example, faster degradation of the D1protein is seen in the presence of elevated levels ofreactive oxygen species. This has been widelyinterpreted as support for the notion that the D1protein becomes damaged and repaired. However,elevated levels of reactive oxygen species also arrestD1 synthesis at multiple levels.
An explosive development is taking place in thegeneral field of oxidative stress. New insights areleading to a reinterpretation of many processes thatused to be regarded as damage, and are now beingrecognized as components of redox-regulated signaltranduction pathways. Furthermore, the distinctionbetween damage and regulation is beginning to blur,and these labels may be merged into a term such asregulation by oxidative modification. It is time thatthe process of photoinhibition of photosynthesis besubjected to the same sort of re-examination.
Reactive oxygen species oxidize proteins, amongwhich the most sensitive to oxidation are signaling
Largesink
Smallsink
Smallsink
Photosyntheticupregulation
Photosyntheticdownregulation
Largesink
Favorable conditions Environmental stress
Evergreens:
Electron transport capacity:Light-harvesting capacity:Sustained thermal dissipation:
DecreasedSameIncreased
Annuals:
Electron transport capacity:Light-harvesting capacity:Sustained thermal dissipation:
DecreasedDecreasedNo
Figure 4 Schematic depiction of the regulation of photosynthesis by demand in different groups of plants.
710 PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition
proteins whose oxidation results in a stress-relatedsignal. Likewise, the most readily oxidized lipids areprecursors of signaling molecules eliciting stressresponses. In both animals and plants, polyunsatu-rated membrane lipids become oxidized to hormone-like stress messengers that orchestrate stressresponses via the regulation of gene expression. It isattractive to speculate that downregulation ofphotosynthesis and development of photoinhibitionunder environmental stress may be triggered by acombination of hormonal signals from sink tissues(Figure 4) with hormonelike and other signalsoriginating in the chloroplast under excess lightstress. Candidates for such stress-triggered signalsoriginating in the chloroplast include reactive oxygenspecies themselves, other redox signals, and messen-gers formed as a result of lipid peroxidation. A moremeaningful way of phrasing the question of damageversus regulation is whether plants would be betteror worse off without photoinhibition. It may be suchthat evergreens, which experience regular seasonalstresses and downregulate photosynthesis periodi-cally, would not be able to preserve their chlorophyllwithout photoinhibition in the form of a loweredenergy conversion efficiency. But virtually no dataare available to address this issue directly. Therehas been but one study with transgenic algaemaintaining a high rate of photosystem II photo-chemistry under environmental stress, which turnedout to die quickly, while the wild-type exhibitingphotoinhibition of photosystem II survived. Studieswith mutant or transgenic higher plants are neededto ascertain whether or not plants that naturallyundergo D1 inactivation and degradation underphotoinhibitory conditions may suffer when forcedto maintain high levels of photochemically active D1under light stress. The only clear conclusion is that,based on the evidence available to date, the tacitassumption that photoinhibition must entail damageshould be abandoned.
Examples for Photoinhibition: Responseto Environmental Stress as Dependent onPlant Species
Decreases in photosynthetic electron transport capa-city can occur with or without lasting decreases insolar energy conversion efficiency (Figure 4). Thetype of response displayed varies among plantspecies. Evergreens maintain green leaves, and thusa high light-harvesting capacity, and exhibit sus-tained thermal energy dissipation when electrontransport capacities are decreased. Annuals oftenlower light-harvesting capacity together with overall
photosynthetic capacity and do not exhibit sustainedthermal energy dissipation.
These two phenomena represent the oppositeextremes of a continuum. Preservation of a highchlorophyll content, i.e., preservation of a highcapacity to collect light during times of diminishedutilization of solar energy, necessitates a highcapacity for harmless removal of excess absorbedlight (Figures 2 and 4). On the other hand, apronounced degradation of chlorophyll lowers thecapacity for light collection and prevents theabsorption of large amounts of excess light. Thislatter strategy puts relatively less emphasis on thethermal dissipation of excess absorbed light duringtimes when overall photosynthetic capacity isstrongly decreased. These two strategies may beviewed as different ways of achieving the same effect,i.e., lowering the levels of excess excitation energy.
In a strict sense, only the combination of decreasedelectron transport capacities with lasting decreases insolar energy conversion efficiency should be termedphotoinhibition. A concomitant decrease of light-harvesting capacity with overall photosyntheticcapacity may be viewed as part of the regulation ofphotosynthesis by demand via concomitant regula-tion of the expression of chlorophyll a/b-binding andCO2-fixing enzymes.
Why don’t all species simply lower the capacity forlight collection? Short-lived species seem to do thismost consistently, whereas many perennial ever-greens take the approach of maintaining dark greenleaves and a high light-collecting capacity (Figures 2and 4). The reason for this may lie in their differentlifespans and growth habits. Evergreen coniferousforests instantly resume maximal photosynthesisrates after spending the winter in a strongly photo-inhibited state. This rapid response is facilitated bythe fact that the complement of light-harvestingcomplexes does not have to be resynthesized. Incontrast, annual species have evolved acceleratedmetabolic responses to complete their life cyclebefore the onset of a season with severe stress.Leaves of these species typically yellow and senescewhen environmental conditions no longer permithigh growth rates.
Photoinhibition Involving Chlorophyll Preservationand Strong, Lasting Decreases in Solar EnergyConversion Efficiency
Photoinhibition can occur as a consequence of hightemperatures, high levels of ultraviolet irradiation, ordrought. Here we will focus on two additionalconditions, i.e., cold temperatures and increases ingrowth light intensity.
PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition 711
Conifers and other winter-hardy evergreens andwinter stress The case of photoinhibition in over-wintering conifers has been studied rather exten-sively. While maintaining their proverbial greenneedles, many conifers do arrest growth during thewinter season and downregulate photosyntheticcapacity. This involves pronounced decreases in thelevel of the D1 protein of the photosystem II reactioncenter but maintenance of considerable levels oflight-harvesting proteins (Figure 5; cf. Figure 2).Furthermore, this preservation of the light-harvestingsystem is accompanied by retention of high levels ofzeaxanthin and antheraxanthin at all times as well aslasting decreases in the efficiency of energy conver-sion into photochemistry in photosystem II (fromFv/Fm) (Figure 5; cf. Figure 1). Once again, maintena-nce of a high light-harvesting capacity in the absenceof a sink for excitation energy in the form ofphotochemistry calls for a drastic lowering of theefficiency of solar energy conversion in the light-collection system, which presumably occurs viasustained high levels of xanthophyll-dependent ther-mal energy dissipation (Figures 1 and 2).
The response of chilling-sensitive species to coldstress will be addressed in a separate section.
Transfer of evergreen shade-grown plants grown indeep shade to high light Tropical evergreen tough-leafed species, like Schefflera arboricola and Mon-stera deliciosa, have been examined extensively inthis respect. Both species occur naturally over a widerange of irradiances and are tolerant of deep shade aswell as full sunlight. When deep shade-grown plantsof these species are suddenly transferred to high lightlevels, an enormous excess of light is absorbed,owing to their dark green leaves with a high light-
collecting capacity and their low maximal capacityfor photosynthesis. The result of such a transfer isstrong photoinhibition, with leaves remaining darkgreen for many days and exhibiting a strong decreasein the efficiency of energy conversion into photo-chemistry in photosystem II. This photoinhibitioninvolves a locking-in of maximal dissipation ofabsorbed energy, with maintenance of the xantho-phyll cycle in a state of maximal conversion to thephotoprotective pigments zeaxanthin and anther-axanthin (cf. Figure 3). As is the case in over-wintering evergreens, these shade-grown leavesstrongly degrade the D1 protein of photosystem IIwhile maintaining the light-harvesting proteins. Inaddition, we have observed a continuing accumula-tion of carbohydrates in these photoinhibited leavesover many days and conclude that carbon-exportcapacity of deep shade-grown leaves of evergreenM. deliciosa is permanently low. Yet, when theseplants are grown for extended periods of time in highlight, new leaves develop with higher capacities forphotosynthesis and no signs of photoinhibition. Inshade-grown herbaceous species with soft leaves,growth of new leaves with higher photosyntheticcapacities and no photoinhibition occurs much morerapidly, i.e., within a few days upon an increase ingrowth irradiance, whereas this takes months in thecase of the evergreens. Furthermore, in herbaceousspecies even the shade-grown leaves themselves showsome capacity for high light acclimation (photosyn-thetic upregulation), whereas this capacity is verylimited in tough-leafed evergreens.
Availability of soil nitrogen can have a strongimpact on the responses during transfer from low tohigh irradiance. Transfer to high irradiance at lowsoil nitrogen levels can result in photoinhibition,while high soil nitrogen levels can prevent photo-inhibition. In the absence of adequate levels of soilnitrogen, plants are presumably unable to growsufficiently rapidly – or upregulate their photosyn-thetic capacity sufficiently – to utilize the muchincreased level of available light energy. In evergreenperennials this tends to induce sustained decreases insolar energy conversion efficiency, whereas in annualspecies a lowering of light-harvesting capacityappears to be the predominant response, as isdescribed below.
Stress-Induced Degradation of Chlorophyll andDecreases in Photosynthetic Capacity on a LeafArea Basis, but No or Little Decrease in EnergyConversion Efficiency
Herbaceous species under nitrogen limitation Inherbaceous species (like spinach (Spinacea oleracea),tobacco (Nicotiana tabacum), corn (Zea mays;
Relaxed solar energy conversion efficiency
D1 protein/electron transport
capacity
Light-harvestingcomplex
Relaxed zeaxanthin + antheraxanthin
Summer Winter
Seasons
Summer
Figure 5 Seasonal changes in the utilization of solar energy in
photosynthesis in conifers that exhibit strong photoinhibition
during the winter season. Relaxed solar energy conversion
efficiency (Fv/Fm) and relaxed zeaxanthinþantheraxanthin levels
are representative of needles collected predawn and kept at
warm temperatures for several hours.
712 PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition
maize), or wheat (Triticum spp.)) nitrogen limitationleads to decreased photosynthetic capacities withdecreased levels of chlorophyll and CO2-fixingenzymes. These features are seen only in sun-exposedbut not in shaded plants. Does that mean thatphotodamage is involved? The most severe limitationunder limiting soil nitrogen is experienced in thegrowing points of the plant, particularly in poten-tially fast-growing sun-exposed individuals. Soilnitrogen limitation affects primarily the growingpoints of the plant and severely limits sink activity(Figure 4). Carbohydrates build up in the leaves as aconsequence, and the rate of synthesis of chloro-phyll-binding and CO2-fixing proteins decreases.This leads to a downregulation of photosyntheticcapacity in response to the imbalance between a highavailability and a low utilization of carbohydrates(Figure 4), at the same time remobilizing some of thenitrogen from photosynthetic enzymes to the grow-ing points of the plant. In the chloroplast, an increasein the capacity for rapidly reversible xanthophyllcycle-dependent thermal energy dissipation is ob-served, but no sustained increase in thermal dissipa-tion or photoinhibition (as a sustained decrease inenergy conversion efficiency). This is likely related tothe strong decrease in chlorophyll content, and thuslight-collection capacity.
Species of tropical origin growing in temperateclimates When plants are grown outside the rangeof environmental conditions (with respect to tempera-tures, water availability, salinity, etc.) to which theyare well adapted, yellowing of leaves is frequentlyobserved together with decreasing photosyntheticcapacities, sometimes over prolonged periods of time.This applies not only to chilling-sensitive annual cropsin areas with, for example, early-season cold spells,but also to tropical evergreen species in cold or dryclimates. Photoinhibition is typically observed in sun-exposed locations and leaves. However, that also doesnot allow the conclusion that photoinhibition iscaused by photodamage since sun exposure hasdramatic effects on parameters such as leaf and planttemperature, soil and plant water deficit, and watertransport throughout the plant.
One likely scenario in the case of water stress is adisruption of water transport throughout the plant inspecies adapted to warm, humid climates by embo-lisms as a result of soil water deficits or frequentfreeze–thaw cycles in cold climates. Either of theseconditions is likely to disrupt water delivery to theshoot of plants, causing stomatal closure and down-regulation of photosynthetic capacity via suppressionof the synthesis of chlorophyll-binding proteins aswell as other photosynthetic proteins. If this were the
sequence of events, any efforts to increase plantproductivity and survival would have to target thelimiting steps of inherent drought or temperaturetolerance. An interesting study for the case oftemperature intolerance is available from cyano-bacteria. Engineering cyanobacteria to contain amembrane lipid desaturase (that increases membranefluidity under moderately low temperatures) drama-tically increased survival of the bacteria at thesetemperatures. In addition, this change dramaticallydecreased D1 degradation and photoinhibition inthese bacteria at the low temperatures. High levels ofD1 degradation under low-temperature stress in thechilling-intolerant wild-type of these cyanobacteriahave been interpreted as a sign of damage, but mightalso be viewed as a protective response to avoidmassive reactive oxygen formation. Removing theapparent source of the chilling intolerance byameliorating membrane fluidity either prevented thedamage to D1 or made the inactivation of photo-chemistry unnecessary.
Maintaining Electron Transport Capacity duringEnvironmental Stresses
The herbaceous weed Malva neglecta is a sun-lovingspecies with high growth rates and high photo-synthetic capacities that are maintained even duringthe harsh winter conditions of a montane climate.Never throughout its lifecycle doesM. neglecta showeither photoinhibition or extremely high capacitiesfor photoprotection (in the form of very highcapacities for thermal energy dissipation). WhereasM. neglecta does not show appreciable levels ofphotoinhibition, this species does maintain photo-system II in a dissipative state with high levels ofretained zeaxanthin and antheraxanthin as long asleaf temperatures remain substantially below freez-ing in the field. This is probably caused bymaintenance of a proton gradient across the photo-synthetic membrane that keeps thermal energydissipation engaged at all times. Since this cold-sustained energy dissipation disappears promptlyupon warming of the leaves, it should not beconsidered photoinhibition. This response offersprotection during cold periods in the winter. Duringintermittent milder periods, photosystem II quicklyreturns to a photochemically highly competent stateand photosynthesis resumes. Whereas biennial andannual herbaceous species, such as M. neglecta,spinach, winter rye (Secale cereale), mullein (Ver-bascum thapsus), and Arabidopsis thaliana, persistthroughout a single winter season and maintaingrowth and high photosynthetic capacities through-out the challenging season, they are unable to
PHOTOSYNTHESIS AND PARTITIONING /Photoinhibition 713
survive the more extreme conditions that someperennial evergreens can tolerate.
Engineering Plants for Enhanced StressTolerance: Where Does PhotoinhibitionFit In?
Correlations exist between high rates of D1 turnoverand a high resistance to photoinhibition amongvarious plant species and varieties. Based on thisobservation, two contrasting hypotheses have beenformulated. One is that the rate of D1 synthesis orD1 turnover is limiting stress resistance in somespecies or varieties. In this case, plants engineered topossess higher rates of D1 synthesis and turnoverwould be expected to exhibit greater stress tolerance.In the alternative hypothesis, the rate of D1 synthesisand turnover is controlled by signals reflecting theopportunity for utilizing solar energy for plantgrowth. In this case, manipulations of growth ratewould automatically modulate D1 synthesis andturnover, but increase of D1 synthesis rate per se,without addressing growth rates, may actually leadto greater rates of membrane lipid peroxidation.Studies with transgenic plants are needed to deter-mine which hypothesis is correct.
Another area of interest is antioxidant systems.Conditions triggering photoinhibition commonlyinvolve an imbalance between light absorption andlight product utilization in the chloroplast. Such animbalance has the potential to increase the levels ofvarious messengers that trigger a net degradation ofphotosynthetic proteins. Future research may clarifywhether these events involve any damage or solelyregulation. However, the consequences may berather similar, in terms of efforts to manipulateplant stress tolerance. Antioxidation and otherprocesses that counterbalance the oxidative pro-cesses may be expected either to prevent damage orto counteract the generation of signals that triggerdegradation. Future research will have to showwhether overexpression of the capacity for thermalenergy dissipation or for antioxidation in thechloroplast will result in less photoinhibition and/or chlorophyll degradation in any plant species. Ithas to be noted that all species examined to dateappear to possess a remarkable plasticity in theirability to increase the capacity of photoprotectiveprocesses. At the same time, the response of differentspecies to conditions that limit growth and sinkactivity varies widely as outlined above. While manycrops respond with massive chlorophyll degradationand premature senescence, evergreens maintaingreen, albeit photosynthetically inactive leaves.Might it be possible to engineer crops able to
maintain dark green and fully protected leavesthroughout intermittent periods with harsh environ-mental conditions that prevent continued sinkactivity? Future research needs to address species-dependent differences in the signaling processes thatresult in contrasting responses.
See also: Abiotic Stresses: Cold Stress; Free Radicals,Oxidative Stress and Antioxidants. Genetic Modification,Applications: Oxidative Stress. Genetic Modification ofPrimary Metabolism: Photosynthesis. Genetic Modifica-tion of Secondary Metabolism: Terpenoids. Photo-synthesis and Partitioning: C3 Plants; Sources andSinks. Primary Products: Oils. Regulators of Growth:Jasmonates. Water Relations of Plants: Drought Stress.
Further Reading
AdamsWW III, Rosenstiel TN, Demmig-Adams B, Ebbert V,and Brightwell AK (2002) Photosynthesis and photopro-tection in overwintering plants. Plant Biology 4: 545–557.
Critchley C (1999) Molecular adaptation to irradiance: thedual functionality of photosystem II. In: Singhal GS,Renger G, Sopory SK, Irrgang K-D, and Govindjee (eds)Concepts in Photobiology: Photosynthesis and Photo-morphogenesis, pp. 571–587. New Dehli: NarosaPublishing House.
Dalton TD, Shertzer HG, and Puga A (1999) Regulation ofgene expression by reactive oxygen. Annual Review ofPharmacology and Toxicology 39: 67–101.
Demmig-Adams B and Adams WW III (1996) The role ofxanthophyll cycle carotenoids in the protection ofphotosynthesis. Trends in Plant Science 1: 21–26.
Koch KE (1996) Carbohydrate-modulated gene expressionin plants. Annual Reviews of Plant Physiology and PlantMolecular Biology 47: 509–540.
Melis A (1999) Photosystem-II damage and repair cycle inchloroplasts: what modulates the rate of photodamage invivo? Trends in Plant Science 4: 130–135.
Osmond B, Badger M, Maxwell K, Bjorkman O, andLeegood R (1997) Too many photons: photorespiration,photoinhibition and photooxidation. Trends in PlantScience 2: 119–121.
Primary Products ofPhotosynthesis, Sucrose andother Soluble CarbohydratesJ D Everard, DuPont Experimental Station,Wilmington, DE, USA
Introduction
Plants are autotrophic organisms and thus havethe capacity to create all the components required
714 PHOTOSYNTHESIS AND PARTITIONING /Primary Products of Photosynthesis
ANRV375-PP60-19 ARI 8 April 2009 16:30
Signal Transduction inResponses to UV-B RadiationGareth I. JenkinsPlant Science Group, Division of Molecular and Cellular Biology, Faculty of Biomedicaland Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom;email: [email protected]
Annu. Rev. Plant Biol. 2009. 60:407–31
The Annual Review of Plant Biology is online atplant.annualreviews.org
This article’s doi:10.1146/annurev.arplant.59.032607.092953
AbstractUV-B radiation is a key environmental signal that initiates diverseresponses in plants that affect metabolism, development, and viability.Many effects of UV-B involve the differential regulation of geneexpression. The response to UV-B depends on the nature of the UV-Btreatment, the extent of adaptation and acclimation to UV-B, andinteraction with other environmental factors. Responses to UV-B aremediated by both nonspecific signaling pathways, involving DNA dam-age, reactive oxygen species, and wound/defense signaling molecules,and UV-B-specific pathways that mediate photomorphogenic responsesto low levels of UV-B. Importantly, photomorphogenic signaling stim-ulates the expression of genes involved in UV-protection and hencepromotes plant survival in UV-B. Photomorphogenic UV-B signalingis mediated by the UV-B-specific component UV RESISTANCELOCUS8 (UVR8). Both UVR8 and CONSTITUTIVEPHOTOMORPHOGENESIS1 (COP1) are required for UV-B-induced expression of the ELONGATED HYPOCOTYL5 (HY5)transcription factor, which plays a central role in the regulation ofgenes involved in photomorphogenic UV-B responses.
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Reactive oxygenspecies (ROS):include singlet oxygen,superoxide anion,hydrogen peroxide,and hydroxyl radical
Ultraviolet-B (UV-B) radiation has an extensiveimpact on the biosphere. In addition to its directeffects on organisms, UV-B impinges on nu-merous processes that affect ecosystem function(5, 25, 26, 113). Most of the UV solar radiation
that reaches the earth is absorbed by the strato-spheric ozone layer (108), and therefore UV-Bwavelengths (280 to 315 nm) are only a mi-nor component of solar radiation at the Earth’ssurface. The ambient level of UV-B is actuallyvery variable and is affected by several factors(108, 113). In particular, the latitude, season,and time of day affect the solar angle and hencethe thickness of the atmosphere that UV-B mustpenetrate. In addition, the release of chloroflu-orocarbons has caused depletion of the ozonelayer, resulting in local elevations in UV-B (5,108). The level of UV-B is also dependent onaltitude, the degree of cloud cover, dispersal orabsorbance by atmospheric aerosols and pollu-tants, surface reflectance, and the thickness ofthe vegetation canopy.
