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IntroductionAll photosynthetic organisms house an elaborately folded
network of photosynthetic membranes responsible for trans-duction of solar energy into biochemically useful forms. Inmost vascular plants and some green algae, the continuousthylakoid membrane network is differentiated into regulardomains of closely appressed membranes in granal stackswhich are interconnected by non-appressed single mem-branes, the stroma thylakoids. Only the outer surface of non-appressed membrane domains, stroma thylakoids and themargins and end membranes of granal stacks are in contactwith the chloroplast stroma. This structural organisation,characteristic of more recently evolved plants and somegreen algae which have light-harvesting chlorophyll a/b-proteins, is not universal (Staehelin 1986). Many of thechlorophyll b-containing green algae have much longer, veryirregular areas of stacking, sometimes of groups of two thyl-akoids, which extend from one end of the chloroplast to theother. For example, in Chlamydomonas there appears to be avery irregular arrangement with thylakoids coming togetherand moving apart, while thylakoids of the Characeae, e.g.Nitella display grana structures virtually indistinguishablefrom those of higher plant chloroplasts. Both cyanobacteria(Cyanophytes) and red algae (Rhodophytes) have singlemembranes with large light-harvesting antennae, the phyco-bilisomes, attached to the outer surface. However, theprokaryotes that contain chlorophyll b have large areas of
somewhat stacked thylakoids which bear no resemblance tothe regular grana stacking of terrestrial plants. In contrast,the large division of the Chromophyta with light-harvestingchlorophyll a/c-proteins possess more morphologicallydiverse classes: with the exception of the cryptomonadswhich have thylakoids typically stacked in groups of two, themembranes of diatoms, dinoflagellates, brown algae(Pheophyceae) and other classes are often stacked in groupsof three which may traverse the entire chloroplast length.Despite membrane stacking, the arrangement of the photo-synthetic apparatus of the Chromophyta differs from that ofthe grana of higher plants and some more recently evolvedgreen algae which are considered here.
A transmission electron micrograph of a shade plantchloroplast (Fig. 1) with extensive grana highlights theintriguing architecture of these folded structures, but suchimages are of course frozen in time. In vivo, granal stackingis highly dynamic, varying in time and tissue locationaccording to the light climate, as well as species adaptationto sun and shade. For example, shade and low-light plantshave many more grana per chloroplast (Fig. 1a), with muchmore appressed relative to non-appressed thylakoids(Fig. 1b), compared to sun and high-light plants. Further, inC4 plants, while chloroplast populations of mesophyll cellsshow extensive granal stacks, the bundle sheath cells of someC4 variants that lack core PSII* and LHCII have no grana. Isall of this happenstance, or does thylakoid stacking into
Abstract. The striking structural architecture of thylakoid membranes of higher plant and some greenalgal chloroplasts that house the light harvesting and energy transducing functions of chloroplasts haveevoked many hypotheses concerning the significance of grana. The differentiation of the thylakoids intograna and stroma membrane regions is a morphological reflection of the non-random distribution of thephotosystems II and I between appressed and non-appressed membrane domains, which became knownas lateral heterogeneity. In this overview, the first section deals with changing concepts regarding the dis-tribution of the photosystems between stacked and unstacked thylakoid domains from a personal histori-cal perspective. The remaining section describes some functional implications of the lateral separation ofmost PSII complexes in appressed membrane regions of grana stacks from PSI complexes, ATP synthaseand auxiliary proteins located in non-appressed membrane domains.
Keywords: grana, lateral heterogeneity, personal perspective, photosynthesis, photosystems II and I,thylakoid stacking, vascular plants.
*Abbreviations used: Chl, chlorophyll; D1 protein, gene product of psbA in the PSII reaction centre heterodimer; LHCII, LHCI, light-harvesting Chl a/b-proteins of PSII and PSI antennae; PS, photosystem.
AJPP Special IssueOverview
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grana carry a selective advantage? This fascinating questionhas evoked many hypotheses concerning the functional sig-nificance of grana for species acclimation, as well as theirstructural importance for functional integration of PSII andPSI.
Towards the functional significance of grana:a personal perspective
My scientific odyssey with the molecular organisation ofthylakoid membranes of higher plant chloroplasts beganwhen I arrived in Canberra to work in CSIRO, Division ofPlant Industry. The hypothesis of Hill and Bendall (1960)that two, not one, light reactions were required for photosyn-thesis which were carried out by two photosystems acting inseries had just been published. I wondered about grana for-mation, greatly inspired by the aesthetic beauty of chloro-plast structure revealed by Sam Wildman and Don Spencerwho were using phase contrast light microscopy. Within leafsections, living plant cells contained chloroplasts surroundedby a mobile phase (cf. Spencer and Wildman 1962) (Fig. 2a).These chloroplasts possessed darker green regions whichglowed a more intense red under ultraviolet light (Fig. 2b).