UV-B has the highest energy of any partof the daylight spectrum and has the potentialto damage macromolecules, including DNA, togenerate reactive oxygen species (ROS), and toimpair cellular processes (13, 21, 54, 79, 85,120). Organisms have therefore evolved mecha-nisms to protect against UV-B and to repair UVdamage (120). One of the most important pro-tective mechanisms in higher plants is the de-position of UV-absorbing phenolic compoundsin epidermal tissues (18, 85, 120). These com-pounds act as a sunscreen, reducing penetrationof UV-B into the leaf (18, 107). Genotypes lack-ing such protection suffer increased injury byUV-B (98, 101, 128). Other protective mech-anisms are also important, including the pro-duction of cuticular waxes and hairs (102, 130)and the enhancement of cellular antioxidantsystems (21, 79). The repair of DNA damagein plants involves similar mechanisms to thosein other organisms, including light-stimulatedrepair via DNA photolyases (19). The systemsof UV-protection and repair are evidently veryeffective because plants in the natural environ-ment rarely show any signs of UV damage.
UV-B is not simply an agent of damage, buta key environmental signal that regulates di-verse processes in a range of organisms (13,21, 54, 79, 80, 113, 120, 141). In plants, UV-Bstimulates the expression of genes involvedin UV protection and damage repair (21, 54,
408 Jenkins
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80–82, 141) and therefore actively promotessurvival in sunlight. Furthermore, responses toUV-B modify plant biochemical composition,influence plant morphology, and help to deterpests and pathogens (13, 24, 54, 78, 79, 131,138). Given the broad significance of regula-tory responses to UV-B, it is important to un-derstand the underlying mechanisms of UV-Bperception and signal transduction. At present,much less is known about these processesthan about perception and signaling involvingthe phytochrome, cryptochrome, and pho-totropin photoreceptors (35). However, it willnot be possible to understand fully how lightcontrols plant development without knowledgeof the regulatory effects of UV-B. This reviewfocuses on the cellular and molecular mecha-nisms of plant responses to UV-B and high-lights the most recent advances. Additionalinformation on this topic can be found in severalprevious reviews (1, 21, 54, 80, 82, 131, 141).
PLANT RESPONSES TO UV-B
Numerous studies have examined the effectsof UV-B on plants. These studies have in-volved diverse species at various stages ofdevelopment, a wide range of growth and treat-ment conditions in the field, glasshouse or con-trolled environment chambers, and a variety ofUV-B sources that provide different spectralqualities, amounts, and durations of UV-Bexposure. From this extensive literature it ispossible to highlight some general principles.
First, UV-B has diverse effects on plants.UV-B influences various developmental pro-cesses and modifies plant architecture. In gen-eral, UV-B reduces extension growth and leafexpansion and promotes branching, but nu-merous other effects have been reported (24,54, 78, 120, 138). In addition, UV-B modifiesmetabolism (104) and promotes the synthesisof a range of secondary metabolites, includ-ing the UV-protective flavonoids (66, 120). Thesignificance of some of the metabolic changesis not understood, although some secondarymetabolites probably help to deter predation byherbivorous insects (24, 76).
Fluence rate: theamount of light,expressed as thenumber of moles ofphotons impinging ona defined area in agiven time, e.g., μmolm−2 s−1
Second, the nature of the response toUV-B is dependent on the fluence rate, du-ration, and wavelength of the UV-B treat-ment (21, 54, 80, 141). In general, exposureto high fluence rates and short wave-lengths of UV-B is likely to cause stressresponses and possibly necrosis. Several stud-ies have reported damage to DNA, pro-teins, and membrane lipids and the inhibi-tion of protein synthesis and photosyntheticreactions (13, 30, 79, 85, 120). The most dam-aging effects are generally observed in plantsexposed to above-ambient levels of UV-B, of-ten in artificial lighting environments where thetotal fluence rate and light quality differ sub-stantially from the natural environment. In con-trast, low fluence rates of UV-B are sufficient toinitiate regulatory responses. For example, lowlevels of UV-B inhibit stem extension, stimulatecotyledon opening, promote the accumulationof flavonoids, and regulate the expression of arange of genes (16, 23, 53, 94, 133, 140). Theseregulatory responses are evidently not stressresponses, as discussed below, and can beconsidered as photomorphogenic in nature,comparable to responses mediated by phy-tochromes, cryptochromes, and phototropins.
Third, differences in the level of adaptationor acclimation to UV-B determine the responseto UV-B exposure. Genotypes within a speciesdiffer in their tolerance and responsiveness toUV-B (41, 72, 90, 139). In addition, for a givengenotype the extent of prior acclimation toUV-B influences the magnitude and nature ofthe response. Plants grown in light lackingUV-B are more likely to suffer stress on first ex-posure whereas plants grown in UV-B are morelikely to tolerate an increase in dose. Plants thatare exposed to elevated UV-B express genes thathelp the plant to counter any stress effect, repairdamage, and establish increased protection (22,29, 93, 140). Following acclimation, the ampli-tude of response to UV-B is diminished becausea certain level of protection is already in place(88). Once acclimated, plants express genes thatmaintain the acclimated state (68).
Fourth, interactions with other environ-mental variables condition the UV-B response
www.annualreviews.org • UV-B Signaling 409
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Transcriptomicanalysis: assay oftranscript abundancesfor the whole genome
and UV-B influences responses to other factors(24, 26). For example, interactions have beenreported between UV-B and water stress (64,116), nutrient status (42), and low temperature(33). In addition, UV-B interacts with otherlight-response pathways in a complex signalingnetwork (16, 143). Little is known about themechanisms underlying interactions betweenUV-B and other stimuli and the use of systems-orientated approaches would help to increaseknowledge in this area.
GENE EXPRESSION UNDERPINSUV-B RESPONSES
Many effects of UV-B on plants involve differ-ential gene expression (22, 23, 29, 31, 32, 93,140, 146). Different types of UV-B exposureregulate different sets of genes (21, 23, 31, 54,141). High fluence rates and short wavelengthsof UV-B induce many genes normally expressedin defense, wound, or general stress responses(1, 21, 93, 141), whereas low fluence rates, briefexposures, and longer wavelengths of UV-B in-duce a variety of genes, a number of which areknown to be involved in UV protection or theamelioration of UV damage (23, 53, 82, 140).Differences in the fluence rate dependence of
0 0.1 0.2
CHS
WRKY30
HY5
0 0.5 1 3 7 12
ACTIN 2
UV-B μmol m–2 s–1
Figure 1Genes differ with respect to the UV-B fluence rate required for induction.Transcripts of the CHALCONE SYNTHASE (CHS), ELONGATEDHYPOCOTYL5 (HY5), WRKY30, and control ACTIN2 genes in Arabidopsis leaftissue were assayed by RT-PCR. Plants were grown in white light lackingUV-B before exposure to the fluence rates shown for 4 hours. For comparison,full sunlight contains approximately 3 μmol m−2 s−1 UV-B (280–315 nm;midsummer, United Kindgom). Data taken from Reference 23. The WRKY30transcription factor is induced by reactive oxygen species (ROS) and woundsignaling pathways (125, 137).
UV-B-induced gene expression are illustratedin Figure 1.
Transcriptomic analyses with maize (15, 28,29, 31) and Arabidopsis (20, 22, 23, 68, 93,141) show that UV-B regulates a large num-ber of genes concerned with a wide range ofcellular processes. Some of these genes are ex-pressed in specific organs and at particular de-velopmental stages (31). Some, such as thoseconcerned with flavonoid biosynthesis, werepredicted from earlier studies, but many werepreviously unconnected to UV-B. Investigationof the functions of these gene products mayprovide important new insights into processesregulated by UV-B. In addition, the transcrip-tomic analyses have provided much detailed in-formation on patterns of gene regulation. Theacute response to elevated UV-B is rapid andmany genes display transient expression kinet-ics (93, 140). Most genes increase in expressionalthough a significant number are repressed (29,31, 93, 140). Plants that have acclimated toUV-B display quite different expression pro-files to those suddenly exposed to elevatedUV-B (68).
Some genes in maize are induced only abovecertain UV-B dose thresholds, suggesting theoperation of different signaling pathways atdifferent fluence rates (31). Furthermore, inArabidopsis some genes show differential ex-pression in shorter and longer wavelengths ofUV-B, again implying different signaling mech-anisms (140). The short-wavelength pathwaynegatively regulates expression of some genesinduced by longer-wavelength UV-B. Differ-ent effects of short- and long-wavelength UV-Bhave also been observed for growth responsesin cucumber (126), where there is evidence thatthe short-wavelength response involves DNAdamage (127). Interestingly, in both maize (31)and Arabidopsis (93), altered gene expressionis observed in tissues not directly exposed toUV-B, including roots, indicating that a sig-nal is transmitted from UV-illuminated tononilluminated tissues. Similar effects are ob-served in response to other abiotic stresses (93)and are well known in pathogen defense re-sponses. It will be interesting to learn whether
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common systemic signaling mechanisms areinvolved.
Transcriptional Control
Most genes regulated by UV-B are likely con-trolled at the level of transcription and thereis direct evidence for this in several cases (34,67, 121, 140). It is therefore important to iden-tify DNA sequence elements and transcrip-tion factors involved in these responses andto understand how transcriptional regulation iscoupled to UV-B signaling pathways. Detailedstudies of DNA sequence elements that regu-late transcription of the gene encoding the keyflavonoid biosynthesis enzyme chalcone syn-thase (CHS) did not identify any UV-B-specificelement (67, 86). However, a UV-B-specific el-ement was identified recently in the Arabidop-sis ANAC13 gene, which encodes a putativeNAC-domain transcription factor (121). Thiselement is necessary for induction by shorter-wavelength, higher–fluence rate UV-B.
A significant proportion of the genes mostrapidly induced by UV-B encode transcrip-tion factors (93, 140) and these proteins un-doubtedly play key roles in UV-B responses.Some UV-B-induced transcription factors areinvolved in controlling the biosynthesis of UV-protective phenolic compounds (37, 38, 114).The AtMYB4 transcription factor represses thebiosynthesis of UV-protective sinapate estersand its expression is downregulated by UV-B(83). The nuclear accumulation of AtMYB4 re-quires the importin β–like protein SAD2; a sad2mutant is impaired in nuclear accumulation ofAtMYB4 and has increased tolerance of UV-B(151).
The Arabidopsis basic leucine-zippertranscription factor ELONGATEDHYPOCOTYL5 (HY5) is induced by UV-B(22, 140) (Figure 1). HY5 is required for theUV-B induction of a substantial number ofgenes (22, 112, 140), including those with vitalroles in UV protection. Hence the hy5 mutanthas much reduced viability when exposed toelevated levels of UV-B (22, 112). The closelyrelated protein, HY5 HOMOLOG (HYH),
CHS: CHALCONESYNTHASE
HY5: ELONGATEDHYPOCOTYL5
HYH: HY5HOMOLOG
acts redundantly with HY5 in the regulationof a number of UV-B-induced genes, but hyhis less sensitive to UV-B than hy5, indicatingthat HYH is of secondary importance (23).Given the pivotal role of HY5, it is criticalto understand how UV-B regulates HY5transcription.
Dynamic Chromatin
Recent studies indicate that the ability of UV-Bto trigger modifications in chromatin structureis crucial in regulating transcription. Evidencefrom maize shows that proteins likely to be in-volved in regulating chromatin organization areimportant in adaptation to high ambient levelsof UV-B (28). Maize genotypes adapted to liv-ing at high altitudes, where they experience in-creased doses of UV-B, have elevated levels oftranscripts that encode putative chromatin re-modeling proteins compared with control lines.Transgenic RNAi lines with knocked-down lev-els of these transcripts showed increased foliardamage following UV-B exposure and alteredexpression of several UV-B-induced genes (28).These findings indicate that the ability to mod-ify chromatin organization is important bothin acute gene expression responses involved inacclimation to UV-B and in evolutionary adap-tation to elevated UV-B.
In eukaryotes, histones are important inrecruiting regulatory proteins to particularregions of chromatin (96, 100). Specificposttranslational modifications of particularhistones facilitate the remodeling of chromatinstructure, regulating interactions with proteinsinvolved in transcription, DNA repair, andreplication. Recent research indicates thatUV-B promotes histone modifications thatcorrelate with altered transcription. FollowingUV-B treatment of Arabidopsis, the promoterregions of several UV-B-regulated genes wereenriched in chromatin containing diacetyl-histone H3 (K9/K14), indicating a correlationbetween this histone modification and in-creased transcriptional activity in response toUV-B (37). Furthermore, proteomic analysisof maize nuclear proteins revealed that UV-B
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Chromatinimmunoprecipitation(ChIP): chromatinfragments areimmunoprecipitatedwith an antibodyrecognizing achromatin-boundprotein and DNA isanalyzed by PCR
causes numerous alterations in histone compo-sition and posttranslational modification (27).UV-B-tolerant lines showed more changesthan did sensitive lines, suggesting that histonesare important in acclimation and adaptationto UV-B. Chromatin immunoprecipitation(ChIP) experiments indicated that acetylationof histones H3 and H4 in chromatin containingUV-B-regulated genes was correlated withincreased expression in response to UV-B. Ingeneral, the strongest correlations were foundfor UV-tolerant lines. Histone modificationappeared to promote a more open chromatinconformation at these gene loci.
It is important to define further the rolesof specific histones and histone modificationsin UV-B responses and to establish how suchmodifications influence the recruitment of tran-scription factors. In addition, it is necessary todetermine the functions of proteins that regu-late chromatin organization and to understandhow chromatin remodeling is regulated byUV-B signaling.
UV-B PERCEPTION AND SIGNALTRANSDUCTION
As explained above, plants show diverseresponses to UV-B, ranging from acute stressresponses to regulatory photomorphogenic re-sponses. To understand fully the effects ofUV-B on plants it is necessary to dissect thecellular and molecular mechanisms that under-pin the different responses. However, this is acomplex task because, firstly, different types ofUV-B responses involve different mechanismsof UV-B perception and signal transductionand, secondly, at present we do not know howmany different UV-B signaling pathways plantspossess. Hence it is necessary to categorize thedifferent types of responses and to define theUV-B perception and signaling processes in-volved in each of them.
Several authors have classified UV-B re-sponses according to the fluence rates requiredto initiate them (21, 54). This is a helpful ap-proach because it highlights fundamental dif-ferences in the nature of the responses and their
underlying signaling pathways. However, un-doubtedly there is considerable overlap in thefluence rates that initiate different types of re-sponses (Figure 1). Moreover, the thresholdfluence rate required to initiate a particular re-sponse is likely to vary according to the devel-opmental stage of the plant, the degree of ac-climation to UV-B, and interaction with otherenvironmental factors.
An alternative way of categorizing UV-Bresponses is according to their function, forinstance, whether the response is an acutestress response that may help the plant surviveexposure to elevated UV-B, or whether it is aphotomorphogenic response that establishesUV-B protection or modifies development.In general, stress responses to UV-B appearto be mediated by signaling pathways that arenot specific to UV-B, and many of the genesinduced can be activated by other stresses (1,21, 77, 93, 131). In contrast, UV-B-specificsignaling is of major importance in mediatingphotomorphogenic acclimation responses (22).However, it would be incorrect to give theimpression that all plant responses to UV-Bfall into these opposing categories and tolabel nonspecific UV-B signaling pathways asconcerned only with stress. For instance, theactivation of defense signaling pathways byUV-B could influence morphogenesis througheffects on ethylene production (3, 115). More-over, ROS production may affect morphology(62). Plants growing in ambient UV-B likelyuse a combination of nonspecific and UV-B-specific pathways to optimize their responses tothe frequently varying level of UV-B radiation.Evidently, it is important to determine thenumber of different UV-B signaling pathwaysand to define their roles and functional re-lationships in plants growing under naturalconditions.
NONSPECIFIC UV-B SIGNALING
DNA Damage Signaling
DNA damage can be caused by several fac-tors, including UV radiation, ROS, genotoxic
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chemicals, and aberrations in cellular processessuch as DNA replication (14). Several typesof damage can occur, from strand breaks andcross-links to various base modifications. Inplants, UV-B exposure principally causes theformation of cyclobutane pyrimidine dimers(CPD) and, to a lesser extent, pyrimidine [6–4] pyrimidinone dimers (6–4 photoproducts)(19). These forms of damaged DNA can be re-paired by DNA photolyases in the presence ofUV-A/blue light (photoreactivation) and bylight-independent processes (19). In yeast andmammalian cells, UV radiation (both UV-Cand UV-B) initiates DNA damage signal-ing pathways that arrest cell cycle progres-sion and promote DNA repair (122). Severalcomponents of these pathways are presentin plants, suggesting at least partial conser-vation of DNA damage signaling processes.For example, Arabidopsis has orthologs ofthe Ataxia telangiectasia-mutated (ATM) andAtaxia telangiectasia-mutated and Rad3-related(ATR) protein kinases that sense DNA double-strand breaks and single-stranded DNA, re-spectively, and initiate signaling (43, 44, 60).Mutants deficient in ATR show hypersensitiv-ity to UV-B in a root growth assay (43), consis-tent with the hypothesis that ATR is importantin regulating cell cycle progression when DNAreplication is impaired, in this case by UV-B.
Evidence shows that a number of UV-Bresponses in plants are initiated by DNA
ATM: Ataxiatelangiectasia-mutated
ATR: Ataxiatelangiectasia-mutatedand Rad3-related
damage signaling. Action spectra for some re-sponses peak at approximately 260 nm, consis-tent with absorption by DNA (11, 47, 99, 129)(Table 1), and correlations have been ob-served between CPD formation and UV-Bresponses (63, 97). Significantly, several re-sponses to short-wavelength UV-B, includ-ing isoflavonoid production (11) and β-1,3-glucanase gene expression (97) in French beanand the suppression of hypocotyl elongationin cucumber (127), are reversed by exposureto light that promotes photoreactivation, indi-cating that the responses are initiated by theformation of damaged DNA. Unfortunately,no information is available about the signalingcomponents involved in these responses so it isunknown whether they involve pathways thatrequire or are independent of ATM and ATR.
Reactive Oxygen SpeciesAccumulation and Signaling
ROS accumulate in response to various abioticand biotic stresses (6, 109). In addition to theirpotential to cause oxidative damage to cellularcomponents, ROS have important roles as sig-naling molecules and can stimulate expressionof a range of genes. Differences in the type ofROS produced and in the spatial and tempo-ral parameters of ROS accumulation are impor-tant in determining which response pathway isactivated (6, 109).
Table 1 A selection of published UV-B action spectra that illustrates the range of peak wavelengthsobtained
Several different types of measurementhave demonstrated that UV-B generates ROSin plants (4, 9, 45, 70, 71). Most commonlydetected was the superoxide radical, which israpidly converted to hydrogen peroxide in cells.The origin of ROS produced by UV-B is notclear. The most likely sources are photosyn-thetic reactions, respiration, and the activity ofenzymes such as peroxidases and oxidases (109).Under conditions where UV-B impairs photo-synthetic electron transport (13, 79, 85), excessROS would likely be generated by a reducedability to dissipate excitation energy. Consistentwith this hypothesis, superoxide production iscorrelated with the inhibition of photosynthe-sis by UV-B (9). However, UV-B is reportedto stimulate NADPH oxidase activity (2, 118),and mutants in the Arabidopsis AtRBOHD andAtRBOHF NADPH oxidase genes showreduced hydrogen peroxide accumulation inresponse to UV-B (91). Thus, it appears thatUV-B can produce ROS by more than onemechanism.
Plants attempt to counter the accumula-tion of ROS by enhancing antioxidant systems.Transcriptome analyses show that UV-B in-creases the expression of various genes con-cerned with reducing oxidative stress (22, 23,29, 31, 93, 140). Consistent with this, the activi-ties of ROS-scavenging enzymes, such as ascor-bate peroxidase and superoxide dismutase, arereported to increase following UV-B treatment,although most increases are observed only atquite high UV-B doses (45, 98, 118, 148). Ascor-bate is an important cellular antioxidant andthe Arabidopsis ascorbate-deficient vtc1 mutantis highly sensitive to UV-B (40, 59).
Evidence exists for the involvement of ROSin some morphological changes and gene ex-pression responses initiated by UV-B. Cotyle-don curling in Brassica napus is stimulated byboth UV-B and hydrogen peroxide and inhib-ited by ascorbate (62). Exposure to relativelyhigh fluence rates of UV-B decreases the abun-dance of transcripts of the Arabidopsis LHCB1gene, which encodes the major chlorophyll-binding protein of chloroplasts, and this re-sponse is inhibited by ascorbate (134) and
by a scavenger of superoxide radicals (2). Ifsuperoxide is involved it probably does notoriginate from NADPH oxidase because theatrbohd and atrbohf mutants are unaltered in theLHCB1 response (91). However, these mutantsare reported to have altered expression of sev-eral other genes in response to UV-B.
Some studies of ROS production andROS-induced gene expression have used veryhigh levels of UV-B and consequently some ofthe findings may have little relevance to plantsgrowing in natural conditions. Although ROSproduction and signaling are likely used byplants to modulate expression of some genesin response to varying ambient levels of UV-B,little information is available on the extentand nature of ROS production under naturalgrowth conditions. Interestingly, ambient UV-B supplementation in greenhouse conditionsincreased CPD formation and reduced leaf areain Gunnera magellanica, but did not cause lipidperoxidation; modulation of ascorbate contentappeared to counter oxidative stress (63).
Activation of Wound/DefenseSignaling
UV-B exposure stimulates expression of aset of genes normally induced in responseto pathogen attack or wounding, includingpathogenesis-related (PR) genes and proteinaseinhibitor genes (1, 21, 131). Moreover, mi-croarray experiments with field-grown Nico-tiana longiflora show that UV-B regulates anumber of genes that are normally induced byinsect predation (77). Prior exposure to UV-B isreported to reduce the level of insect herbivoryin a range of species, probably because of theincreased production of secondary metabolites,proteinase inhibitors, and other molecules thatdeter herbivorous insects (24, 76, 131).
An explanation for the overlap in responsesto UV-B, wounding, and pathogenesis is thatUV-B stimulates accumulation of signalingmolecules that mediate wound/defense re-sponses, including jasmonic acid ( JA), ethy-lene, salicylic acid (SA), and ROS (see above).Exposure of Arabidopsis to relatively high levels
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of UV-B promotes rapid increases in the levelsof JA (3) and ethylene (3, 115), but a slower in-crease in SA levels (134). Evidence shows thatthese molecules mediate some UV-B-regulatedgene expression responses. Altered UV-B in-duction of defense-related genes was observedin the ethylene insensitive etr-1 mutant, theJA-insensitive jar1 mutant (3), and a tomatomutant defective in JA production (39). Trans-genic Arabidopsis plants expressing the salicylatehydroxylase, NahG, were unable to accumu-late SA and showed reduced UV-B induction ofseveral PR genes (134).
The involvement of ROS in defense signal-ing is well established and superoxide, gener-ated by plasma membrane NADPH oxidase,plays a key role (6). Evidence shows that ROSare involved in the UV-B induction of severaldefense genes (1, 2, 65, 134) and pharmaco-logical experiments suggest that NADPH oxi-dase and peroxidase enzymes may be responsi-ble for ROS production (2). Superoxide appearsto be involved in the UV-B regulation of somedefense genes, either directly or through theproduction of H2O2 (1, 2).
Although it is clear that UV-B stimulates de-fense and wound signaling, there is little in-formation on how it activates components ofthe signaling pathways. Experiments in tomatoindicate that UV-B initiates similar signalingprocesses to systemin, the peptide that stimu-lates the wound response (131). It has been re-ported that systemin interacts with a receptor-like kinase (131). Initial data suggested that thetomato systemin receptor might also functionas the brassinosteroid receptor (144), althoughrecent evidence indicates otherwise (74a). Theinteraction of systemin with its receptor in-duces rapid apoplastic alkalinization and acti-vates two specific MAP kinases. UV-B treat-ment initiates the same processes and activatesan additional MAP kinase (74, 147). The in-hibitor suramin blocks the interaction betweensystemin and its receptor and prevents down-stream alkalinisation and MAP kinase activation(132). Suramin also inhibits the UV-B response(147), indicating that UV-B activates either thesystemin receptor or a related suramin-sensitive
receptor through a ligand-independent pro-cess. Ligand binding to receptor-like kinasesoften promotes autophosphorylation and het-erodimerization with a related receptor (61, 84).It would be interesting to know whether UV-Binitiates the same processes and, if so, how thisoccurs independently of ligand binding.
PHOTOMORPHOGENIC UV-BPERCEPTION AND SIGNALING
As discussed above, some UV-B responses canbe defined as photomorphogenic in character.The most extensively studied examples are thesuppression of hypocotyl extension by low flu-ence rates of UV-B (7, 8, 16, 94, 126, 133)and the UV-B induction of genes involvedin flavonoid biosynthesis, such as CHS (53,81, 82). Together the results from a numberof studies show that these UV-B responsesare not mediated by DNA damage signaling,stress/wound/defense signaling, or the knownphotoreceptors, but instead involve distinctphotomorphogenic signaling processes.
The threshold UV-B doses that initiate pho-tomorphogenic responses are much lower thanthose that cause detectable DNA damage orinduce stress/defense/wound gene expression.Less than 0.1 μmol m−2 s−1 UV-B (approxi-mately 1/40 of the fluence rate of UV-B in sun-light) is sufficient both to suppress hypocotylextension (16, 94) and to induce CHS expressionin Arabidopsis (23) (Figure 1). Several genesassociated with stress pathways require at leastan order of magnitude higher fluence rate forUV-B induction (21, 23) (Figure 1). Moreover,less than five minutes’ exposure to UV-Bincreases CHS transcript abundance in Ara-bidopsis (82) and subsecond illumination isreportedly sufficient to stimulate transcriptionfrom the CHS promoter in parsley cells (53).The photomorphogenic induction of geneexpression does not correlate with CPDformation (53, 88). Moreover, mutants defec-tive in DNA repair, which would be expectedto show increased levels of responses mediatedby DNA damage signaling, do not show alteredsuppression of hypocotyl extension, promotion
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of cotyledon opening, or induction of severalgenes by low-fluence UV-B (16, 94, 140). Fur-thermore, coillumination with light that wouldrepair DNA damage by photoreactivationdoes not reduce the UV-B induction of CHSbut actually enhances it through a synergisticinteraction (55, 143).
The very brief, low-fluence UV-B treat-ments that are sufficient to induce genes such asCHS are very unlikely to cause detectable accu-mulation of ROS or signaling molecules suchas ethylene, SA, and JA. It is therefore very un-likely that these molecules mediate photomor-phogenic UV-B signaling. Consistent with thishypothesis, whereas the UV-B stimulation ofdefense gene expression is reduced in the JAand ethylene signaling mutants jar1 and etr1(3), the UV-B induction of CHS is unaltered(C.M. Pidgeon & G.I. Jenkins, unpublisheddata). Similarly, although the UV-B inductionof defense genes is inhibited by antioxidants(65, 134), these compounds do not impair theUV-B induction of CHS in Arabidopsis cells (82).CHS expression shows little or no stimulationby ROS in either Arabidopsis cells (82) or plants(46, 56), and hydrogen peroxide accumulationactually reduces the level of CHS expression(142). Nevertheless, it has been reported thatthe UV-B induction of CHS is reduced in theArabidopsis atrbohdf double mutant, leading tothe suggestion that NADPH oxidase quanti-tatively affects the response (91). Although nosuch difference between wild type and atrbo-hdf was observed in the author’s laboratory (T.Wang & G.I. Jenkins, unpublished data), thisdiscrepancy may be explained by the particu-lar growth and treatment conditions employed.Thus, taken together, the above studies indi-cate that the photomorphogenic UV-B induc-tion of CHS does not require either ROS orwound/defense signaling molecules.
Further research has demonstrated thatphotomorphogenic UV-B responses are notmediated by the known photoreceptors. Al-though phytochromes, cryptochromes, andphototropins are able to absorb UV-B andtherefore have the potential to mediateUV-B responses, various mutants lacking these
photoreceptors retain low fluence UV-B in-duction of CHS and a number of other genes(21, 23, 140, 143). Similarly, the suppressionof hypocotyl extension by UV-B is presentin mutants that lack phytochromes and cryp-tochromes (8, 16, 133; but see Reference 94).However, photomorphogenic UV-B signalingis not independent of other light signalingpathways; UV-B and phyB interact to regu-late cotyledon opening (16), and UV-B-inducedCHS expression is negatively regulated by PhyBand synergistically enhanced by UV-A and bluelight detected by unknown photoreceptor(s)(143).
Is There a UV-B Photoreceptor?
The possibility that plants possess a UV-B-specific photoreceptor to initiate photo-morphogenic responses has been discussedfor many years (54, 80, 81, 145). The ideais attractive both because other low-fluence,photomorphogenic responses are initated byphotoreceptors and because no alternativeUV-B-absorbing molecules appear to mediatephotomorphogenic UV-B responses. Severalfactors have hindered the search for the pu-tative photoreceptor, not least that many cel-lular components absorb UV-B (e.g., proteins,nucleic acids, and phenolic compounds) andthat UV-B responses do not have a charac-teristic signature equivalent to, for example,phytochrome photoreversibility to facilitatephotoreceptor identification. Moreover, fewUV-B-specific responses can be used in sim-ple genetic screens to isolate a putative UV-Bphotoreceptor mutant.
The construction of action spectra, basedon careful measurements of dose-response re-lationships, can provide important insightsinto the nature of photoreception. Sev-eral action spectra for photomorphogenicUV-B responses, anthocyanin and flavonoidaccumulation in particular, have been published(47, 145) (Table 1) and most have maximabetween 280 and 300 nm. Given the rangeof different species, responses, and methodsof action spectrum production used in these
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experiments, variation is inevitable. There-fore, whether there is a single mechanism ofphotoreception or whether different systemsmediate the responses remains unclear. Experi-ments with Arabidopsis suggest that some genesare differentially responsive to the 280–290and 300–310 wavelength regions (89, 140), sothere could be multiple UV-B photoreceptionmechanisms.
Chromophores that could enable a pho-toreceptor to absorb UV-B wavelengths exist.Pterins or flavins in the reduced form are can-didates (57, 58), and there is some experimentalsupport for their involvement in UV-B percep-tion. Compounds that antagonize flavins andpterins impair the UV-B induction of antho-cyanin synthesis in maize (92) and the UV-Bsuppression of hypocotyl extension in tomato(7). Also, the introduction of riboflavin to pars-ley cells increases CHS protein and flavonoidaccumulation in response to UV-B but notblue light (48). Another possible chromophoreis a phenolic molecule. A p-coumaric acidchromophore enables the Photoactive YellowProtein (PYP) photoreceptor found in pur-ple photosynthetic bacteria to absorb in theUV-A and blue region of the spectrum (75).Therefore, a different phenolic molecule ab-sorbing at shorter wavelengths could act as aUV-B chromophore, although Arabidopsis mu-tants that lack UV-absorbing flavonoids andsinapate esters retain the UV-B induction ofHY5 gene expression (B.A. Brown & G.I.Jenkins, unpublished data). Thus at presentthere is no direct evidence for the existence ofa specific chromophore-bound UV-B photore-ceptor equivalent to the known photoreceptors.
An alternative possibility is that UV-B issensed through some form of direct activa-tion of a cellular component. One mecha-nism of UV-B perception in mammalian cellsinvolves the ligand-independent activation ofplasma membrane receptor kinases. Activationof receptor tyrosine kinases normally involvesbinding to a ligand such as a cytokine. How-ever, ROS produced by UV-B exposure arereported to cause temporary inactivation of ty-rosine phosphatases, leading to activation of
the receptor either by autophosphorylation orphosphorylation by a separate kinase (12, 69).Receptor activation, whether by cytokines orUV-B, leads to stimulation of genes in the in-flammatory response. Although plants do notpossess receptor tyrosine kinases they do havea large number of other receptor kinases (84).In principle, UV-B could activate such a re-ceptor by a similar mechanism to that re-ported in mammalian cells. However, it seemsunlikely that sufficient ROS would be gen-erated by brief or low fluence UV-B expo-sure to cause receptor activation leading tophotomorphogenic responses, and presumablyactivation by ROS would not be UV-B spe-cific. The brassinosteroid receptor BRASSI-NOSTEROID INSENSITIVE1 (BRI1) is areceptor-like kinase (61) and, interestingly, thebri1 mutant is reported to have reduced expres-sion of some UV-B induced genes (124). How-ever, it is not clear whether this phenotype isspecific to UV-B and, moreover, if BRI1 is re-sponsible for UV-B photoreception one wouldexpect the response to be eliminated in themutant rather than reduced. Multiple receptorkinases could act redundantly in UV-B percep-tion, at least in some responses, and this possi-bility merits investigation.
Further studies with mammalian cells haveimplicated the arylhydrocarbon receptor (AhR)in UV-B signaling. UV-B irradiation of trypto-phan in solution produces the AhR ligand FICZand FICZ is formed in cells following UV-B ex-posure (50). Depletion of cytosolic tryptophanimpairs the UV-B stimulation of both geneexpression and plasma membrane receptor ac-tivation mediated by AhR. It will be interestingto see if a comparable mechanism operates inplants for UV-B responses.
The ability of aromatic amino acids, notablytryptophan, strongly to absorb UV-B raisesthe possibility that direct UV-B absorption bya protein could initiate UV-B responses (7, 47,62). All proteins absorb UV-B, so for a partic-ular protein to act as a UV-B photoreceptor,UV-B absorption would have to cause a specificphysical or chemical change to the protein thatinitiates signaling. However, the maximum
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UVR8: UVRESISTANCELOCUS8
absorption of tryptophan occurs at 280 nm andit is not clear whether the protein environmentsurrounding a tryptophan residue wouldenable it to absorb strongly up to 300 nm, thepeak wavelength of some photomorphogenicUV-B action spectra (Table 1). If not, a UV-B-absorbing chromophore would be required.
In summary, the identity of the putative pho-tomorphogenic UV-B photoreceptor remainselusive. Genetic or biochemical studies may oneday uncover a chromophore-binding photore-ceptor, but the mechanism of UV-B sensing alsomay not involve a classical photoreceptor or in-deed one specific class of molecule.
Some researchers have attempted to gain in-sights into photomorphogenic UV-B signalingby using pharmacological and cell physiologi-cal approaches (82). Inhibitor studies with cellsuspension cultures indicated that the UV-Bsignaling pathway regulating CHS gene ex-pression involves calcium ions, calmodulin, andprotein phosphorylation (36, 51, 52). The path-way is distinct from phytochrome and cryp-tochrome signaling pathways that regulate thesame gene. The effects of calcium-channelantagonists and Ca2+-ATPase inhibitors sug-gested the involvement of an intracellular cal-cium pool rather than flux across the plasmamembrane (36, 105). Further experiments witha cell-impermeable electron acceptor and aflavoprotein antagonist suggested that redoxprocesses at the plasma membrane are alsoinvolved (105). Additional evidence from ex-periments in parsley cells shows that redoxstatus can modulate the UV-B signalingpathway (106).
Although the above experiments were care-fully undertaken using appropriate controls,they do not provide unequivocal evidence forthe involvement of calcium fluxes and re-dox activity in UV-B signaling. Direct mea-surements of these processes are required tocomplement the pharmacological experiments
and, unfortunately, this evidence is lacking. Al-though UV-B was reported to elevate cytoso-lic calcium concentration in parsley cells (53),the increase was small and gradual and did notresemble in either magnitude or kinetics thecalcium signatures initiated by other stimuli.Thus, further research is needed to examine theinvolvement of calcium fluxes and redox pro-cesses in photomorphogenic UV-B signaling.
Studies of UV-B signaling processes thatregulate expression of genes concerned withcatharanthine biosynthesis in Catharanthusroseus cells suggest the activation of cell surfacereceptors, similar to the situation with the sys-temin receptor (117). Direct evidence was pre-sented for medium alkalinization, ROS produc-tion, and kinase activation.
Genetic Approaches
Genetic approaches have great potentialto identify components involved in UV-Bperception and signaling and have alreadybeen responsible for some significant advances(80). Genetic variation exists in UV toleranceand responsiveness to UV-B in various species(41, 90, 123, 139) and examination of thebasis of these differences may provide valuableinsights into UV-B responses. For instance,sequence variation in CPD photolyase is a keydeterminant of the differential UV sensitivityof rice cultivars (72). In addition, mutantscreens can be used to identify componentsin UV-B perception and signaling. Screens inArabidopsis for altered sensitivity to UV-B haveidentified various mutants either defective inDNA repair or altered in accumulation ofUV-protective phenolic compounds (80).Kliebenstein and coworkers (95) used such ascreen to isolate the uv resistance locus8 (uvr8)mutant, which has reduced flavonoid contentand CHS expression in UV-B. Screens for mu-tants altered specifically in the UV-B inductionof gene expression using transgenic linesexpressing CHS or HY5 promoter–luciferasefusions identified additional uvr8 alleles (22)and a mutant in the CONSTITUTIVELY
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PHOTOMORPHOGENIC1 (COP1) gene(112). Both UVR8 and COP1 play crucialroles in photomorphogenic UV-B signaling, asdiscussed below.
A screen was undertaken to isolate Ara-bidopsis mutants altered in the photomor-phogenic suppression of hypocotyl extensionby UV-B (133). Several UV light insensitive(uli ) mutants were identified that had reducedhypocotyl growth suppression in UV-B but nor-mal hypocotyl lengths in other light qualities.In addition, the uli3 mutant had reduced ex-pression of some genes in response to supple-mentary UV-B but, with the exception of PR1,transcript levels were also reduced in UV-A.The predicted protein encoded by ULI3 haslimited sequence identity with a human diacyl-glycerol kinase but no information is availableon its cellular function. Thus, further researchis needed to establish the significance of ULI3in the regulation of hypocotyl extension andPR1 expression by UV-B.
COP1 Is a Positive Regulatorof UV-B Responses
Recent research has revealed a novel functionfor the Arabidopsis COP1 protein in UV-B re-sponses. COP1 is a key negative regulator ofseedling photomorphogenesis (35, 150). COP1represses photomorphogenic gene expressionand development in darkness and hence dark-grown cop1 mutants display several featuresof light-grown plants. COP1 acts as an E3ubiquitin ligase in darkness, targeting HY5and other positive regulators of photomor-phogenic gene expression for destruction bythe proteasome (35, 150). Following illumina-tion, COP1 is inactivated and moves slowly outof the nucleus, enabling HY5 and other tran-scription factors to accumulate and promotephotomorphogenesis.
In marked contrast to its function as a neg-ative regulator of photomorphogenesis, COP1is a positive regulator of UV-B responses. Bothflavonoid accumulation (112) and the suppres-sion of hypocotyl elongation in response to
UV-B (94, 112) are impaired in cop1 mutantplants. In addition, Oravecz and colleagues(112) discovered that cop1-4 seedlings had muchreduced expression of many of the genes in-duced by a low-fluence UV-B treatment ofwild type. Among the genes positively regulatedby COP1 is HY5. In fact, nearly half of thegenes regulated by COP1 are also controlledby HY5, indicating that HY5 is a key effectorof the COP1 pathway. Thus, COP1 and HY5act together in the nucleus in UV-B responses,whereas COP1 degrades HY5 in the nucleusin darkness. The cop1-4 mutant showed in-creased chlorosis under UV-B stress comparedwith wild type, consistent with a reduced abil-ity to induce genes required for UV protection.The mutant is much less UV-sensitive than hy5,although this might be explained by a higherbasal level of expression of UV-protective genesin cop1-4. In contrast to the response of dark-grown seedlings to white light, UV-B stimu-lates the nuclear accumulation of COP1 taggedwith yellow fluorescent protein. However, nu-clear accumulation occurs more slowly than theinduction of gene expression by UV-B, indi-cating that other processes are involved in theresponse.
The above research reveals that COP1 hascontrasting functions in UV-B responses andin the repression of seedling photomorpho-genesis. However, the positive role of COP1appears not to be specific to UV-B becauseevidence shows a comparable function in someresponses to red light that require phytochromeB (17). Whether COP1 acts positively in otherlight responses remains unknown. The mecha-nism of COP1 function in the UV-B responseis not understood. If COP1 acts as an E3ubiquitin ligase it would presumably effect theremoval of a negative regulator of UV-B re-sponses, although no such component has beendescribed. Alternatively, COP1 may act via adifferent, unknown mechanism. The latter isperhaps more likely because COP1 appears tohave different requirements, both structurallyand for interaction with SUPPRESSOR OFPHYTOCHROME A (SPA) proteins in the
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SPA1:SUPPRESSOR OFPHYTOCHROME A
responses to UV-B and white light (112). COP1has distinct functional domains that interactwith numerous proteins so its cellular role maynot be restricted to E3 ligase activity. Thereis some evidence for this, because COP1 isrequired for the nuclear accumulation of thePHYTOCHROME INTERACTING FAC-TOR3 (PIF3) transcription factor in darknessbut does not mediate its destruction followingred and far-red illumination (10).
UVR8: A UV-B-SPECIFICREGULATOR
UVR8 Function
Recent research has demonstrated that Ara-bidopsis UVR8 acts specifically to mediate re-sponses to UV-B, including the gene expres-sion responses that establish UV protection.Brown and coworkers (22) showed that uvr8mutants lack the UV-B induction of CHStranscripts but retain stimulation of CHS ex-pression by several other light and nonlightstimuli. Similarly, uvr8 is defective in theinduction of HY5 transcripts specifically byUV-B. UVR8 mediates gene expression re-sponses at low fluence rates of UV-B (downto 0.1 μmol m−2 s−1), consistent with its in-volvement in photomorphogenic UV-B sig-naling (23). No other component is knownto act specifically in photomorphogenic UV-Bresponses.