These regions of enhanced chlorophyll content (Fig. 2a, b)correspond to granal stacks observed by electronmicroscopy.
My challenge then was two-fold. (i) To determine if therewere indeed two discrete photosystems as Hill and Bendall’s(1960) hypothesis implied, and (ii) to ascertain whether thesephotosystems were uniquely associated with appressed vsnon-appressed domains of vascular plant thylakoids.
(i) Differentiation of function and structure
My first experimental approach to prove the reality of thephotosystems was to follow the development of the photo-systems during the greening of etiolated bean leaves, whichinitially had neither chlorophyll, photosynthesis nor granastructures. (Keith Boardman had used etiolated beans tocharacterise protochlorophyllide reductase during chloro-phyll synthesis.) It was widely thought that PSI would have
Fig. 1. (upper) Electron micrograph of an Alocasia macrorrhiza chloro-plast from an extreme shade forest (X 15 500). (lower) A giant granal stackthat extended across the Alocasia chloroplast. (Both from Goodchild et al.(1972)).
Fig. 2. (upper) Phase contrast light micrograph of a portion of a livingspinach cell showing chloroplasts containing grana and a few starch grains,and stroma surrounded by a mobile phase (cf. Spencer and Wildman 1962).(lower) Chloroplasts in a spinach cell illuminated in the light phase micro-scope with fluorescent light. The very regular areas of enhanced chlorophyllfluorescence correspond to granal stacks. Both photos taken in 1961 werevery kindly supplied by Dr Don Spencer, CSIRO, Division of Plant Industry,Canberra.
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evolved prior to the more complicated PSII; naively wehoped this might happen during chloroplast developmentthereby allowing us to characterise the properties of onephotosystem. It was an impossible task. The photochemicalactivities of isolated bean etioplasts and greening chloro-plasts were vanishingly low, they were heavily contaminatedwith mitochondria and, although PSI appeared to developbefore oxygen evolution (Anderson and Boardman 1964), wehad little idea if this was so with leaves.
Our next strategy involved detergent fragmentation ofchloroplasts. Luckily spinach, then unavailable commer-cially in Australia unlike USA, was being grown hydroponic-ally for trace element studies within the Division of PlantIndustry. I gladly turned to spinach from which functionalchloroplasts had been isolated overseas. Digitonin fragmen-tation of spinach chloroplasts followed by differential centri-fugation yielded a heavier fraction enriched in chlorophyll band PSII activity, while the lighter fraction depleted inchlorophyll b contained mainly PSI activity (Boardman andAnderson 1964; Anderson and Boardman 1966). JohnThorne had built a spectrofluorometer capable of correctingfor the wavelength dependence of instrument response, so wewere able to characterise the fluorescence properties of PSII-enriched and PSI membrane fragments at room and low tem-perature; to our surprise at room temperature most of thechlorophyll fluorescence was emitted by PSII (Boardman etal. 1966). Thus, this partial physical separation of the photo-systems proved there were indeed two photosystems.
In 1966, Izawa and Good (1966) presented an amazingelectron micrograph of an envelope-free spinach chloroplast(Fig. 3) that no longer had the characteristic areas of stackedmembranes; instead the thylakoid membrane network wascompletely unstacked. This thylakoid destacking which wasachieved by suspension of envelope-free chloroplasts in low-salt zwitterionic buffers (just invented by Good et al. (1966)),resulted in no loss of photochemical activities. Remarkablythe addition of mono- or di-valent cations restored granalstacks almost indistinguishable from the original profiles(Izawa and Good 1966). Curiously, when unstacked spinachthylakoids suspended in Tricine buffer were fragmented bydigitonin, the submembrane fractions contained the sameamount of total chlorophyll as obtained from control chloro-plasts with the conventional stacked structure, but all mem-brane fractions had the same Chl a/Chl b ratios and both PSIIand PSI activities (Anderson and Vernon 1967): the signifi-cance of this finding was not understood. As yet there is nouniversally agreed model for the three-dimensional organiza-tion of the continuous thylakoid membrane network consis-tent with dynamic destacking and restacking of thylakoidmembranes (Izawa and Good 1966). However, a recentmodel for the topology of thylakoid membranes presented byArvidsson and Sundby (1999) is more convincing.