Transcriptome analysis (22) identified a setof approximately 70 genes stimulated by UV-Bunder the control of UVR8. Among these genesare several known to have key roles in UV pro-tection, including those encoding the principalenzymes of flavonoid biosynthesis, the DNAphotolyases, and enzymes involved in ameliora-tion of oxidative stress and photooxidative dam-age. These data explain why the uvr8 mutantshows severe necrosis when exposed to levelsof UV-B found in bright sunlight, whereas itis indistinguishable from wild type in the ab-sence of UV-B (22, 95). The above findingsdemonstrate that UVR8 is a key regulator of
UV protection and therefore helps to promotesurvival of plants exposed to UV-B.
Interestingly, the role of UVR8 is not con-fined to UV protection because it also medi-ates morphological responses to UV-B. Theuvr8 mutant lacks the photomorphogenic sup-pression of hypocotyl extension in response toUV-B (49). This phenotype probably resultsfrom altered gene expression in the uvr8 mu-tant, although the genes involved have not beenidentified.
Effectors of UVR8 Signaling
UVR8 regulates the expression of the HY5and HYH transcription factors at low UV-Bfluence rates (23). Transcriptome analysis (22)indicated that approximately half of the genesregulated by UVR8 are also regulated by HY5,but this is an underestimate and does not takeinto account functional redundancy betweenHY5 and HYH (23). Gene expression studieswith hy5,hyh double mutant plants show thatsome UVR8-regulated genes that appear notto be regulated by HY5 alone in the transcrip-tome experiments are in fact regulated by eitherHY5 or HYH (23). Further analysis suggeststhat these transcription factors may regulate allthe UVR8 pathway genes and are therefore piv-otal downsteam effectors of UVR8 signaling(23). HY5 is evidently a very important regula-tor of UV-B responses because the hy5 mutantis very sensitive to UV-B, similar to uvr8 (22,112). The hyh mutant is less sensitive, indicatingthat it has a subsidiary role (23).
Structure and Activity
UVR8 is 35% identical in amino acidsequence to the human REGULATOROF CHROMATIN CONDENSATION1(RCC1) protein (95). RCC1 is a guaninenucleotide exchange factor (GEF) for the smallGTP-binding protein Ran, which is involvedin nucleocytoplasmic transport, regulation ofthe cell cycle, and mitosis (110). RCC1 hasa seven-bladed propeller structure (119) and
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amino acids that help to maintain this structureare highly conserved in UVR8. However,the predicted structural similarity is rathermisleading because several lines of evidenceshow that RCC1 and UVR8 differ in activityand function. In particular, UVR8 has verylittle Ran-GEF activity and whereas yeast andmammalian cells lacking RCC1 fail to grow(110), Arabidopsis uvr8 grows normally in theabsence of UV-B. This phenotype is unlikely tobe explained by redundancy because no otherArabiodopsis gene encodes a protein sufficientlysimilar in sequence to UVR8 to be a functionalhomolog.
Likely orthologs of UVR8 are found in otherspecies. Several higher plants have protein se-quences that are approximately 70% identicalto UVR8. Interestingly, the moss Physcomitrellapatens has sequences that are 65% identical toUVR8, raising the intriguing possibility thatUVR8 has played an important role in UV pro-tection throughout land plant evolution.
One similarity between UVR8 and RCC1 isthat they associate with chromatin via histones.UVR8 interacts most strongly with histoneH2B (37). Initial ChIP experiments showed thatUVR8 binds to chromatin that contains thepromoter region of the HY5 gene (22), consis-tent with the observation that UVR8 is requiredfor UV-B induction of HY5 expression. FurtherChIP assays show that UVR8 associates with 5′
noncoding, coding, and 3′ noncoding regions atthe HY5 locus, but not with sequences approx-imately 5 kb upstream or downstream of thegene (37). It is proposed that UVR8 interactswith nucleosomes associated with HY5 and thatits binding mediates the recruitment or activa-tion of transcription factors required for HY5transcription specifically in response to UV-B(Figure 2).
Further ChIP assays indicate that UVR8 as-sociates with other genes it regulates, not onlyHY5. However, UVR8 may not associate withall its target genes, because several were not de-tected among the ChIP products (37). A morecomprehensive analysis is required to estab-lish which genes UVR8 associates with. ChIP
UV-B
UVR8
COP1
TF
TFTF
UVR8
UVR8
HY5
Cytosol Nucleus
Figure 2A model for the regulation of transcription by UV RESISTANCE LOCUS8(UVR8). UVR8 associates with nucleosomes on chromatin in the region oftarget genes such as ELONGATED HYPOCOTYL5 (HY5) (22, 37). UVR8 isproposed to facilitate the activation or recruitment of transcription factorsrequired for the UV-B induction of transcription. CONSTITUTIVELYPHOTOMORPHOGENIC1 (COP1) is also required for the UV-B inductionof HY5 (112). UV-B promotes the rapid nuclear accumulation of UVR8 and isrequired for UVR8 function in the nucleus (87). UV-B stimulates interactionbetween UVR8 and COP1 (49). TF, transcription factor.
experiments indicate that UV-B is not requiredfor binding of UVR8 to chromatin, which sug-gests that UV-B either modifies UVR8 associ-ated with chromatin or regulates UVR8 activityby causing a secondary activator to interact withthe protein.
Nuclear Accumulation
A fusion of UVR8 with green fluorescent pro-tein (GFP) expressed in the uvr8 mutant accu-mulates in the nucleus following UV-B treat-ment. In plants grown in white light lackingUV-B, GFP-UVR8 is present in both the nu-cleus and cytoplasm (22). However, as littleas five minutes of UV-B exposure promotesa substantial increase both in the number ofnuclei that contain GFP-UVR8 and in thebrightness of nuclear fluorescence (87). Nuclear
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FHY1: FAR-REDELONGATEDHYPOCOTYL1
FHL: FHY1 LIKE
accumulation is not initiated by other lightqualities and occurs at low fluence rates ofUV-B, consistent with the features of UVR8-regulated gene expression. The GFP-UVR8fusion is functional in promoting HY5 and CHSexpression in response to UV-B.
Further experiments showed that a 23-amino-acid region at the N terminus of UVR8is required for efficient nuclear accumulation(87). This region does not contain a nuclearlocalization signal (NLS) and could be re-quired for interaction with other componentsthat mediate the response. One possibility isthat UVR8 is translocated into the nucleusthrough association with a protein contain-ing a NLS, similar to the nuclear importof phytochrome A mediated by FAR-REDELONGATED HYPOCOTYL1 (FHY1) andFHY1 LIKE (FHL) (73). However, the mecha-nism of UVR8 accumulation remains unknown.Addition of a nuclear export signal (NES) toGFP-UVR8 causes the fusion to be localizedin the cytoplasm, but brief UV-B exposureoverrides the effect of the NES, indicating aconcerted mechanism for nuclear accumulation(87). Addition of a NLS to GFP-UVR8 causesthe fusion to be constitutively nuclear but, in-terestingly, the presence of GFP-UVR8 in thenucleus is not sufficient to initiate expression ofHY5 in the absence of UV-B. Evidently, UV-Bexposure is required to activate UVR8 function,either directly or indirectly, in the nucleus.Thus, UV-B has the dual effect of promotingnuclear accumulation of UVR8 and activationof its function in the nucleus (Figure 2).
Relationship with COP1
UVR8 and COP1 regulate many of the samegenes and are both required for the UV-B in-duction of HY5. Therefore, UVR8 and COP1appear to function in the same pathway, al-though little information is available to ex-plain their functional relationship. If COP1mediates the destruction of a negative regula-tor of photomorphogenic UV-B signaling, thenit would not target UVR8 or HY5 because
they are positive regulators of the response.COP1 may remove a negative regulator ofUVR8 or HY5 but, as discussed above, COP1might not function as an E3 ubiquitin ligase inphotomorphogenic UV-B responses. BecauseUVR8 is a UV-B-specific component it maydirect the action of COP1 in UV-B responses.One possibility is that UVR8 regulates the nu-clear accumulation of COP1 or vice versa, butno information is available on this point. An-other possibility is that UVR8 recruits COP1into a complex involved in the UV-B regula-tion of transcription. This possibility is sup-ported by a recent report that UVR8 colocalizeswith and directly interacts with COP1 in vivoin a UV-B-dependent manner (49). Clearly,further research is required to address thesepossibilities.
Could UVR8 Be a UV-BPhotoreceptor?
Several features of UVR8 raise the intriguingpossibility that it could function as a UV-B pho-toreceptor. UVR8 is UV-B specific and actsto mediate low fluence photomorphogenic re-sponses. Genetic screens have failed to identifycomponents that function upstream of UVR8(22, 49). Moreover, the rapid accumulation ofUVR8 in the nucleus in response to induc-tive illumination (87) is reminiscent of phy-tochromes. There is no evidence that UVR8binds a chromophore but it has 14 tryptophanresidues, more than most proteins of its size(e.g., RCC1 has three). The UVR8 trypto-phans are highly conserved in other species, in-cluding the moss Physcomitrella, suggesting thatthey are functionally important. Perhaps oneor more of these tryptophans enable UVR8 toabsorb UV-B and initiate signaling. However,it is not known whether this mechanism wouldenable the protein to act over the range of wave-lengths that initiate photomorphogenic UV-Bresponses or whether a UV-B-absorbing chro-mophore is required. Further research shouldresolve this point.
UV-B clearly activates a number of distinctsignaling pathways in plants, as illustrated inFigure 3. Each pathway initiates particularresponses, although there is overlap betweensome pathways. Progress has been made inidentifying components of the pathways butmuch remains to be done. It will not be pos-sible to establish how many distinct pathwaysmediate UV-B responses until we know moreabout their components. Furthermore, little isknown about the relative importance of thedifferent UV-B signaling pathways in plantsgrowing in the natural environment. Evidenceexists for interactions between short- and long-wavelength UV-B pathways (126, 140), for in-teractions between photomorphogenic UV-Band other light signaling pathways (16, 143),for negative regulation of photomorphogenicUV-B signaling by defense signaling pathways(103), and for interactions between UV-B andother environmental stimuli (24, 26). However,little is known about the functional integrationof these different pathways in plants growing innatural conditions.
The nonspecific UV-B signaling pathwaysshould not be thought of entirely as stress-related pathways activated by nonphysiologicalexposure to UV-B. These pathways are likelyto function in plants at normal ambient lev-els of UV-B, although relatively little informa-tion is available on this point. UV-B is a veryvariable environmental signal and fluctuationsin fluence rate will probably modulate the lev-els of CPDs, ROS, and wound/defense signal-ing molecules. ROS levels are likely to changetransiently through alterations in the balance ofproduction and scavenging (6, 109). Moreover,there is evidence that some plants maintain alow level of DNA damage (72), which could ac-tivate signaling. The nonspecific pathways reg-ulate numerous genes and could influence mor-phogenesis by affecting cell cycle progression(43, 44), the amounts of plant growth regula-tors (e.g., ethylene; 3, 115), or responsiveness
Adaptation
AcclimationOther signals
Photomorphogenic signaling
Photoreceptor(s)?
UVR8
COP1
HY5 HYH
UVR8/COP1/HY5independent
Responses
DNAdamage ROS
JA SAC2H4
Targetgenes
UV-Bwavelength, fluence rate, duration
Nonspecific signaling
Figure 3UV-B signal transduction pathways. UV-B stimulates distinct nonspecific andphotomorphogenic signal transduction pathways, depending on thewavelength, fluence rate, and duration of exposure, leading to the induction ofspecific sets of target genes and downstream responses. Signaling and responsewill be influenced by the extent of plant adaptation and acclimation to UV-Band interactions with other stimuli. ROS, reactive oxygen species; JA, jasmonicacid; SA, salicylic acid, UVR8, UV RESISTANCE LOCUS8; COP1,CONSTITUTIVELY PHOTOMORPHOGENIC1; HY5, ELONGATEDHYPOCOTYL5; HYH, HY5 HOMOLOG.
to these compounds (e.g., brassinosteroids; 124,131).
The photomorphogenic UV-B signalingpathways act at low ambient fluence rates ofUV-B to regulate UV protection and morpho-genesis but also operate at high ambient levels(Figure 1). Whether there are multiple pho-tomorphogenic pathways remains unclear, butdifferences in action spectra (Table 1) and re-sponsiveness to different wavelengths of UV-B(89, 140) suggest this might be the case. Fur-thermore, some genes are induced at low flu-ence rates of UV-B independently of UVR8 andCOP1 (L. Headland & G.I. Jenkins, unpub-lished data). Hence, much remains to be done todefine the different UV-B signaling pathways,to establish their functions, and to understandhow they are integrated in plants growing in thenatural environment.
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SUMMARY POINTS
1. UV-B is a key environmental signal that regulates diverse responses in plants.
2. UV-B promotes UV protection and plant survival in sunlight and influences metabolism,development, and plant defense. Many of the effects of UV-B involve the differentialregulation of gene expression.
3. Plant responses to UV-B depend on the nature of the UV-B treatment, the extent ofadaptation and prior acclimation to UV-B, and interaction with other environmentalfactors.
4. UV-B responses are mediated by both nonspecific signaling pathways, which involveDNA damage, reactive oxygen species (ROS), and wound/defense signaling, and UV-B-specific pathways that mediate photomorphogenic responses.
5. Photomorphogenic UV-B signaling is mediated by the UV-B-specific componentUV RESISTANCE LOCUS8 (UVR8). Both UVR8 and CONSTITUTIVELYPHOTOMORPHOGENIC1 (COP1) are required for UV-B induction of theELONGATED HYPOCOTYL5 (HY5) transcription factor, which regulates targetgenes involved in photomorphogenic UV-B responses, including those required for UVprotection.
FUTURE ISSUES
1. What is the relative importance of different UV-B signaling pathways in plants growingin the natural environment, and how are the pathways functionally integrated?
2. What mechanisms are involved in the regulation of morphogenesis by UV-B?
3. How is UV-B detected in photomorphogenic responses—is there a UV-B-specific pho-toreceptor?
4. How is UVR8 regulated by UV-B, and how does UVR8 act in transcriptional regulation?
5. What is the functional relationship of UVR8 and COP1? How do they regulate tran-scription of the key HY5 transcription factor?
DISCLOSURE STATEMENT
The author is not aware of any biases that might be perceived as affecting the objectivity of thisreview.
ACKNOWLEDGMENTS
I am grateful to Dr. Bobby Brown and Dr. John Christie for their helpful comments on themanuscript and to members of the Jenkins and Christie laboratories for productive discussions ofUV-B research.
LITERATURE CITED
1. A-H-Mackerness S. 2000. Plant responses to UV-B (UV-B: 280–320 nm) stress: What are the keyregulators? Plant Growth Regul. 32:27–39
424 Jenkins
Ann
u. R
ev. P
lant
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l. 20
09.6
0:40
7-43
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om a
rjou
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s.an
nual
revi
ews.
org
by U
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Sevi
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n 05
/04/
09. F
or p
erso
nal u
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ANRV375-PP60-19 ARI 8 April 2009 16:30
2. A-H-Mackerness S, John CF, Jordan B, Thomas B. 2001. Early signaling components in UV-B responses:distinct roles for different reactive oxygen species and nitric oxide. FEBS Lett. 489:237–42
3. A-H-Mackerness S, Surplus SL, Blake P, John CF, Buchanan-Wollaston V, et al. 1999. Ultraviolet-B-induced stress and changes in gene expression in Arabidopsis thaliana: role of signalling pathwayscontrolled by jasmonic acid, ethylene and reactive oxygen species. Plant Cell Environ. 22:1413–23
4. Allan AC, Fluhr R. 1997. Two distinct sources of elicited reactive oxygen species in tobacco epidermalcells. Plant Cell 9:1559–72
5. Andrady A, Aucamp PJ, Bais AF, Ballare CL, Bjorn LO, et al. 2005. Environmental effects of ozonedepletion and its interactions with climate change: Progress report, 2004. Photochem. Photobiol. Sci. 4:177–84
6. Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction.Annu. Rev. Plant Biol. 55:373–99
7. Ballare CL, Barnes PW, Flint SD. 1995. Inhibition of hypocotyl elongation by UV-B radiation in de-etiolating tomato seedlings. 1. The photoreceptor. Physiol. Plant. 93:584–92
8. Ballare CL, Barnes PW, Kendrick RE. 1991. Photomorphogenic effects of UV-B radiation on hypocotylelongation in wild-type and stable-phytochrome-deficient mutant seedlings of cucumber. Physiol. Plant.83:652–58
9. Barta C, Kalai T, Hideg K, Vass I, Hideg E. 2004. Differences in the ROS-generating efficacy of variousUV wavelengths in detached spinach leaves. Funct. Plant Biol. 31:23–28
10. Bauer D, Viczıan A, Kircher S, Nobis T, Nitschke R, et al. 2004. Constitutive photomorphogenesis 1and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcriptionfactor required for light signaling in Arabidopsis. Plant Cell 16:1433–45
11. Beggs CJ, Stolzer-Jehle A, Wellmann E. 1985. Isoflavonoid formation as an indicator of UV stress inbean (Phaseolus vulgaris L.) leaves. Plant Physiol. 79:630–34
12. Bender K, Blattner C, Knebel A, Iordanov M, Herrlich P, Rahmsdorf HJ. 1997. UV-induced signaltransduction. J. Photochem. Photobiol. B-Biol. 37:1–17
13. Bjorn LO. 1996. Effects of ozone depletion and increased UV-B on terrestrial ecosystems. Int. J. Environ.Stud. 51:217–43
14. Blueyard J-Y, Gallego ME, White CI. 2006. Recent advances in understanding of the DNA double-strandbreak repair machinery of plants. DNA Repair 5:1–12
15. Blum JE, Casati P, Walbot V, Stapleton AE. 2004. Split-plot microarray design allows sensitive detec-tion of expression differences after UV radiation in the inbred parental lines of a key maize mappingpopulation. Plant Cell Environ. 27:1374–86
16. Boccalandro HE, Mazza CA, Mazzella MA, Casal JJ, Ballare CL. 2001. Ultraviolet B radiation enhancesa phytochrome-B-mediated photomorphogenic response in Arabidopsis. Plant Physiol. 126:780–88
17. Boccalandro HE, Rossi MC, Saijo Y, Deng XW, Casal JJ. 2004. Promotion of photomorphogenesis byCOP1. Plant Mol. Biol. 56:905–15
18. Bornman JF, Reuber S, Cen Y-P, Weissenbock G. 1997. Ultraviolet radiation as a stress factor andthe role of protective pigments. In Plants and UV-B: Responses to Environmental Change, ed. P Lumsden,pp. 157–68. Cambridge: Cambridge Univ. Press
19. Britt AB. 2004. Repair of DNA damage induced by solar UV. Photosynth. Res. 81:105–1220. Brosche M, Schuler MA, Kalbina I, Connor L, Strid A. 2002. Gene regulation by low level UV-B
radiation: identification by DNA array analysis. Photochem. Photobiol. Sci. 1:656–6421. Brosche N, Strid A. 2003. Molecular events following perception of UV-B radiation by plants. Physiol.
Plant. 117:1–1022. Showed that UVR8is a UV-B-specificregulator of HY5
expression and thatUVR8 and HY5 regulateUV-protective genes.
22. Brown BA, Cloix C, Jiang GH, Kaiserli E, Herzyk P, et al. 2005. A UV-B-specific signalingcomponent orchestrates plant UV protection. Proc. Natl. Acad. Sci. USA 102:18225–30
23. Brown BA, Jenkins GI. 2008. UV-B signalling pathways with different fluence-rate response profiles aredistinguished in mature Arabidopsis leaf tissue by requirement for UVR8, HY5, and HYH. Plant Physiol.146:576–88
24. Caldwell MM, Ballare CL, Bornman JF, Flint SD, Bjorn LO, et al. 2003. Terrestrial ecosystems, increasedsolar UV radiation and interactions with other climatic change factors. Photochem. Photobiol. Sci. 2:29–38
www.annualreviews.org • UV-B Signaling 425
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
25. Caldwell MM, Bjorn LO, Bornman JF, Flint SD, Kulandaivelu G, et al. 1998. Effects of increased solarUV radiation on terrestrial ecosystems. J. Photochem. Photobiol. B Biol. 46:40–52
26. Wide-rangingrecent review of theimpact of UV-B onplants.
26. Caldwell MM, Bornman JF, Ballare CL, Flint SD, Kulandaivelu G. 2007. Terrestrial ecosystems,increased solar UV radiation, and interactions with other climate change factors. Photochem.
Photobiol. Sci. 6:252–66
27. Highlighted the roleof chromatinremodeling inregulation oftranscription by UV-B.
27. Casati P, Campi M, Chu F, Suzuki N, Maltby D, et al. 2008. Histone acetylation and chromatinremodelling are required for UV-B-dependent transcriptional activation of regulated genes inmaize. Plant Cell 20:827–42
28. Casati P, Stapleton AE, Blum JE, Walbot V. 2006. Genome-wide analysis of high-altitude maizeand gene knockdown stocks implicates chromatin remodeling proteins in response to UV-B.Plant J. 46:613–27
29. Casati P, Walbot V. 2003. Gene expression profiling in response to UV radiation in maize genotypeswith varying flavonoid content. Plant Physiol. 132:1739–54
30. Casati P, Walbot V. 2004. Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B andits effects on translation. Plant Physiol. 136:3319–32
31. Casati P, Walbot V. 2004. Rapid transcriptome responses of maize (Zea mays) to UV-B in irradiated andshielded tissues. Genome Biol. 5:R16
32. Casati P, Zhang X, Burlingame AL, Walbot V. 2005. Analysis of leaf proteome after UV-B irradiationin maize lines differing in sensitivity. Mol. Cell. Proteomics 4:1673–85
33. Chalker-Scott L, Scott JD. 2004. Elevated UV-B radiation induces cross-protection to cold in leaves ofRhododendron under field conditions. Photochem. Photobiol. 79:199–204
34. Chappell J, Hahlbrock K. 1984. Transcription of plant defence genes in response to UV light or fungalelicitor. Nature 311:76–78
35. Chen M, Chory J, Fankhauser C. 2004. Light signal transduction in higher plants. Annu. Rev. Genet.38:87–117
36. Christie JM, Jenkins GI. 1996. Distinct UV-B and UV-A/blue light signal transduction pathways inducechalcone synthase gene expression in Arabidopsis cells. Plant Cell 8:1555–67
37. Cloix C, Jenkins GI. 2008. Interaction of the Arabidopsis UV-B-specific signaling component UVR8 withchromatin. Mol. Plant 1:118–28
38. Cominelli E, Gusmaroli G, Allegra D, Galbiati M, Wade HK, et al. 2008. Expression analysis of an-thocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. J. Plant Physiol.165:886–94
39. Conconi A, Smerdon MJ, Hope GA, Ryan CA. 1996. The octadecanoid signalling pathway in plantsmediates a response to UV radiation. Nature 383:826–29
40. Conklin PL, Williams EH, Last RL. 1996. Environmental stress sensitivity of an ascorbic acid-deficientArabidopsis mutant. Proc. Natl. Acad. Sci. USA 93:9970–4
41. Correia CM, Areal ELV, Torres-Pereira MS, Torres-Pereira JMG. 1999. Intraspecific variation in sensi-tivity to UV-B radiation in maize grown under field conditions. II. Physiological and biochemical aspects.Field Crop. Res. 62:97–105
42. Correia CM, Pereira JMM, Coutinho JF, Bjorn LO, Torres-Pereira JMG. 2005. Ultraviolet-B radiationand nitrogen affect the photosynthesis of maize: a Mediterranean field study. Eur. J. Agron. 22:337–47
43. Culligan K, Tissier A, Britt A. 2004. ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsisthaliana. Plant Cell 16:1091–104
44. Culligan KM, Robertson CE, Foreman J, Doerner P, Britt AB. 2006. ATR and ATM play both distinctand additive roles in response to ionizing radiation. Plant J. 48:947–61
45. Dai QJ, Yan B, Huang SB, Liu XZ, Peng SB, et al. 1997. Response of oxidative stress defense systems inrice (Oryza sativa) leaves with supplemental UV-B radiation. Physiol. Plant. 101:301–8
47. Ensminger PA. 1993. Control of development in plants and fungi by far-UV radiation. Physiol. Plant.88:501–8
48. Ensminger PA, Schafer E. 1992. Blue and UV-B light photoreceptors in parsley cells. Photochem. Photobiol.55:437–47
426 Jenkins
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
49. Provides evidencefor direct interactionbetween UVR8 andCOP1.
49. Favory J, Stec A, Gruber H, Rizzini L, Oravecz A, et al. 2009. Interaction of COP1 and UVR8regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J.
28:591–60150. Fritsche E, Schafer C, Calles C, Bernsmann T, Bernshausen T, et al. 2007. Lightening up the UV
response by identification of the arylhydrocarbon receptor as a cytoplasmic target for UV B radiation.Proc. Natl. Acad. Sci. USA 104:8851–56
51. Frohnmeyer H, Bowler C, Schafer E. 1997. Evidence for some signal transduction elements involved inUV-light-dependent responses in parsley protoplasts. J. Exp. Bot. 48:739–50
52. Frohnmeyer H, Bowler C, Zhu JK, Yamagata H, Schafer E, Chua NH. 1998. Different roles for calciumand calmodulin in phytochrome- and UV-regulated expression of chalcone synthase. Plant J. 13:763–72
53. Frohnmeyer H, Loyall L, Blatt MR, Grabov A. 1999. Millisecond UV-B irradiation evokes prolongedelevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures. PlantJ. 20:109–17
54. Frohnmeyer H, Staiger D. 2003. Ultraviolet-B radiation-mediated responses in plants. Balancing damageand protection. Plant Physiol. 133:1420–28
55. Fuglevand G, Jackson JA, Jenkins GI. 1996. UV-B, UV-A, and blue light signal transduction pathwaysinteract synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell 8:2347–57
56. Gadjev I, Vanderauwera S, Gechev TS, Laloi C, Minkov IN, et al. 2006. Transcriptomic footprintsdisclose specificity of reactive oxygen species signalling in Arabidopsis. Plant Physiol. 141:436–45
57. Galland P, Senger H. 1988. The role of flavins as photoreceptors. J. Photochem. Photobiol. B Biol. 1:277–9458. Galland P, Senger H. 1988. The role of pterins in the photoreception and metabolism of plants.
Photochem. Photobiol. 48:811–2059. Gao Q, Zhang L. 2008. Ultraviolet-B-induced oxidative stress and antioxidant defense system responses
in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana. J. Plant Physiol. 165:138–4860. Garcia V, Bruchet H, Camescasse D, Granier F, Bouchez D, Tissier A. 2003. AtATM is essential for
meiosis and the somatic response to DNA damage in plants. Plant Cell 15:119–3261. Gendron JM, Wang ZY. 2007. Multiple mechanisms modulate brassinosteroid signalling. Curr. Opin.
Plant Biol. 10:436–4162. Gerhardt KE, Wilson MI, Greenberg BM. 2005. Ultraviolet wavelength dependence of photomor-
phological and photosynthetic responses in Brassica napus and Arabidopsis thaliana. Photochem. Photobiol.81:1061–68
63. Giordano CV, Galatro A, Puntarulo S, Ballare CL. 2004. The inhibitory effects of UV-B radiation (280–315 nm) on Gunnera magellanica growth correlate with increased DNA damage but not with oxidativedamage to lipids. Plant Cell Environ. 27:1415–23
64. Gitz DC, Liu-Gitz L. 2003. How do UV photomorphogenic responses confer water stress tolerance?Photochem. Photobiol. 78:529–34
65. Green R, Fluhr R. 1995. UV-B-induced PR-1 accumulation is mediated by active oxygen species. PlantCell 7:203–12
66. Hahlbrock K, Scheel D. 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu.Rev. Plant Physiol. Plant Mol. Biol. 40:347–69
67. Hartmann U, Valentine WJ, Christie JM, Hays J, Jenkins GI, Weisshaar B. 1998. Identification ofUV/blue light-response elements in the Arabidopsis thaliana chalcone synthase promoter using a homol-ogous protoplast transient expression system. Plant Mol. Biol. 36:741–54
68. Hectors K, Prinsen E, De Coen W, Jansen MAK, Guisez Y. 2007. Arabidopsis thaliana plants acclimatedto low dose rates of UV B radiation show specific changes in morphology and gene expression in theabsence of stress symptoms. New Phytol. 175:255–70
69. Herrlich P, Bohmer FD. 2000. Redox regulation of signal transduction in mammalian cells. Biochem.Pharmacol. 59:35–41
70. Hideg E, Barta C, Kalai T, Vass I, Hideg K, Asada K. 2002. Detection of singlet oxygen and superoxidewith fluorescent sensors in leaves under stress by photoinhibition or UV radiation. Plant Cell Physiol.43:1154–64
71. Hideg E, Vass I. 1996. UV-B induced free radical production in plant leaves and isolated thylakoidmembranes. Plant Sci. 115:251–60
www.annualreviews.org • UV-B Signaling 427
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
72. Hidema J, Kumagia T. 2006. Sensitivity of rice to UV-B radiation. Ann. Bot. 97:933–4273. Hiltbrunner A, Tscheuschler A, Viczian A, Kunkel T, Kircher S, Schafer E. 2006. FHY1 and FHL
act together to mediate nuclear accumulation of the phytochrome A photoreceptor. Plant Cell Physiol.47:1023–34
74. Holley SR, Yalamanchili RD, Moura DS, Ryan CA, Stratmann JW. 2003. Convergence of signalingpathways induced by systemin, oligosaccharide elicitors, and UV-B radiation at the level of mitogen-activated protein kinases in Lycopersicon peruvianum suspension-cultured cells. Plant Physiol. 132:1728–38
74a. Holton N, Cano-Delgado A, Harrison K, Montoya T, Chory J, Bishop GJ. 2007. Tomato BRASSINO-STEROID INSENSITIVE1 is required for systemin-induced root elongation in Solanum pimpinellifoliumbut is not essential for wound signaling. Plant Cell 19:1709–17
75. Imamoto Y, Kataoka M. 2007. Structure and photoreaction of photoactive yellow protein, a structuralprototype of the PAS domain superfamily. Photochem. Photobiol. 83:40–49
76. Izaguirre MM, Mazza CA, Svatos A, Baldwin IT, Ballare CL. 2007. Solar UV-B radiation and insectherbivory trigger partially overlapping phenolic responses in Nicotiana attenuata and Nicotiana longiflora.Ann. Bot. 99:103–9
77. Izaguirre MM, Scopel AL, Baldwin IT, Ballare CL. 2003. Convergent responses to stress. Solar UV-Bradiation and Manduca sexta herbivory elicit overlapping transcriptional responses in field-grown plantsof Nicotiana longiflora. Plant Physiol. 132:1755–67
78. Jansen MAK. 2002. Ultraviolet-B radiation effects on plants: induction of morphogenic responses. Physiol.Plant. 116:423–29
80. Jenkins GI, Brown BA. 2007. UV-B perception and signal transduction. In Light and Plant Development,ed. GC Whitlam, KJ Halliday, pp. 155–182. Oxford: Blackwell
81. Jenkins GI, Fuglevand G, Christie JM. 1997. UV-B perception and signal transduction. In Plants andUV-B: Responses to Environmental Change, ed. P Lumsden, pp. 135–56. Cambridge: Cambridge Univ.Press
82. Jenkins GI, Long JC, Wade HK, Shenton MR, Bibikova TN. 2001. UV and blue light signalling:pathways regulating chalcone synthase gene expression in Arabidopsis. New Phytol. 151:121–31
83. Jin HL, Cominelli E, Bailey P, Parr A, Mehrtens F, et al. 2000. Transcriptional repression by AtMYB4controls production of UV-protecting sunscreens in Arabidopsis. EMBO J. 19:6150–61
84. Johnson KL, Ingram GC. 2005. Sending the right signals: regulating receptor kinase activity. Curr. Opin.Plant Biol. 8:648–56
85. Jordan BR. 1996. The effects of UV-B radiation on plants: a molecular perspective. Adv. Bot. Res. 22:97–162
86. Kaiser T, Emmler K, Kretsch T, Weisshaar B, Schafer E, Batschauer A. 1995. Promoter elements of themustard CHS1 gene are sufficient for light regulation in transgenic plants. Plant Mol. Biol. 28:219–29
87. Showed the dualrole of UV-B inregulating UVR8nuclear accumulationand function.
87. Kaiserli E, Jenkins GI. 2007. UV-B promotes rapid nuclear translocation of the UV-B-specificsignalling component UVR8 and activates its function in the nucleus. Plant Cell 19:2662–73
88. Kalbin G, Hidema J, Brosche M, Kumagai T, Bornman JF, Strid A. 2001. UV-B-induced DNA damageand expression of defence genes under UV-B stress: tissue-specific molecular marker analysis in leaves.Plant Cell Environ. 24:983–90
89. Kalbina I, Li S, Kalbin G, Bjorn LO, Strid A. 2008. Two separate UV-B radiation wavelength regionscontrol expression of different molecular markers in Arabidopsis thaliana. Funct. Plant Biol. 35:222–27
90. Kalbina I, Strid A. 2006. Supplementary UV-B irradiation reveals differences in stress responses betweenArabidopsis thaliana ecotypes. Plant Cell Environ. 29:754–63
91. Kalbina I, Strid A. 2006. The role of NADPH oxidase and MAP kinase phosphatase in UV-B-dependentgene expression in Arabidopsis. Plant Cell Environ. 29:1783–93
92. Khare M, Guruprasad KN. 1993. UV-B-induced anthocyanin synthesis in maize regulated by FMN andinhibitors of FMN photoreactions. Plant Sci. 91:1–5
93. Transcriptomicstudy showing theoverlap between UV-Band stress-induced geneexpression.
93. Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, et al. 2007. The AtGenExpress global stressexpression data set: protocols, evaluation and model data analysis of UV-B light, drought andcold stress responses. Plant J. 50:347–63
428 Jenkins
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
94. Kim BC, Tennessen DJ, Last RL. 1998. UV-B-induced photomorphogenesis in Arabidopsis thaliana. PlantJ. 15:667–74
95. Kliebenstein DJ, Lim JE, Landry LG, Last RL. 2002. Arabidopsis UVR8 regulates UV-B signal trans-duction and tolerance and contains sequence similarity to human Regulator of Chromatin Condensation 1.Plant Physiol. 130:234–43
96. Kouzarides T. 2007. Chromatin modifications and their function. Cell 128:693–70597. Kucera B, Leubner-Metzger G, Wellmann E. 2003. Distinct UV-signaling pathways in bean leaves.
DNA damage is associated with β-1,3-glucanase gene induction, but not with flavonoid formation. PlantPhysiol. 133:1445–52
99. Langcake P, Pryce RJ. 1977. The production of resveratrol and the viniferins by grapevines in responseto UV radiation. Phytochemistry 16:1193–96
100. Li B, Carey M, Workman JL. 2007. The role of chromatin during transcription. Cell 128:707–19101. Li JY, Oulee TM, Raba R, Amundson RG, Last RL. 1993. Arabidopsis flavonoid mutants are hypersen-
sitive to UV-B irradiation. Plant Cell 5:171–79102. Liakopoulos G, Stavrianakou S, Karabourniotis G. 2006. Trichome layers versus dehaired lamina of Olea
europaea leaves: differences in flavonoid distribution, UV-absorbing capacity, and wax yield. Environ. Exp.Bot. 55:294–304
103. Logemann E, Hahlbrock K. 2002. Crosstalk among stress responses in plants: pathogen defense overridesUV protection through an inversely regulated ACE/ACE type of light-responsive gene promoter unit.Proc. Natl. Acad. Sci. USA 99:2428–32
104. Logemann E, Tavernaro A, Schulz W, Somssich IE, Hahlbrock K. 2000. UV light selectively coin-duces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.Proc. Natl. Acad. Sci. USA 97:1903–7
105. Long JC, Jenkins GI. 1998. Involvement of plasma membrane redox activity and calcium homeostasisin the UV-B and UV-A/blue light induction of gene expression in Arabidopsis. Plant Cell 10:2077–86
106. Loyall L, Uchida K, Braun S, Furuya M, Frohnmeyer H. 2000. Glutathione and a UV light-induced glu-tathione S-transferase are involved in signaling to chalcone synthase in cell cultures. Plant Cell 12:1939–50
107. Mazza CA, Boccalandro HE, Giordano CV, Battista D, Scopel AL, Ballare CL. 2000. Functional signif-icance and induction by solar radiation of UV-absorbing sunscreens in field-grown soybean crops. PlantPhysiol. 122:117–26
108. McKenzie RL, Bjorn LO, Bais A, Ilyasd M. 2003. Changes in biologically active UV radiation reachingthe earth’s surface. Photochem. Photobiol. Sci. 2:5–15
109. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. 2004. Reactive oxygen gene network of plants.Trends Plant Sci. 9:490–98
110. Moore JD. 2001. The ran-GTPase and cell-cycle control. Bioessays 23:77–85111. Ng YL, Thimann KV, Gordon SA. 1964. The biogenesis of anthocyanins. X. The action spectrum for
anthocyanin formation in Spirodela oligorrhiza. Arch. Biochem. Biophys. 107:550–58112. Identified thepositive function ofCOP1 in UV-Bresponses.
112. Oravecz A, Baumann A, Mate Z, Brzezinska A, Molinier J, et al. 2006. CONSTITUTIVELYPHOTOMORPHOGENIC1 is required for the UV-B response in Arabidopsis. Plant Cell
18:1975–90113. Paul ND, Gwynn-Jones D. 2003. Ecological roles of solar UV radiation: towards an integrated approach.
Trends Ecol. Evol. 18:48–55114. Piazza P, Procissi A, Jenkins GI, Tonelli C. 2002. Members of the c1/pl1 regulatory gene family mediate
the response of maize aleurone and mesocotyl to different light qualities and cytokinins. Plant Physiol.128:1077–86
115. Predieri S, Krizek DT, Wang CY, Mirecki RM, Zimmerman RH. 1993. Influence of UV-B radiation ondevelopmental changes, ethylene, CO2 flux and polyamines in cv. Doyenne d’Hiver pear shots grown invitro. Physiol. Plant. 87:109–17
116. Poulson ME, Boeger MRT, Donahue RA. 2006. Response of photosynthesis to high light and droughtfor Arabidopsis thaliana grown under a UV-B enhanced light regime. Photosynth. Res. 90:79–90
www.annualreviews.org • UV-B Signaling 429
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
117. Ramani S, Chelliah J. 2007. UV-B-induced signaling events leading to enhanced-production of catha-ranthine in Catharanthus roseus cell suspension cultures. BMC Plant Biol. 7:61
119. Renault L, Nassar N, Vetter I, Becker J, Klebe C, et al. 1998. The 1.7 A crystal structure of the regulatorof chromatin condensation (RCC1) reveals a seven-bladed propeller. Nature 392:97–101
120. Rozema J, van de Staaij J, Bjorn LO, Caldwell M. 1997. UV-B as an environmental factor in plant life:stress and regulation. Trends Ecol. Evol. 12:22–28
121. Safrany J, Haasz V, Mate Z, Ciolfi A, Feher B, et al. 2008. Identification of a novel cis-regulatory elementfor UV-B-induced transcription in Arabidopsis. Plant J. 54:402–14
122. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. 2004. Molecular mechanisms of mammalianDNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73:39–85
123. Sato T, Ueda T, Fukuta Y, Kumagai T, Yano M. 2003. Mapping of quantitative trait loci associated withUV-B resistance in rice (Oryza sativa L.). Theor. Appl. Genet. 107:1003–8
124. Savenstrand H, Brosche M, Strid A. 2004. Ultraviolet-B signalling: Arabidopsis brassinosteroid mutantsare defective in UV-B regulated defence gene expression. Plant Physiol. Biochem. 42:687–94
125. Scarpeci TE, Zanor MI, Carrillo N, Mueller-Roeber B, Valle EM. 2008. Generation of superoxide anionin chloroplasts of Arabidopsis thaliana during active photosynthesis: a focus on rapidly induced genes. PlantMol. Biol. 66:361–78
126. Shinkle JR, Atkins AK, Humphrey EE, Rodgers CW, Wheeler SL, Barnes PW. 2004. Growth and mor-phological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonousseedlings. Physiol. Plant. 120:240–8
127. Shinkle JR, Derickson DL, Barnes PW. 2005. Comparative photobiology of growth responses to twoUV-B wavebands and UV-C in dim-red-light- and white-light-grown cucumber (Cucumis sativus)seedlings: physiological evidence for photoreactivation. Photochem. Photobiol. 81:1069–74
128. Stapleton AE, Walbot V. 1994. Flavonoids can protect maize DNA from the induction of UV-radiationdamage. Plant Physiol. 105:881–89
129. Steinmetz V, Wellmann E. 1986. The role of solar UV-B in growth regulation of cress (Lepidium sativumL.) seedlings. Photochem. Photobiol. 43:189–93
130. Steinmuller D, Tevini M. 1985. Action of UV radiation (UV-B) upon cuticular waxes in some crop plants.Planta 164:557–64
131. Stratmann J. 2003. Ultraviolet-B radiation co-opts defense signaling pathways. Trends Plant Sci. 8:526–33132. Stratmann J, Scheer J, Ryan CA. 2000. Suramin inhibits initiation of defense signaling by systemin,
chitosan, and a β-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells. Proc. Natl. Acad.Sci. USA 97:8862–67
133. Suesslin C, Frohnmeyer H. 2003. An Arabidopsis mutant defective in UV-B light-mediated responses.Plant J. 33:591–601
134. Surplus SL, Jordan BR, Murphy AM, Carr JP, Thomas B, A-H-Mackerness S. 1998. Ultraviolet-B-induced responses in Arabidopsis thaliana: role of salicylic acid and reactive oxygen species in the regula-tion of transcripts encoding photosynthetic and acidic pathogenesis-related proteins. Plant Cell Environ.21:685–94
135. Takeda J, Abe S. 1992. Light-induced synthesis of anthocyanin in carrot cells in suspension. IV. Theaction spectrum. Photochem. Photobiol. 56:69–74
136. Takeda J, Ozeki Y, Yoshida K. 1997. Action spectrum for induction of promoter activity of phenylalanineammonia-lyase gene by UV in carrot suspension cells. Photochem. Photobiol. 66:464–70
137. Taki N, Sasaki-Sekimoto Y, Obayashi T, Kikuta A, Kobayashi K, et al. 2005. 12-Oxo-phytodienoicacid triggers expression of a distinct set of genes and plays a role in wound-induced gene expression inArabidopsis. Plant Physiol. 139:1268–83
138. Tevini M, Teramura AH. 1989. UV-B effects on terrestrial plants. Photochem. Photobiol. 50:479–87139. Torabinejad J, Caldwell MM. 2000. Inheritance of UV-B tolerance in seven ecotypes of Arabidopsis
thaliana L. Heynh. and their F-1 hybrids. J. Hered. 91:228–33
430 Jenkins
Ann
u. R
ev. P
lant
Bio
l. 20
09.6
0:40
7-43
1. D
ownl
oade
d fr
om a
rjou
rnal
s.an
nual
revi
ews.
org
by U
nive
rsity
of
Sevi
lla o
n 05
/04/
09. F
or p
erso
nal u
se o
nly.