The concept that photosynthesis involves PSII and PSIcooperating in series, immortalised in Hill and Bendall’s Z
Insights into consequences of appressed grana thylakoids
Fig. 3. Spinach chloroplasts suspended in low-salt hypotonic buffer afterglutaraldehyde fixation have unstacked membranes. Section through chloro-plast perpendicular to the thylakoid membrane sheets showing completeabsence of grana and the continuous sheets of double membranes (3a);(from Izawa and Good 1966).
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scheme (Hill and Bendall 1960), defines neither structuralnor spatial organisation of the photosynthetic apparatus (Fig.4). Initially it was thought that PSI might be located towardsthe outer membrane surface and PSII, being less accessible,towards the inside. However, following spectral and bio-chemical characteristics of membrane fractions fragmentedby mechanical means instead of detergents, Sane et al.(1970) suggested that PSII and PSI were located together instacked granal membranes, with some PSI also located innon-appressed stromal thylakoids (Fig. 4). Biophysicalstudies supported the idea that most PSII and PSI were incontact in appressed granal membranes, and light excitationregulation between PSII and PSI was thought to be controlledby spillover with a common pool of LHCII being in contactwith both photosystems (e.g. Butler 1978). This proposaldominated the ideas of molecular organisation in the 1970s,but searches then for heterogeneity in PSI were unsuccessful.
(ii) Extent of grana stacking is regulated by lightacclimation
My next adventure into grana stacking came with OlleBjörkman’s visit from Stanford to Canberra in 1970/71where he galvanised nine scientists from three differentlaboratories to collaborate and determine the characteristicsof sun and shade chloroplasts. David Goodchild demon-strated that chloroplasts from plants adapted to extremeshade in a tropical rainforest that received very low irradi-ance are almost filled with thylakoid membranes (Fig. 1a)with granal stacks of ‘prodigious proportions’, over 100 thyl-akoids per granum (Fig. 1b) (Goodchild et al. 1972). Such anorganisation leaves little space for stromal enzymes, but
greatly decreased amounts of Rubsico and other carbon fix-ation enzymes are sufficient for the very low rates ofmaximal photosynthesis achieved by shade plants.
Plants grown in cabinets in light with different lightintensities of fixed quality also showed accclimation to lightintensity. For example, Atriplex patula, a sun plant accli-mated to different light intensities during growth, displayed avariable stacking profile, with low-light chloroplasts havingmore stacked membranes than medium-light and in turnhigh-light chloroplasts. Photosynthetic capacity increasedwith reduced extent of appressed thylakoids (Björkman et al.1972). While these classical studies showed that the struc-ture, composition and function of the photosynthetic appara-tus were greatly modulated in response to both light qualityand quantity, we did not understand why chloroplasts withhuge grana stacks had low photochemical activities.
Lateral heterogeneity of thylakoid complexes
(i) Proteins and lipids in lively membranes
Knowledge of thylakoid proteins which made up abouthalf the membrane mass was minimal, and ideas of thearrangement of the electron transport chains as they weretermed was also rudimentary in the 1960s to mid-1970s. Incontrast, the composition of thylakoid lipids with highcontent of galactolipids and polyunsaturated fatty acids wasrather well characterised (Benson 1974).
On sabbatical leave at Cambridge, amazingly, serendipitycoincided with the coalminers’ strike of 1973. With initiallylittle, then no, electricity by day, experiments were impossi-ble in Derek Bendall’s laboratory. I retired to a perfect ivorytower, my snug attic room at Newnham College, to considermembrane architecture, inspired by reading the superbarticle of Singer and Nicholson (1972) on fluid mosaic mem-branes which changed forever concepts of membrane struc-ture. The astonishing economy of placing moleculesasymmetrically across thin membranes imparts order byremoving random three-dimensional movement allowing aspectacular enhancement of effective concentration: forexample, this is important for light regulation of the manychlorophylls and carotenoid molecules so exquisitelyordered by their proteins. Gone was the rigid sandwich struc-ture with proteins at the outer surfaces with a lipid filling;now multiprotein complexes that spanned the entire mem-brane danced in the fluid lipid bilayer. ‘This organisationallows for rapid changes in the conformation and the distri-bution of the macromolecular complexes which are essentialfor the function and structure of the intricate chloroplastmembrane’ I confidently asserted in ‘The molecular organi-sation of chloroplast thylakoids’ (Anderson 1975), probablymy most significant article. Filled with speculations galore,due in large part to the extreme lack of knowledge of manythylakoid proteins as well as molecular structure of knownthylakoid proteins, it was only possible to glimpse any
Fig. 4. Schema of changing concepts for the molecular organisation ofPSII and PSI between appressed and non-appressed membrane domains.