ANRV375-PP60-19 ARI 8 April 2009 16:30
140. Highlighteddifferential geneexpression in differentUV-B wavelengths andthe role of HY5.
140. Ulm R, Baumann A, Oravecz A, Mate Z, Adam E, et al. 2004. Genome-wide analysis of geneexpression reveals function of the bZIP transcription factor HY5 in the UV-B response ofArabidopsis. Proc. Natl. Acad. Sci. USA 101:1397–402
141. Ulm R, Nagy F. 2005. Signalling and gene regulation in response to UV light. Curr. Opin. Plant Biol.8:477–82
142. Vanderauwera S, Zimmermann P, Rombauts S, Vandenabeele S, Langebartels C, et al. 2005. Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-inducedtranscriptional cluster involved in anthocyanin biosynthesis. Plant Physiol. 139:806–21
143. Wade HK, Bibikova TN, Valentine WJ, Jenkins GI. 2001. Interactions within a network of phytochrome,cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression inArabidopsis leaf tissue. Plant J. 25:675–85
144. Wang ZY, He JX. 2004. Brassinosteroid signal transduction—choices of signals and receptors. TrendsPlant Sci. 9:91–96
145. Wellmann E. 1983. UV radiation in photomorphogenesis. In Encyclopedia of Plant Physiology, New Series,ed. OL Lange, PS Nobel, CB Osmond, H Ziegler, 16B:745–56. New York: Springer
146. Xu C, Sullivan JH, Garrett WM, Caperna TJ, Natarajan S. 2006. Impact of solar UV-B on the proteomein soybean lines differing in flavonoid contents. Phytochemistry 69:38–48
147. Yalamanchili RD, Stratmann JW. 2002. Ultraviolet-B activates components of the systemin signalingpathway in Lycopersicon peruvianum suspension-cultured cells. J. Biol. Chem. 277:28424–30
149. Yatsuhashi H, Hashimoto T, Shimizu S. 1982. Ultraviolet action spectrum for anthocyanin formationin broom sorghum first internodes. Plant Physiol. 70:735–41
150. Yi C, Deng XW. 2005. COP1—from plant photomorphogenesis to mammalian tumorigenesis. TrendsCell Biol. 15:618–25
151. Zhao J, Zhang W, Zhao Y, Gong X, Guo L, et al. 2007. SAD2, an importin β-like protein, is requiredfor UV-B response in Arabidopsis by mediating MYB4 nuclear trafficking. Plant Cell 19:3805–18
Seminis (Oxnard, CA, USA) announced thatit would eliminate 2000 varieties or 25% of itstotal product line as a cost-cutting measure.Seminis, a subsidiary of the Mexican con-glomerate Savia and controlling nearly a fifthof the worldwide fruit and vegetable seedmarket, gained .8000 varieties comprising60 species of fruits and vegetables by acquiringa dozen or so seed companies, most notably, thegarden seed division of Asgrow, Petoseed andRoyal Sluis. The company has 79 issued orallowed patents, and is seeking patents relatedto beans, bean sprouts, broccoli, cauliflower,celery, corn, cucumber, eggplant, endive, leek,lettuce, melon, muskmelon, onion, peas, pumpkin,radish, red cabbage, spinach, squash, sweetpepper, tomato, watermelon and white cabbage.
Agritope analysesphytochemicals
Agritope, Inc. (Portland, OR, USA) is developingnew plant varieties containing increasedlevels of naturally occurring phytochemicals.Agritope’s MetaGene™ Metabolic GenomicsTechnology facilitates the identification ofspecific genes that regulate the levelsof phytochemicals such as carotenoids,lycopene, flavonoids, isoflavones, vitamins,folic acid and various elements and minerals.
Rice genome newsA new website http://www.rice-research.orgprovides access to the Monsanto Rice GenomeSequence data at no charge. Monsanto is collaborating with the International RiceGenome Sequencing Project (IRGSP) to com-plete and publish the entire genome sequence.Information on the IRGSP program can befound at http://rgp.dna.affrc.go.jp/Seqcollab.html
Fusarium consideredto kill coca plants
Colombia has reluctantly agreed to take stepsin developing a Fusarium oxysporum myco-herbicide against the coca and heroin-poppyfields. A proposal will be made to the UnitedNations that would include testing for the pres-ence of the fungus in coca. Environmentalistsand other activists in both countries object toany field tests of the fungus, arguing thatit is virtually a biological weapon – one thatmight upset Colombia’s ecology or endan-ger farmers, animals and food crops.
New generationselection genes
Marker genes enable scientists to select a raretransformed plant cell after co-introducing thedesired gene along with the marker. Beyond thelaboratory, these markers have no role and thustheir presence in crops and food has provokedmuch public concern, especially because thesegenes either code for antibiotic or herbicide resis-tance. Solutions that have been proposed includethe removal of marker genes using the Cre-loxsystem or transposable elements, or new positiveselectable systems. The manAgene encoding PMIwas cloned from E. coli by researchers at DaniscoBiotechnology (Copenhagen, Denmark) Plantcells transformed with this gene can uniquelyconvert mannose-6-phosphate to fructose-6-phosphate, which is then easily metabolized. Anincreased transformation efficiency might occurbecause transformed cells are actively encouragedto grow rather than just allowed to survive.Researchers interested in obtaining the manAgene or who require more information aboutthis Positech system can contact: Andy Beadleat [email protected]://www.seedquest.com/News/releases/europe/Novartis/n2687.htm
UV-B susceptibilityin plants
Barbara Hohn and colleagues (FriedrichMiescher Institute, Basel, Switzerland) reportthat excessive UV-B radiation damages DNAin plants and stunts their growth, that the plants’susceptibility accumulates over generationsand could worsen if the depletion of the earth’sprotective ozone layer continues at its currentpace. http://www.fmi.ch/reports/Hohnb.htm
Fusarium control byFusarium
To control Fusarium wilt disease in an environmentally friendly way, beneficial strainsof Fusarium are being used to control plantpathogenic strains of Fusarium oxysporum.Deborah R. Fravel (Agriculture ResearchService, Beltsville, MD, USA) and GeorgeLazarovits (Agriculture and Agri-Food Canada,Ontario, Canada) found one strain, CS-20,which reduced wilt by 49.6%. Mixing benefi-cial strains of a fungus (Trichoderma virensstrain G1-3) and a bacterium (Burkholderia viet-namiensis strain Bc-F) also reduced wilt inci-dence by 41.6%. The mechanism of biocontrolmight rely on induced systemic resistance.http://www.ars.usda.gov/is/pr/2000/000714.htm
Floral scent researchImproving floral scent is one of the goals of the $20billion per year horticulture industry. The aromaof a flower can contain as few as seven differentoils, such as in snapdragon (Antirrhinum majus),or as many as 100 such as in orchids. In snapdragonflowers, the volatile ester methyl benzoate is themost abundant scent compound. Natalia Dudareva(Purdue University, West Lafayette, IN, USA) iso-lated a novel S-adenosyl-L-methionine:benzoicacid carboxyl methyltransferase (BAMT), the finalenzyme in the biosynthesis of methyl benzoate,and characterized its corresponding cDNA.www.plantcell.org/cgi/content/full/12/6/949;www.hort.purdue.edu/hort/people/faculty/dudareva.html
Internet news media, edited by Gert E. de Vries.
Plants that produce hydrogen cyanide gas toprotect themselves against predators can do soby the enzymatic breakdown of a class ofcompounds known as cyanogens, such ascyanogenic glycosides. In a study by HeleneEngler and colleagues (University of Texas,Austin, TX, USA), a neotropical butterfly,Heliconius sara, can avoid the harmful effectsof the cyanogenic leaves of Passiflora auricu-lata (passion vine) by a a unique enzymaticconversion. The mechanistic details of thispathway might suggest new ways to makecyanogenic crops more useful as a food source.http://uts.cc.utexas.edu/~gilbert/research/butterflies/
Oxidative Stress andRedoxSignalling in PlantsPer Muhlenbock, Stockholm University, Stockholm, Sweden
Barbara Karpinska, University College of Sodertorns, Huddinge, Sweden
Stanislaw Karpinski, Institute of Plant Physiology, Polish Academy of Sciences, Krakow,
Poland
Plants are due to their constitution unable to escape from environmental stress and are
constantly at the risk of being exposed to several stress factors simultaneously that
usually are associatedwith oxidative stress. We are presenting some genetic, molecular
and physiological examples that plants functionally integrate varieties of intra- and
intercellular signalling in response to such stresses. It is concluded that fine control of
cellular redox homeostasis as a function of the interaction of hormones and reactive
oxygen species (ROS) signalling is important for integrated regulation of plant defence
and acclimatory responses.
Reactive Oxygen Species
Reactive oxygen species (ROS) such as singlet oxygen,superoxide anion and hydrogen peroxide are formed as adirect consequence of diverse biochemical processes inmany subcellular compartments (Gechev et al., 2006).ROSare highly reactive molecules and may interact with a widevariety of other molecules such as deoxyribosenucleic acid(DNA), pigments, lipids, proteins and other essential cel-lular molecules which lead to a destructive chain of events(Lamb andDixon, 1997). Overreduction of the respiratorychain leads to ROS in the mitochondrion. Amine oxidases,cell wall peroxidases and extracellular peroxidases areapoplastic ROS producers and are involved in severalstress responses (Bolwell et al., 2002; Kawano, 2003). Thelargest producer of ROS during the photoperiod in plantcells is the chloroplast together with the peroxisome duringphotosynthesis and photorespiration, respectively (Asada,1999; Foyer and Noctor, 2005).
At optimal levels, ROS perform important housekeep-ing functions (Lam, 2004; Mori and Schroeder, 2004) butROS are also the major players in processes ranging fromregulation of cell cycle, acclimatory and defence responsesand programmed cell death (PCD) (Dat et al., 2003; Laloiet al., 2004; Gechev et al., 2006). The ability of the plant totolerate different levels of ROS is dependent on the effi-ciency of the plants antioxidant systems and ultimately oncellular metabolism (Couee et al., 2006).
Antioxidant Systems
Antioxidants can be generally divided into enzymatic,nonenzymatic and water or lipid soluble. Carotenoids,xanthophylls and tocopherols are examples of low-molecular-weight lipid soluble nonenzymatic antioxidants.
Glutathione (GSH) and ascorbate (AsA) are the majorlow-molecular-weight water-soluble antioxidants andmake up the foundation of the redox control within thecellular compartments, since they provide the ability toscavenge ROS and change the redox status of the cell (Ballet al., 2004; Gomez et al., 2004; Mateo et al., 2004).Various enzymes that catalyse ROS scavenging reac-
tions use AsA and GSH as cofactors (Allan and Fluhr,1997; Foreman et al., 2003; Lund et al., 1998; Wingsle andKarpinski, 1996). These enzymes have different subcellularlocalizations and their activity influence redox states of thedifferent subcellular compartments. Redox signals regulateprocesses like metabolism, morphology and development(Foyer and Noctor, 2005) and it has been suggested thatcellular redox changes contribute to the signal transductionthat results from excess excitation energy (EEE) stress(Karpinski et al., 1997, 1999; Karpinska et al., 2000; Ballet al., 2004). Cellular redox homeostasis is regulated bya tight equilibrium between ROS producing and ROS-scavenging reactions (Foyer and Noctor, 2005). In redoxreactions, cystein residues of proteins play a very importantfunction since their thiol groups can easily be oxidized orreduced under physiological conditions (Cooper et al.,2002). Redox reactions between thiols are the basis for theenergy transfers of the antioxidant regeneration systemsand they also serve as regulators of gene expression andprotein functions in several processes such as in PCD(Cooper et al., 2002).
Photooxidative Stress
One of the major fluctuating factors in the natural envi-ronment is the amount of incident light. The amount of
Article Contents
Advanced article
. Reactive Oxygen Species
. Antioxidant Systems
. Photooxidative Stress
. Cell Death in Response to Oxidative Stress in Plants
. Regulation of Cell Death in Response to EEE
. Hormonal Regulation of Stress Responses
. Ethylene
. Salicylic Acid
. Auxin
. Mutants in Arabidopsis with Deregulated Oxidative
Stress Responses and PCD
. LSD1 Integrates Chloroplastic EEE Signals
. SAA is Associated with HR-like Cell Death
. Conclusions
. Acknowledgements
doi: 10.1002/9780470015902.a0020135
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absorbed light energy in excess of what is needed for pho-tosynthetic metabolism, termed EEE, causes increasedproduction of ROS (Demmig-Adams and Adams, 1992;Mullineaux and Karpinski, 2002).
When plants are exposed to high light intensities, anoverreduction of the photosynthetic electron carriers oc-cur. Then, the excitation energy that is absorbed by thechlorophyll in light-harvesting complexes leads to in-creased production of electrons from the water-splittingcomplex, and a subsequent increase in formation of ROS(Asada, 1999; Karpinski et al., 1999). EEE usually resultsfrom increasing light intensities, but it can also be gener-ated at low light intensities during different stresses whichlimit carbon dioxide supply, such as drought, chilling orfreezing stress and pathogen infection (Mullineaux andKarpinski, 2002). The ensuing production of ROS mayprovoke photoinhibition (decreased efficiency in photo-synthesis) or if prolonged, also permanent photodamage(Karpinski et al., 1999; Niyogi, 1999). The purpose of lightacclimation is therefore mainly focused on avoiding andcontrolling EEE. Therefore, plants have evolved severalEEE avoidance mechanisms such as rapid leaves andchloroplasts movements that reduced absorption of light(e.g. Karpinski et al., 2003).
The main EEE direct dissipatory processes can be di-vided into either photochemical or nonphotochemicalquenching processes (NPQ). NPQ processes directlyquench EEE and disperse it in the form of heat with thehelp of xanthophylls and other carotenoids. The photo-chemical quenching processes provide alternative electronsinks along the photosynthetic electron transport, mainlythrough the Mehler reaction, water–water cycle and pho-torespiration (Willekens et al., 1997; Asada, 1999; Ort,2001; Mullineaux and Karpinski, 2002). Photorespirationand the water–water cycle lead to a higher production ofH2O2 in the peroxisome and the chloroplast, respectively(Willekens et al., 1997; Asada, 1999; Mateo et al., 2004).
Long-term acclimation processes in leaves consist ofadjustment and optimizing of light absorbance by PSII andPSI antennae, optimizing levels of the antioxidants such asglutathione (GSH) and ascorbate (AsA) (Willekens et al.,1997; Bailey et al., 2001; Karpinski et al., 2003; Tausz et al.,2004;Walters, 2005), optimizing levels of the componentsofphotosynthetic electron transport (morphological changesin the chloroplasts structure) and induction of stressproteins such as, for example, the early light inducible pro-teins (ELIPs) (Heddad and Adamska, 2000; Walters, 2005;Becker et al., 2006). Distant parts of the plant that are notdirectly exposed to EEE also induce acclimation responsesby optimizing antioxidant defences and by adjustment andoptimizing photosynthetic electron transport efficiency.This response is termed systemic acquired acclimation(SAA) (Karpinski et al., ). Stomatal conductance, whichis precisely regulated by ROS, hormones, carbondioxide concentration, temperature, water pressure andlight quality, is particularly important in the regulation ofphotosynthesis and responses to EEE. This is because theenzyme that catalyses the first reaction of the Calvin cycle,
Rubisco, also has a high affinity to oxygen. Closure ofstomata can therefore induce photorespiration by limitingthe availability of carbon dioxide in comparison to oxygen(Wingler et al., 2000; Noctor et al., 2002; Fryer et al., 2003).Because of this, activities of many enzymes of the Calvincycle are regulated by redox changes in the photosyntheticelectron transport (Kaiser, 1979).Plants perceive EEE stress through different redox sen-
sors and secondary redox changes (Escoubas et al., 1995;Pfannschmidt et al., 1999; Mullineaux and Karpinski,2002; Karpinski et al., 2003). Redox changes in theplastoquinonepool, in other photosynthetic electron trans-port components and other redox chloroplastic sensorsseems to bemore dominant in the retrograde chloroplast tonucleus signalling and responses to EEE, whereas lightsensing proteins such as phytochromes, cryptochromesand phototropins regulate processes such as germination,plant circadian rhythms, shadow avoidance, chloroplastmovements, phototropism, seasonal acclimation and de-velopment changes (Escoubas et al., 1995; Pfannschmidtet al., 1999; Smith, 2000; Karpinski et al., 2003).
Cell Death in Response to OxidativeStress in Plants
Many environmental stresses may induce cell death symp-toms. Abiotic stresses induce lesions, physiological leafspots and accelerated senescence (Buchanan-Wollastonet al., 2003;Wu and vonTiedemann, 2004). Themajor partof these types of cell death are considered to be necrotic butvery few studies present data on this topic (Huh et al., 2002;Wu and von Tiedemann, 2004). Aerenchyma formationand accelerated senescence have been shown to be associ-ated with attributes of PCD in relation to abiotic stresses(Munne-Bosch and Alegre, 2004). Biotic stresses inducecell death during development of disease symptoms or asthe result of specific plant pathogen interactions, so calledgene-for-gene interactions (McDowell and Dangl, 2000).Plants have evolved several means of defence against
pathogen attack that involve genetically induced signallingpathways leading to the formation of antimicrobial com-pounds, strengthening of cell walls, stomata closure andPCD (McDowell and Dangl, 2000). An example of plants’biotic defences is the gene-for-gene induction of the hyper-sensitive response (HR), a burst of ROS leading to theinduction of PCD during which a limited number of cellsdie at the site of pathogen infection (Lamb and Dixon,1997). This process is accompanied by a set of defence re-actions, including activation of defence genes and the onsetof systemic acquired resistance (SAR) (Lamb et al., 1989).The properties of HR vary between different plant–pathogen interactions (Heath, 2000) but HR has been pro-posed to be similar to apoptosis in animals with hallmarkssuch as Ca2+ signals, extracellular production of ROS byreduced form of nicotinamide adenosine diphosphate(NADPH) oxidases, activation of a genetic programme,
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cell shrinkage and DNA laddering (McDowell and Dangl,2000). However, comparative studies rather point to thatboth disease development and HR responses have somedegree of necrosis and PCD (Heath, 2000; Beers andMcDowell, 2001; Dat et al., 2003; Greenberg and Yao,2004). Additionally, not all kinds of PCD in animals areapoptotic and this range of phenotypes of different kinds ofcell death has led to the idea of a continuous spectrumbetween PCD and necrosis (Schwartz et al., 1993; Levinet al., 1999). Then, why do plants have a system to inducecell death in response to environmental stresses if the cellsmay die anyway?HRand lysogenic aerenchyma formationin response to roots hypoxia are both typical examples ofthe altruistic purpose of environmentally induced PCD.HR is thought to prevent pathogens from proliferatingand aerenchyma supplies suffocating roots with oxygenalthough the actual benefit of this has beendifficult to provein vivo (Hammond-Kosack and Jones, 1996). Alterna-tively, altruistic PCD in animals proves that this process isresponsible for removing cells that have become dangerousor malignant, thereby contributing to an increased fitnessof the organism (Jacobson et al., 1997; Gilchrist, 1998;Johnstone et al., 2002). Importantly, PCD also preventsnecrosis and the dangers of infection and toxicity thatresult from necrotic tissues (Davies, 2000; Munne-Boschand Alegre, 2004). There are many examples of altruisticcell death in developmental PCD (e.g. flower senescence,xylem formation, developmental aerenchyma, aleuron andendosperm cell death, dioic cell death, etc.) and in PCDevoked as a result of householding functions (root cap celldeath, petal senescence, etc.) (Jones, 2001; Kuriyama andFukuda, 2002). Since developmental cell death is outside ofthe scope of this article, however, only environmental celldeath will be considered further.
Regulation of Cell Death in Responseto EEE
Several environmental factors have been shown to influ-ence the signalling pathways of PCD (Gan and Amasino,1997). Studies have shown, that EEE may have a chlorop-last-dependent signalling effect on plant PCD (Samuilovet al., 2003; Mateo et al., 2004; Bechtold et al., 2005). ROSare necessary for the induction of cell death pathways andplant respiratory burst homologues to the animalNADPHoxidases have been shown to be active in the plant PCDpathways (Lamb and Dixon, 1997; Van Breusegem andDat, 2006). ROS-dependent cytochrome c release, a hall-mark trait of animal PCD has also been shown in plants(Vacca et al., 2006). Another molecule involved in PCD isnitric oxide (NO) that acts both as an oxidant and anti-oxidant in biotic and abiotic stress responses (Delledonneet al., 2001; Beligni et al., 2002; Zago et al., 2006).