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molecular organisation of thylakoid components. Never-theless, this article (Anderson 1975) provided a focus for anew era in photosynthesis concerning the proposed vectorialdistribution of electron transport components across themembrane, rejection of the then prevalent idea that PSI waslocated towards the outer membrane surface and PSIItowards the inner surface, and emergence of the concept thatthe photosystems must span the membrane. It also providedtentative assignment of an asymmetric location of complexesalong the membrane, the highly dynamic nature of fluidthylakoid membranes seen for example in the dramaticnarrowing of thylakoids in light versus dark (Murakami andPacker 1970), and the molecular forces involved in mem-brane stacking.
(ii) Partitioning the hylakoid complexes
Application of the elegant method of aqueous-polymertwo-phase partition pioneered by Per-Åke Albertsson (1971)to a granal thylakoid subfraction produced right-side-out andinside-out vesicles derived from non-appressed andappressed membranes, respectively. Further, PSII activitywas very enriched in the appressed granal fraction(Andersson 1978). Following my novel non-denaturing‘green gel’ procedure which permitted most of the pigmentsto remain attached to apoproteins, and analysis of resolvedchlorophyll-proteins, we demonstrated that less than 5% ofPSI was located in the granal fraction, which was greatlyenriched in PSII and LHCII complexes (Andersson andAnderson 1980). We proposed an extreme lateral hetero-geneity of distribution of the photosystems (Andersson andAnderson 1980) (Fig. 5). If true this meant that the lateraldistribution of the photosystems was the opposite to that
earlier thought, with PSI being excluded from appressedmembrane domains where most PSII-LHCII complexes werelocated (Fig. 4). Initially this was a startling, even hereticalidea as it had been widely assumed then that most of LHCIIwas in contact with both PSII and PSI located mainly inappressed granal regions for the regulation of light excitationdistribution between the photosystems.
With the resolution of the location of cytochrome bfcomplex in both stacked and unstacked regions (Cox andAndersson 1981; Anderson 1982a) it is plastocyanin ratherthan plastoquinone that carries electrons between PSII inappressed membranes and PSI in non-appressed granamargins (Fig. 6; Anderson and Andersson 1982). The struc-tural separation of linear and non-linear electron transport invivo has long been considered (Sane et al. 1970), althoughthe membrane domains involved have altered considerably(Fig. 4). With the demonstration that stroma-exposed granamargins indeed contain proteins, PSI, cytochrome bfcomplex and ATP synthase (Webber et al. 1988; Albertsson1995), linear electron transport and non-cyclic photo-phosphorylation is confined to granal stacks, while stromathylakoids carry out cyclic photophosphorylation as pro-posed earlier (Sane et al. 1970; Anderson 1982a).
Some aspects of the functional significance of granastacking: consequences of lateral heterogeneity in the
distribution of most PSII from PSI
The consequences of the exclusion of PSI from thestacked granal membrane domains (Andersson andAnderson 1980) refocussed attention on the significance ofgrana stacking. If the bulk of the antenna pigments of PSIIand PSI were in direct contact with the reaction centres of
Insights into consequences of appressed grana thylakoids
Fig. 5. Lateral heterogeneity in the distribution of PSII, PSI and ATP synthase between appressed membranes of grana stacks and non-appressed membranedomains (stroma thylakoids, grana margins and grana end membranes), with cytochrome bf complex uniformly distributed along both appressed and non-appressed thylakoid membranes.
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both photosystems as favoured in early continuous arraymodels (e.g. Butler 1978), most of the light energy would endup in PSI which contains longer-wavelength absorbing com-ponents than PSII. However, there can only be limitedspillover of absorbed light excitation energy from PSII to PSIin grana-containing chloroplasts (Anderson 1981, 1982b).
(i) Segregation of most PSII in the appressed domains ofgrana limits spillover of light excitation energy from PSII toPSI
PSII complexes consist of more than 26 polypeptidescomprising the heterodimeric D1/D2 proteins which bind theredox components necessary for photosynthetic charge sepa-ration and charge recombination, cytochrome b-559 and Chla-proteins, CP43 and CP47, together with minor, monomericLHCII complexes, LHCIIa (CP29), LHCIIc (CP 26) andLHCIId (CP 24), surrounded by two or more major trimers ofLHCIIb which bind at least 60% of PSII chlorophyll.Dimeric PSIIα complexes are located in appressed granalmembranes: only those PSIIα complexes at the outer rim ofthe appressed grana domains are in direct contact with PSIαcomplexes (Figs 4 and 5). Since appressed grana membranedomains allow closer packing of chlorophyll and carotenoidmolecules and the proteins to which they are bound per unit
membrane area, it is more economical in shade plant chloro-plasts to have more non-appressed domains to organise effec-tively their enhanced chlorophyll content (Fig. 1). The closepacking of these large PSII dimers into appressed mem-branes, mediated by LHCIIb, facilitates good connectivity ofLHCII and PSII both along the membrane, and across thenarrow partition gap between adjacent appressed mem-branes. At limiting light, grana formation facilitates trappingof excitation energy in PSII, particularly in the extensivegranal stacks of shade plant chloroplasts which maximisetrapping of the highly attenuated PSII light of deep shade.