Several hormones and signalling components such asethylene (ET), salicylic acid (SA), jasmonic acid (JA) andabscisic acid (ABA) have also been shown to control PCD
(Kangasjarvi et al., 2005; Overmyer et al., 2005). In oneproposed model, SA and ET have been suggested tocooperate for the induction and spreading of the PCD. Inother studies, auxins (indole acetic acid, IAA) have alsobeen shown to regulate cell cycle and cell death (Kovtunet al., 2000).Theproposed effect of light andofETas agentscontributing to the spreading of PCDmentioned above hasnever been shown in correlation although it is known thatET can be induced by several kinds of stresses (MehlhornandWellburn, 1987). Importantly, also redox signals play afundamental part in PCD (Foyer and Noctor, 2005).
Hormonal Regulation of StressResponses
ABA, JA, ET, SA and IAA are key hormonal players instress responses and there is a considerable amount of cross-talk between the signal transduction pathways that they in-itiate (Hirt, 2000; Kovtun et al., 2000; Glazebrook, 2001;Jonak et al., 2002; Bostock, 2005). Chloroplasts could be asite for this regulation since many stress-response hormonesare produced entirely or partially in the chloroplast-locatedpathways (Mauch-Mani and Slusarenko, 1996; CreelmanandMullet, 1997;Mullineaux andKarpinski, 2002).ABA isawell-documented inducer of stomata closure and regulatorof bothabiotic andbiotic defence responses (Bostock, 2005).JA is a signallingmolecule derived from linoleic acid and
the biosynthetic pathway for this compound containsbiologically active intermediates (Stintzi andBrowse, 2000;Kachroo et al., 2001). JA has roles in stress, developmentand PCD and interferes both with ET and IAA (Tiryakiand Staswick, 2002; Turner et al., 2002; Kangasjarvi et al.,2005). However, it has recently been shown that theJA-mediated signalling is not involved in regulation ofASCORBATE PEROXIDASE 2 gene expression (Changet al., 2004) which depends on specific redox changes in thechloroplast.
Ethylene
ET plays vital roles in several aspects of plant growth anddevelopment and is a particularly important regulator ofstress responses (Wang et al., 2002). It is synthesized via aclearly defined and tightly regulated pathway that respondsto several developmental and environmental stimuli. Fur-thermore, ET has been reported to be an important regu-latorof cell death (HadfieldandBennett, 1997), aerenchymaformation (Drew et al., 2000), disease development and inROS-induced cell death (Kangasjarvi et al., 2005). Addi-tionally, ET interacts with SA signalling indicating that itplays an important role also in biotic stress responses. ET istightly regulated together with ROS (Moeder et al., 2002;Kangasjarvi et al., 2005) and it has been shown that ETmay be necessary for H2O2 release during PCD and that itamplifies the oxidative burst in plant–pathogen interactions
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(Lawton et al., 1994;Chamnongpol et al., 1998;Kangasjarviet al., 2005). It was also reported that ethylene can inhibitphotosynthesis (Kays and Pallas, 1980) and that ET signal-ling may be affected by nutrient status and leaf age. Thesereports indicate an important function of ET in controllingcrosstalk between EEE and PCD signalling pathways.
Salicylic Acid
SA is a compound that can be found in all parts of a plantwhere it has a diversity of functions. It is a phenyl-propanoid derived from phenylalanine from the Shikimatepathway in the chloroplast (Mauch-Mani and Slusarenko,1996). SA is considered to be one of themost determinativeplant hormones in biotic interactions since it is required forSAR. However, it has also been reported that biotic stressresistance is controlled by both SA-dependent and SA-in-dependent pathways, and that the SA-independent path-way may be regulated by both JA and ET (Clarke et al.,2000; Overmyer et al., 2000).
Theamplificationof theoxidativeburst of biotic defencesis dependent on increases in SA and it has been proposedthat SAandROS function as a feedback loop sinceROSarealso involved in key steps of the SA synthesis (Lamb andDixon, 1997; VanCamp et al., 1998). Both SAandROS arerequired to induce HR (McDowell and Dangl, 2000) andCAT (Catalaze) and APX (Ascorbate peroxidase) tran-scriptionwas inhibitedbySA.Consequently, a large varietyof mutants have lesion phenotypes indicating that there is atight link between SA and general cell death responses(Lorrain et al., 2003). Additionally, it has been shown thatSA signalling is affected by EEE and that SA effects bothphotosynthesis and stomatal conductance (Karpinski et al.,2003;Zeier et al., 2004) and itwas shown that SA is involvedin long-term light acclimatory processes and regulation ofcellular redox homeostasis (Karpinski et al., 2003). Theseobservations suggest that SA is important molecule
involved in crosstalk between the signalling pathways ofacclimatory and defence responses. Recently, it was dem-onstrated that SA, GSH and hydrogen peroxide are genet-ically and functionally linked (Mateo et al., 2006).SA also causes stomatal closure (Figure 1a), inhibition of
photosynthesis, long-term EEE acclimation and inducesthe uncontrollable cell death in lsd1 (Jabs et al., 1996;Moriet al., 2001; Karpinski et al., 2003). In Mateo et al. (2006)paper we showed that mutants that are inhibited in SAproduction are unable to acclimate toEEE, underlining theimportance of SA in acclimation responses.We also showed that SA contributes to lesion formation
in combination with EEE and that acclimation to EEE canrevert this lesion formation (Figure 1b). The growth retar-dation in the SA mutants can be reversed by transferringplants to higher light intensities (Mateo et al., 2006) andhigh SA levels are correlated with reduced maximum effi-ciency of photosynthesis and increased respiration rates inthe leaves. These data indicate that SA induces EEE relatedstress and photorespiration. Previously, it has been shownthat SA may reduce ROS scavenging by inhibiting CAT(Chen et al., 1993). We therefore analysed H2O2 produc-tion in several lines of mutants that overproduce SA andsome that have reduced levels of SA. This analysis showedthat H2O2 production is high in SA accumulating mutantsand low in those that are SA-deficient (Figure 2a).We also found a clear link between the levels of SA and
GSH through the regulation of glutathione reductase (GR)activity (Figure 2b). Since high levels of GSH were also as-sociatedwith EEEacclimation (Karpinski et al., 1997), thisindicates that the observed increase in ROS may be due toEEE.Additionally, we andothers have shown thatLSD1 isregulated by GSH (Senda and Ogawa, 2004) and that theLSD1 protein, in turn, regulates important enzymes suchas CuZnSOD, CAT1 and NADPH thioredoxin reductase(NTR) (Mateo, 2005). The lsd1, eds1 and pad4mutants canbe considered to be conditional regulators of SA, this isbecause when the plant is exposed to biotic stress the gene
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Figure 1 SA impairs acclimation to EEE in low light acclimated plants. (a) Relative stomatal conductance (RSC) inwild-type leaves of rosette grown in SD treated
with SA (0.4mM) in comparison to control leaves treatedwithwater (p50.001***). (b) Low light (LL)- andhigh light (HL) acclimated leaves treatedwith 0.4mM
SA for several hours and exposed to excess light (EL; 2200+200uE, 90min exposure). From Mateo et al. (2004).
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functions are to inhibit (lsd1) or promote (eds1 and pad4)the SA pathway (Kliebenstein et al., 1999; Wiermer et al.,2005).We propose that this is reflected in the production ofH2O2 in lsd1, eds1 and pad4mutants when exposed to EEE(Figure 1) and conclude that LSD1 constitutes a rheostat ofEEE-induced ROS and redox signalling that consequentlycontributes to the regulation of SA.
Auxin
Auxins, together with cytokinins, differ from other plantsignalling molecules in that they are required for cell viabil-ity. The prevalent form of auxin is IAAwhich is synthesizedfrom tryptophan through the action of several pathways.Auxin signalling takes place through transportation andbinding/release of active IAA, Ca2+ signalling and protongradients. Recently, the enigmatic and illusive receptor forauxinwas identified inArabidopsis, a significant advance forthe study of this intriguing hormone (Kepinski and Leyser,2005). Auxins are involved in responses such as cell elon-gation, stomatal opening, plant growth, hypoxia responsesand root formation (Gehring et al., 1998; Guilfoyle et al.,1998;Reed, 2001). They are also involved in stress responsesand in the regulation of ROS homeostasis (Kovtun et al.,2000;GuanandScandalios, 2002;Winkel-Shirley, 2002; Jooet al., 2005). Moreover, auxins interact with ET signallingand are required for stress ET production (Mehlhorn andWellburn, 1987; Romano et al., 1993). Importantly, auxinsare also required for cell cycle progression, and recently ithas been reported that they may control PCD and bioticdefence responses (Hirt, 2000;KuriyamaandFukuda, 2002;Gechev et al., 2004).
The regulation by auxins illustratively exemplifies thecomplex situation of homeostatic control of inter- and in-tracellular signals in plants. Depending on tissue, hormonelevels and environmental signals, auxins together withother signalling compounds exert different effects in differ-ent physiological and developmental situations. It is
therefore clear that a certain balance (homeostasis) of sig-nalling molecules is required for different cellular redoxstates or responses (Voesenek and Blom, 1996; O’Donnellet al., 2003).
Mutants in Arabidopsis withDeregulated Oxidative StressResponses and PCD
In Arabidopsis, several mutants have been isolated for de-regulation of genes that are involved in ROS and hormonesignalling and control of PCD (Overmyer et al., 2000; Raoet al., 2002; Lorrain et al., 2003). Somemutants initiate celldeath as the result of an external stimulus, andmutants thatoverproduce SA or ET usually develop lesions or acceler-ated senescence at some point during ageing (Rate et al.,1999;Kirik et al., 2001). accelerated cell death (acd), radicalinduced cell death1 (rcd1) and lesion simulating disease (lsd)(Overmyer et al., 2000; Rusterucci et al., 2001; Greenbergand Yao, 2004) are three reported examples of gene mu-tations where the induction of PCD results in uncontrolledspread of PCD.ACD genes are controlling a SA-dependentPCD pathway (Greenberg et al., 2000). The RCD1 geneproduct is a negative regulator of ozone induced (contrib-utes to extracellular accumulation ofROS like inHR)PCDthat also coordinates a hormone signalling network of SA,ET, JA and ABA (Kangasjarvi et al., 2005). Here, SA wasnecessary for the initiation of the cell death and ET for thecontinued spreading of the cell death (Overmyer et al.,2000).The lsd1mutantswith defectedLESIONSIMULATING
DISEASE1 gene initiate spontaneous lesions in response toageing, changing of light conditions and when exposed tovirulent and avirulent pathogens. The lesions propagatethroughout the leaves, subsequently killing most of theolder leaves. Prediction ofLSD1 structure reveals that it is aprotein with zinc finger domains grouping it with the C2C2class of transcription factors (Epple et al., 2003). Recently it
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Figure 2 SA disrupts redox regulation. (a) Mutants with constitutive accumulation of SA had strongly increased H2O2 levels and in SA-deficient lines H2O2 was
decreased, indicating a strong correlation between SA levels and H2O2 content in the cell. (b) NADPH-dependent glutathione reductase (GR) activity. (a:
significantly different from wt, p50.05). From Mateo et al. (2006).
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was also demonstrated that LSD1 has a thioredoxin-binding domain (Mateo, 2005). Additionally, several of theplantmetacaspases contain zinc-finger domains resemblingthose of LSD1 and it has been shown that LSD1 controlsHR (Uren et al., 2000; Rusterucci et al., 2001). Conse-quently, the LSD1 is a negative regulator of PCD and ahighly conserved paralogue of this gene has been found topositively control PCD (Epple et al., 2003). In lsd1mutants,extracellular accumulation of ROS precedes spreading oflsd1 lesions and it has been shown that inhibition of SAsynthesis was able to revert the lsd1 phenotype (Jabs et al.,1996; Aviv et al., 2002). Furthermore, ROS in combinationwith SA was reported to induce lsd-like lesions in wild-typeplants (Mazel and Levine, 2001). However, lesions are alsoable to form in lsd1 mutants independently of SA (Huntet al., 1997) and there are differences between the inductionofHR signalling and the uncontrolled cell death in lsd1 thatmay provide clues to how the spread of PCD is regulated(Kliebenstein et al., 1999; Rusterucci et al., 2001; Torreset al., 2006).
The lipase (ENHANCEDDISEASESUSCEPTIBILITY1) EDS1 and its interacting lipase-like partner (PHYTOA-LEXINDEFICIENT4) PAD4,were shown to be needed forthe initiation of unchecked cell death in lsd1 and are essentialregulators of SAR (Feys et al., 2005; Wiermer et al., 2005;Bartsch et al., 2006). EDS1 forms several different proteincomplexes with PAD4 and (SENESCENCE ASSOCI-ATED GENE 101) SAG101, which constitute regulatorynodes for SA, JA and ET pathways (Wiermer et al., 2005).
During pathogen response these genes are active upstreamofSA signalling (Wiermer et al., 2005). In the lsd1mutant, theyseem to function in a positive feedback loop of ROS accu-mulation. The double mutants lsd1/eds1 and lsd1/pad4are reverted in the lsd phenotype, i.e. they do not formspontaneous lesions or unchecked cell death (Rusterucciet al., 2001).
LSD1 Integrates ChloroplasticEEE Signals
One of the responses common to both biotic and abioticstress is that stomata closes (McDonald and Cahill, 1999;Apel andHirt, 2004;Laloi et al., 2004;Desikan et al., 2005).The role of stomata in regulating gas exchange is thereforeintegral in the regulation of foliar cellular redox status sincethe limitation of gas exchange inevitably induces photo-respiratory H2O2 accumulation (Karpinski et al., 1999;Wingler et al., 2000).We found that lsd1 leaves had a lowerstomatal conductance thanwild-type leaves and that trans-ferring low light acclimated lsd1 plants to high-light con-ditions induced unchecked cell death (Figure 3a). Thisobservation indicates that lsd1 is defective in controllingphotorespiratory ROS. Artificially limiting gas exchangeprovoked accumulation of ROS in wild-type plants within24 h and was enhanced in the lsd1mutant, initially withoutbeing associated with any visible lesion formation
∗∗∗
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−1 s
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Ws-0
T0 T24 T48
lsd1
eds1-1/lsd1
pad4-5/lsd1
(c)(b)
(a)
Figure 3 Effects of lower stomata conductance and forced limitation of foliar gas exchange in lsd1 are reverted in pad4-5/lsd1and eds1-1/lsd1. (a) Relative
stomatal conductance (RSC) and (b) CAT activity in leaves of Ws-0, lsd1, pad4-5/lsd1, and eds1-1/lsd1 in short day (SD) permissive conditions (p50.001***,
p50.05*) in lsd1 and the recovery of wild-type phenotype in the doublemutants. (c) DCF-2 yellow-green fluorescence (H2O2) monitored after 24 h treatment
by limitation of foliar gas exchange. Runaway cell death was observed in lsd1 but not in Ws-0 nor in pad4-5/lsd1 and eds1-1/lsd1 after 48h. Representative
pictures of treated leaves are shown. From Mateo et al. (2004).
Oxidative Stress and Redox Signalling in Plants
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net6
(Figure 3c). Subsequently, after 48 h, theROS accumulationinduced PCD in the lsd1 mutant in local and systemicleaves (Figure 3c). In eds1-1/lsd1 and pad4-5/lsd1 mutants,H2O2 accumulation was also observed when limiting gasexchange, but it was lower in comparison to that observedin lsd1. These data correlate with the observation that thelsd1 conditioned cell death was blocked in these mutants.We also showed that LSD1 positively regulates catalase(CAT) which is the main scavenger of photorespiratoryROS (Figure 3b). We concluded that LSD1 functions notonly as a negative regulator of pathogen defences but alsoof EDS1- and PAD4-conditioned downregulation of theEEE acclimation response. Furthermore, we concludedthat the formation of uncontrolled cell death in the lsd1mutant is dependent on chloroplast mediated ROS signal-ling and that it is a suitable genetic marker for deregulatedEEE acclimation (Mateo et al., 2004).
SAA is Associated with HR-likeCell Death
Previously we show that EEE-induced acclimatory re-sponses, like SAA are characterized not only by redoxchanges in PET and antioxidant defences but also aremanifested by a specific appearance of cell death (Mateoet al., 2004). This cell death was characterized by the for-mation of microlesions (spot-like lesions made up of a fewcells) when low light adapted Arabidopsis thaliana leaveswere exposed to excess light, so we chose to refer to thesesymptoms as EEE-induced cell death (EEE-CD). EEE-CDwas also detected in systemic tissues (Muhlenbock, 2006),indicating that the lesion formation may be occurringthrough an active process rather than being a toxic effect.These symptoms are functionally and phenotypically sim-ilar to the HR that is induced in association with SAR.Limiting gas exchange, either by physically blocking sto-matal pores or by spraying leaves with ABA also inducedEEE-CD (Figure 4) and also caused uncontrolled spreadingof cell death following the induction of HR (Muhlenbock,2006). Accumulation of ROS was detected prior to theformation of the cell death associated with limiting gasexchange (Muhlenbock, 2006) in all of these cases. Thisindicates that, while EEE can induce cell death signals it
also feeds into general parts of PCD pathways where itcontributes to spreading of the cell death. We concludedthat EEE-CD is dependent on photorespiratory ROS andredox signals originating from the chloroplast.
Conclusions
We were able to link PCD in lsd1 to the activity of PSII(PCD was induced by light-2 (680 nm) but not by light-1(700 nm)), stomatal conductance and ultimately to photo-respiratory H2O2 and redox changes of the plastoquinonepool (Mateo et al., 2004).A cross of lsd1with the chaos (cao)mutant that has a reduced PSII antenna, led to a reducedlesion formation in lsd1/cao double mutants. The lsd1mutant had reduced stomatal conductance and catalaseactivity in short-day permissive conditions and increasedH2O2 accumulation followed by uncheck PCD when sto-matal gas exchange was further impeded. Importantly,these traits depended on the defence regulators EDS1,PAD4 (Mateo, 2005; Muhlenbock, 2006). Mutations inPAD4 andEDS1 block lsd1-conditioned PCD triggered bylong photoperiods, pathogen inoculation, ROS provision,or supply of the phenolic signalling molecule, SA. Further-more, nonphotorespiratory conditions retarded propaga-tion of lesions in lsd1 (Mateo et al., 2004). The results pointto amultiple role of LSD1 in reducing cellularROS content(1) by controlling PAD4- and EDS1-dependent stomatalclosure and consequent foliar (photorespiratory) H2O2
production during EEE and (2) by regulating the H2O2
scavenging capacity (Mateo et al., 2004) Therefore, un-check PCD in lsd1 is resulting from limitation of carbondioxide supply and higher ROS accumulation. We hypoth-esize thatLSD1,PAD4andEDS1are important integrativeregulators of pathogen defence and light-acclimation proc-esses. The above data emphasize the importance of exam-ining multiple physiological conditions when investigatingthe function of genes.
Acknowledgement
This work was supported by project from the SwedishResearch Council (VR) to SK and Polish Ministry of Sci-ence and Higher Education grant N301 075 31/2414.
C 50 µm ABA R.G.
Figure 4 Limitation of gas exchange induces EEE-CD. Representative trypan blue stained dead cells in leaves treated for 24 h with 50 mM ABA and physical
restriction for 24h of gas exchange (R.G.) (C=control).
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ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 7
References
Allan AC and Fluhr R (1997) Two distinct sources of elicited
reactive oxygen species in tobacco epidermal cells. Plant Cell 9:
1559–1572.
Apel K and Hirt H (2004) Reactive oxygen species: metabolism,
oxidative stress, and signal transduction. Annual Review of
ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net 11
Ozone and Reactive OxygenSpeciesAndrew C Allan, HortResearch, Auckland, New Zealand
Robert Fluhr, Plant Sciences, Weizmann Institute of Science, Rehovot, Israel
Increasing levels of the pollutant ozone are likely to have a dramatic effect on future plant
productivity. Ozone’s damaging effect on the plant is mediated primarily via an induction
of cellular reactive oxygen species (ROS).
Introduction
Ozone (O3) is an ever-present pollutant gas, formed by theinteraction of nitrogen oxides with hydrocarbons and UVradiation. Ozone has useful UV-B screening effects; itsdepletion from the stratosphere is therefore the cause ofsome alarm. However, despite these reductions in totalatmospheric ozone, it is predicted that over the next50 years, the tropospheric (from the surface to about 10 kmabove sea level) levels of ozone will increase by 1% peryear. The source of this ozone is anthropogenic emissionsof nitrogen oxides that after photolysis react with carboncompounds to yield ozone. Ambient ozone concentrationsgenerally range from 20 to 60 nanolitres (nL) per litre of air(20–60 ppb), but local increases of up to 250 ppb have beenobserved. Problematic levels of ozone are not restricted totheir source in cities and industrial sites; because of dis-persal patterns it is often rural areas that suffer the highestincreases in ozone. Western Europe, mid-western andeastern USA, and eastern China are currently beingexposed to some of the highest background levels of ozone(Morgan et al., 2006). Current ozone levels can have dam-aging effects on plants at levels above 40 ppb, depending onduration of exposure and the sensitivity of the plant spe-cies. A reported increase of 13 ppb in mean daytime ozonecauses a 20% decrease in soybean seed yield. This increasein ozone concentration could be reached as soon as 2020in south Asia. Thus, the effect of ozone on the health ofour major crops must be of major concern. See also: AcidRain; Biogeochemical Cycles
Effects of Ozone on Plant Performance
Ozone is 12 timesmore soluble inwater than is oxygen. It isunstable and decomposes to highly oxidizing free radicals.The type of damage that ozone exerts on the plant can bedivided into two categories: (1) acute effects, involvingelicitation of plant defence responses and switching on ofeither hypersensitive responses or necrosis, resulting innecrotic areas of brown or white colour appearing in
punctate areas on leaves and fruit; (2) chronic effects,including reduction in growth and fitness, with resultingpigmentation change and chlorosis, the outcome of accel-erated senescence. It is difficult to distinguish chronic fromacute effects. Some parts of the plant may be undergoingacute effects while the plant in general is experiencing asystemic chronic effect. See also: Enzymatic Free RadicalReactions; Plant Stress PhysiologyCertain plant species are ozone tolerant, while others are
hypersensitive. It has been proposed that damage resultingfrom a 4-h exposure to ozone at 50ppb indicates a sensitivespecies; damage due to exposure to 100ppb indicates anintermediate plant; and 150ppb indicates a tolerant species.The so-called ‘sentinels’ are sensitive plants that are intro-duced into an area to serve as early warning devices or aschecks on the efficiency of abatement practices. Examples ofsentinels include sensitive cultivars of tobacco. Importantly,sensitivity to ozone in tobacco and other species is clearlyunder complex genetic control, implying that tolerance isthe result of a combination of traits. In practice, under con-trolled conditions, yield reductions of up to 30% areobserved in such common crops as potato, bean and wheatwhen exposed to local ambient ozone concentrations.Recently, controlled laboratory experiments have beenextrapolated to field conditions. In a free-air gas concen-tration enrichment (FACE)field experiment for the effect ofozone fumigation, a 23% increase in ozone concentrationdecreased seed yield by 20% (Morgan et al., 2006).Being gaseous, water soluble and highly reactive, ozone
will have many effects on the plant’s physiology. For sim-plicity only, wewill divide the probable causes of decreasedplant performance into discrete areas.
Ozone effects on components ofphotosynthesis
Both light and dark photosynthetic reactions are affectedby ozone. Ozone decreases carbon dioxide assimilation in
Article Contents
Advanced article
. Introduction
. Effects of Ozone on Plant Performance
. Induction by Ozone of Plant Stress Responses
. Ozone, ROS Generation and Programmed Cell Death
. ROS during Photosynthesis
. ROS during Senescence
. Ozone and ROS during Pathogenesis
. Ozone and ROS Detoxification in Normal and in
Transgenic Plants
doi: 10.1002/9780470015902.a0001299.pub2
1ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net
Vicia faba by inhibiting guard cellK+ channels, which thendrives the closing of stomates (Torsethaugen et al., 1999).However, the resultant stomatal closure is eventually ben-eficial as it limits further ozone entry.
Exposure to ozone has been shown to decrease the levelsof mRNA for Rubisco small subunit, chlorophyll a/b pro-tein, and glyceraldehyde-3-phosphate dehydrogenase(Glick et al., 1995) either by specifically switching offtranscription or by increased messenger ribonucleic acid(mRNA) instability. Rubisco protein, the major solubleprotein in leaf extracts, was shown to degrademore rapidlywhen exposed to ozone. See also: Photosynthesis; Rubisco
Ozone effects on primary metabolism andhormones
Anumberofmetabolic processes are switchedonor alteredfollowing exposure to ozone. At the whole-plant level,carbohydrate assimilation and carbon allocation to theroots of large trees (mostly studied in conifer species) aredramatically reduced following ozone exposure. The directincrease in stomate resistance and desensitization to ABAnoted above is one mode of action of ozone that will effectmetabolism by limiting carbon dioxide. However, theinduction by ozone of the plant hormone ethylene plays anadditional critical role in this process. Inappropriateinduction of ethylene by ozone appears to trigger featuresof senescence damaging to plant metabolism. Ethyleneproduction in the plant occurs via the enzyme 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase, whichcatalyses the conversion ofS-adenosylmethionine toACC.ACC is then converted into ethylene by ACC oxidase.Increases in the conversion of ACC into ethylene, due tothe presence of ozone, appear to result from damagedmembranes, perhaps allowing normally sequestered subst-rate and enzyme to mix. Moreover, ozone also increasesACC levels by altering ACCmetabolism; levels of mRNAfor ACC synthase and ACC oxidase increase rapidly uponexposure to ozone (Tuomainen et al., 1997). In addition,ozone and ethylene can interact directly, producing super-oxide (O22) and reactive aldehydes,which drivemembranedamage. See also: Plant Growth Factors and Receptors;Stress-induced Premeture Senescence(SIPS)
Induction by Ozone of Plant StressResponses
Microarray experiments have revealed a fuller extent ofplant transcriptional response to ozone; when Arabidopsisis treatedwith 350 ppbozone (3 and 6 h), 2385 genes showadifferential expression equal or greater than 2-fold change(Ludwikow et al., 2004).Many of these genes are part of anethylene and jasmonic acid (JA)-induced stress-response
pathway indicating that damage has occurred and thetranscriptomehas adjusted to induce aprotective response.Indeed, increased ozone-induced foliar lesions wereobserved in jar1 mutant plants that are insensitive to JAproduction (Tamaoki et al., 2003). These responses are inturn antagonizedby the elicited production of salicylic acid(SA) as ozone promotes SA synthesis via ethylene. In npr1mutants of Arabidopsis that cannot promote the SAresponse the upregulation of protective genes is furtherenhanced (Tamaoki et al., 2003). A complex scenario hasemerged in which ozone-induced spreading of cell death isstimulated by early, rapid accumulation of ethylene, whichthen suppresses the protective actions of JA. Further, celldeath induces late accumulation of JA, which inhibits thefurther propagation of cell death via inhibition of the eth-ylene pathway.The fluorescent (flu) mutant of Arabidopsis accumulates
protochlorophyllide in the dark. This protochlorophyllidethen generates singlet oxygen when the plants are returnedto the light. Therefore, transfer of dark grown plants intothe light causes a massive, and specific increase, in singletoxygen within the chloroplast (op den Camp et al., 2003).Microarray analysis of transcript expression inArabidopsisfollowing exogenous application of oxidative stress-caus-ing agents including ozone treatment, and the flu mutant,shows which gene transcripts are specifically altered intheir expression by a particular type of ROS and which aremore generally responsive to ROS (Gadjev et al., 2006).For example, a group of 66 transcripts are commonbetween ozone and methyl viologen treatments and the flumutant singlet oxygen response. Five transcription factorsappear to be ozone specific, including AtWRKY70, whichshows a 37-fold increase in transcript levels after ozonetreatment. This transcription factor has been implicated inSA-mediated suppression of JA-induciblePDF1. 2 expres-sion (Li et al., 2004), lending a molecular basis for thecomplex scenario of cell death and subsequent suppressionof the protective actions of JA described above.Enzymes that function to dissipate ROS are induced by
ozone. Among these are glutathione S-transferase, whichaids in the restoration of molecules oxidized by free rad-icals; superoxide dismutase (SOD), which converts O2
2
into H2O2; and catalase isoform 2, which dissipates H2O2.Secondary effects associated with cell death are the elici-tation of pathogenesis-related proteins (PR proteins) andincreases in SA, phytoalexins, flavonoids, lignin and cal-lose. These responses may aid in plant defence against thepollutant (e.g. production of flavonoids acting as antioxi-dants) or be part of a more general response to stress. ROSare used by the plant as secondary messengers for plantpathogen response (see later sections), so it is not surprisingthat ozone inadvertently switches on pathogenesisresponse pathways. For example, ozone exposure oftobacco results in enhanced resistance to tobacco mosaicvirus infection (Yalpani et al., 1994). In contrast, the dam-age caused by ozone can allow a second abiotic or biotic
Ozone and Reactive Oxygen Species
2
stress to become more damaging. For example, pine treessuffering nutrient deficiency (Mg2+, Ca2+orK+) aremoreaffected by the presence of ozone than those not deficient(Siefermann-Harms et al., 2005). See also: Ecophysiologi-cal Responses of Plants to Air Pollution
Ozone, ROS Generation andProgrammed Cell Death
Ozone enters the plant through open stomata and diffusesthrough the apoplast. The low permeability and strongreactivity of ozone mean that the cytosol and other organ-elles of the plant probably do not experience free ozone.Instead, ozone appears to degrade rapidly into other ROSwithin the apoplast or plasma membrane (Figure 1). ROSare the primarymediators of the oxidative damage in plantcells. They include superoxide radicals (O2
2), .OH, singletoxygen (1O2) and H2O2. ROS, and in particular O2
2, arestrong oxidizing species that can rapidly react with othermolecules, including deoxyribonucleic acid (DNA), lead-ing to metabolic changes and hypersensitive response(HR). Ozone also reacts directly with the cell membrane toproduce aldehyde and organic radicals. Ozone elicitationof ROS has characteristic bimodal kinetics; an early
apoplastic peak in ROS has been observed in response toacute ozone exposure (in the first few hours), with a secondlonger lasting burst (after 15–72 h) observed in ozone-sen-sitive tobacco cultivars in association, both temporally andspatially, with HR. It has been shown, using electron spinresonance, that hydroxyl radicals (.OH) appear in plantcells during exposure to ozone. This conversion is aided bythe presence of free iron. See also: Oxidative StressProbably, the earliest cellular response to ozone is an
elevation in cytosolic-free calcium, which occurs withinseconds of exposure (Evans et al., 2005). The presence of aparticular calcium ‘signature’ and ROS activates a mito-gen-activated protein kinase (MAPK) kinase (MAPKK)signalling pathway.A tobaccoMAPKkinase,NtMPK4, isexpressed in the epidermis. Plants with less NtMPK4 showenhanced sensitivity to ozone and an abnormal regulationof stomatal closure in anABA-independentmanner (Gomiet al., 2005).Ozone-induced ROS is not restricted to the cytosol;
when tobacco cv Bel W3 plants are fumigated with acuteozone levels there is an accumulation of H2O2 inmitochondria and chloroplasts, as well as an early accu-mulation of nitric oxides (NO) in leaf tissues (Ederli et al.,2006). During ozone exposure, H2O2 continues to accu-mulate in the apoplast, apparently because of the activa-tion of both the plasma membrane-bound NAD(P)H
RbohO2
−
O2
H2O2
G-proteins amplification
O3
Ascorbic acid scavenging
Low concentrationchronic exposure
High concentrationshort exposure (acute)
Apoplast
Membrane
Scavengers of ROS andantioxidants
Induction of PR genesAntioxidant pathwaysPAL, flavonoids
Figure 1 A model for plant response to ozone. Elicitation of different plant responses ranging from senescence to rapid necrosis can be generallyexplainedby the relative dose of ozone that theplant receives.Ozone is dissipated in the apoplast to other free radicals andROS. These act asmore diffusiveagents, entering the cytosolwhere they are either scavengedor act as secondarymessengers for a number of plant responses(seeboxes). Cytosolic calciumsignalling is elicited, via influx of Ca2+ from the apoplast. The balance of ROS concentration versus antioxidant scavenging potential helps decide the cellfate. Good evidence exists for an amplification of ozone-elicited ROS, in susceptible plant varieties, by triggering of the plasmamembrane enzymeNADPHoxidase tomake superoxide. This candrive programmed cell death (e.g. hypersensitive response). PR, pathogen response; PAL, phenylalanine lyase; Rboh,respiratory burst oxidase homologues.
Ozone and Reactive Oxygen Species
3
oxidase complex and cell-wall located NAD(P)Hperoxidases. Using Arabidopsis mutants in the a and bsubunits of heterotrimeric G proteins, Joo et al. (2005)dissected out the pathwayproducing the bimodal oxidativeburst elicited by ozone. The early component of the oxi-dative burst, arising primarily from chloroplasts, requiressignalling through the G b–g complex to the membrane-bound nicotinamide-adenine-dinucleotide phosphatereduced (NADPH) oxidase. The late, tissue damage-asso-ciated component of the oxidative burst requires only theG a protein and arises from multiple cellular sources.Necrotic lesions have been seen to appear in tobacco after asingle acute dose of ozone (5 h of 150 ppb). In this case, thelocal concentration of ozone could be high enough for anunregulated cell necrosis to occur as a result of ozone-induced oxidation of membrane fatty acids, leading tomembrane rupture. Alternatively, ROS build-up in theapoplast and the cytosol can be below acute levels andmayelicit HR. HR is a highly regulated process of cell death.The cell undergoes characteristic developmental steps, in-cluding chromosome condensation, release of cytochromeC from the mitochondria, regulated DNA cleavage andvacuolar degradation. These characteristic markers of HR(or programmed cell death, PCD) are seen in ozone-treatedtobacco cv Bel W3 plants (Pasqualini et al., 2003). Tran-sient application of ozone causes the formation of celldeath lesions on the leaves of the ozone-sensitive radical-induced cell death1 (rcd1)mutant ofArabidopsis. The dyingcells exhibited several of the typical morphological char-acteristics of the hypersensitive response and PCD. Dou-ble-mutant analyses indicated a requirement for SA andthe function of the cyclic nucleotide-gated ion channelAtCNGC2 in cell death (Overmyer et al., 2005). See also:Plant Programmed Cell Death
ROS during Photosynthesis
The photosynthetic electron transport chain and the chlo-rophyll pigments themselves can, under certain conditions,produce singlet oxygen and O2
2; this is termed photo-oxi-dative stress. During photosynthesis, any ROS inadvert-ently generated is rapidly removed by antioxidativemechanisms. However, this removal can be compromised(e.g. by exposure to ozone). The balance between produc-tion and removal of ROS in the chloroplast may be per-turbed by abiotic stresses such as ozone or extremetemperature (e.g. low temperatures and sunny conditions).As a result, intracellular ROSmay rapidly rise. One sourceof ROS during photosynthesis is the direct photoreductionof oxygen to superoxide by reduced electron transportcomponents associated photosystems I or II. Furthermore,during photoinhibition, which occurs when plants areexposed to high light intensities, singlet oxygen can becontinuously produced by PSII. Thus, in the presence of
ozone and during excess light the damage wrought bysinglet oxygen and superoxide production drasticallyincreases due to acute overloading of the scavengingcapacity of the cell. See also: Photosynthesis: DarkReactions; Photosynthesis: Light Reactions
ROS during Senescence
Senescence involves a directed general degradation of thecellular components such as proteins and the mobilizationof the products of degradation to other parts of the plant.During both naturally induced senescence and ozone-pro-moted senescence there is a cessation of photosynthesis,disintegration of organelle structures, loss of chlorophylland certain proteins, and increases in lipid peroxidation,membrane leakiness, ethylene and jasmonic acid.Adrivingforce behind senescence is due to the enhanced release ofO2
2 from the peroxisome. The relationship to ozone is notclear. For example, yellowing of Arabidopsis leaf is accel-erated by ozone application that is accompanied by theappearance ofmanybut not all of the naturally senescence-associated genes (Miller et al., 1999). See also: PlantPeroxisomes and Glyoxysomes
Ozone and ROS during Pathogenesis
An oxidative burst is often induced when the plant firstdetects the presence of a pathogen. Pathogen-induced oxi-dative bursts are harnessed by the plant cell both as ameans of ‘poisoning’ an invading pathogen (a massiveburst that drives cell death) and as an intra- and extracel-lular message of this invasion. Therefore, ROS play a cen-tral role in the plant defence against pathogens. H2O2 andO2
2 appear to be agents of this burst and can be generatedby activation of a number of enzymatic sources: a plasmamembrane NAD(P)H oxidase, cell wall-located peroxi-dases, or amine, xanthine or aldehyde oxidase activities. Inaddition, both the activity and level of theROSdetoxifyingenzymes such as ascorbate peroxidase (APX) and catalaseare suppressed. The simultaneous production of ROS anddownregulation of ROS scavenging mechanisms can thentriggerPCD.Asone effect of ozone,mentioned above, is animmediate production of ROS via activation of NADPHoxidase (Joo et al., 2005) its influence on plant resistance todisease may be to short-circuit and jumble the disease-response signal. Necrotroph-style pathogens may be at anadvantage in this state as they utilize the plants investmentin ROS metabolites for their own benefit. An ozone-induced oxidative burst results in a cell death processsimilar to pathogen-induced PCD. However, the ROSgenerated by ozone can later serve to elevate ROS scav-enging enzymes that may compromise an appropriatedefence to biotic attack. This would explain why, in many
Ozone and Reactive Oxygen Species
4
instances, plants exposed to ozone suffer from a secondbiotic infection.
Ozone and ROS Detoxification inNormal and in Transgenic Plants
The elevation of cytoplasmic levels of ROS triggers stresssignalling pathways. However, the plant possesses efficientantioxidative defence systems, including SODs, catalases,peroxidases, ascorbate (vitamin C), glutathione, tocophe-rol (vitamin E), carotenoids and flavonoids.Under normalconditions, these antioxidative enzymes and small mole-cules provide protection against ROS, but when the mag-nitude of the oxidative stress is large, or when defences arepurposely downregulated (e.g. duringHR), large increasesof ROS in the cell can cause acute damage. Thus, attemptshave been made to engineer increased resistance to oxida-tive stresses, such as from ozone, through overexpressionof enzymes that dissipate ROS. Tolerance to ozone hasbeen achieved by targeting these enzymes to the cytoplasm.For example, overexpression of pea cytosolic Cu,ZnSODin tobacco conferred partial protection against ozonedamage, while overexpression of petunia Cu,Zn-SOD inthe chloroplast had no effect (Pitcher and Zilinskas, 1996).Reduced peroxisomal catalase increases the sensitivityof Arabidopsis to both ozone and photorespiratory H2O2-induced cell death. See also: Arabidopsis thaliana as anExperimental Organism; Transgenic Plants
Apparently, apoplastic ascorbic acid levels are a keydeterminant to sensitivity or resistance to ozone; distinctdifferences in apoplastic ascorbate are found in specieswith differences in ozone sensitivity. When expression lev-els of APX are modified in transgenic plants, a 10-foldincrease of APX detected in the chloroplast led to noincrease in ozone tolerance (Torsethaugen et al., 1997).Conversely, plants expressing antisense RNA of cytosolicAPX have an increase in sensitivity to ozone (Orvar andEllis, 1997). Both spinach and beech responded to ozonetreatment by exporting ascorbate into the apoplast in anapparent attempt to detoxify radicals formed before theirentry into the cytoplasm. The role of ascorbate in thedefence against ozone-induced damage is further high-lighted by analysis of Arabidopsis mutants. Arabidopsisplants containing the soz1mutation accumulate only 30%of normal ascorbate levels and are hypersensitive to ozonewhileVitamin c-1 (vtc1) is anozone-sensitivemutantwhichis deficient in ascorbic acid. The mutated locus was clonedand found to be GDP-Man pyrophosphorylase (Conklinet al., 1999), an enzyme which converts D-mannose-1-PintoGDP-mannose, a step in the ascorbic acid biosyntheticpathway.
Increasing the endogenous level of ascorbic acid hasbeen achieved by overexpressing dehydroascorbate red-uctase (DHAR). DHAR-overexpressing plants have a
lower oxidative stress, a lower level of oxidative-relatedenzyme activities, a higher level of chlorophyll and pho-tosynthetic activity following acute (2 h of 200 ppb) andchronic ozone exposure (30 d–100 ppb). Suppression ofDHAR expression had the opposite effect (Chen andGallie, 2005). There are threeDHARgenes inArabidopsis,but it is only cytosolic DHAR that increases with ozoneexposure. A mutant with no cytosolic DHAR activity ishighly ozone sensitive. Although total amounts of ascor-bate are not reduced in this mutant, the apoplastic ascor-bate is 60% lower. It therefore appears that apoplasticascorbate, generated through the reduction of dihydro-ascorbate by cytDHAR, is important for ozone tolerance(Yoshida et al., 2006). However, by elevating the ascorbatelevel, the guard cell is rendered less responsive to ABA andozone. Nevertheless, the harmful effects of ozone on theplant is reduced, mainly due to enhanced protectionagainst ROS.These findings show that engineering ozone tolerance
through a transgenic approach will have to take intoaccount the complex physiology of the ozone effect.Perhaps, the goal of removing the ozone problem wouldbe the discovery of an ‘ozonase activity’ that, like super-oxide dismutase, would dissipate the oxidant. Alterna-tively, breeding of crops with natural tolerance will also beaided through understanding genetic variation in the keymajor genes (e.g. cytDHAR). Indeed, naturally tolerantsoybean cultivars have been shown to have elevatedascorbate levels (Chernikova et al., 2000). Given the mul-titiered nature of the plant response, stacking of multipletransgenes or themanipulationof hierarchical gene controlelements are also reasonable genetic approaches.
References
Chen Z andGallie DR (2005) Increasing tolerance to ozone by elevating
foliar ascorbic acid confers greater protection against ozone than