Trissl and Wilhelm (1993) proposed that granum forma-tion is also one strategy developed by nature to separate phys-ically ‘slow’ PSII from ‘fast’ PSI. This non-synchronousphoton processing by PSII compared to PSI is due to thekinetics of exciton trapping in PSII being some 3-fold slowerthan in PSI. It is also advantageous to pack slow PSII instacked granal membranes for maximum connectivity ofLHCIIs and PSII reaction centres along the membrane andacross the partition gap (Trissl and Wilhelm 1993).
Although the complex molecular organisation of lightharvesting in PSII is often considered in terms of the highquantum efficiency required for photosynthesis, it is equallyimportant to consider regulation of light harvesting which
Fig. 6. Cartoon drawn by the incomparable TAB of TIBS (Trends in Biochemical Sciences) depicting the separation of PSII on Mount Appression (appressedthylakoid domain) from PSI on Mount Stroma (non-appressed thylakoid domain), while mountaineering cytochrome bf complexes scale and descend bothmountains. Electrons are transferred from PSII to PSI by the lateral shuttling of plastocyanin which carries electrons from cytochrome bf complex to PSI andreturns empty-handed (from Anderson and Andersson 1982: with permission). Bertil Andersson had the general idea for this cartoon to be depicted as moun-tains separated by a lake when he visited Queenstown, New Zealand (where JMA grew up).
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allows excess excitation energy to be dissipated as heat in theantennae before or after reaching the reaction centres(Demmig-Adams and Adams 1992; Chow 1994; Osmond1994; Horton et al. 1996). Light excitation energy absorbedby the leaf is partitioned between utilisation by PSII and PSI,and energy loss in heat dissipation. Energy use by PSII ismonitored by the photochemical fluorescence quenchingcoefficient qP, derived from modulated steady-state fluores-cence measurements, while events associated with non-pho-tochemical dissipation of excess excitation energy aremeasured by non-photochemical quenching (NPQ). Leavesunder steady-state photosynthesis are able to balance theexcitation energy used by PSII (1 – qP), which is equivalentto the amount of reduced QA (Huner et al. 1996), with energydissipation by non-photochemical quenching (NPQ) over awide range of irradiance from very low light to sunlight,since the value of the quotient (1 – qP)/NPQ is roughly con-stant (Anderson et al. 1997a). The values of this quotient,however, are different for various sun and shade plants(Anderson et al. 1997a). Given that non-stressed leaves areable to balance energy utilisation by energy dissipation underall irradiances, and that grana stacking in plants is a means toenhance photosynthetic electron and proton transport by PSIIand plastoquinol/cytochrome bf complex, we need also toconsider the other side of the coin: the complementary pro-cesses of excess excitation energy dissipation (Osmond1994; Horton et al. 1996; Osmond et al. 1999).
Does grana stacking contribute to non-photochemicalquenching? Brugnoli et al. (1998) compared non-photo-chemical quenching in sun and shade leaves of several C3 andC4 plants: they clearly demonstrated that both non-photo-chemical quenching and the proportion of photoconvertibleviolaxanthin to zeaxanthin are linearly related to Chl a/Chl bratios, although the slope of the relationship varied betweenspecies. Chl a/Chl b ratios depend on different contents ofchlorophyll-proteins of PSII and PSI reaction centre proteins,as well as minor and major LHCIIs and LHCIs (Andersonet al. 1988): they are inversely linearly correlated with theextent of grana stacking (Anderson and Aro 1994). Thisimplies that non-photochemical quenching and the amountof photoconvertible violaxanthin depend on grana stacking:while it may only reflect differences in the content of chloro-phyll-proteins in grana stacks of sun and shade leaves, theremay also be a structural effect. A spectacular contraction ofgrana stacks in isolated thylakoids occured in light comparedto dark, due to a decrease in the width of the partition gap andlumen which may be up to 30%, as well as a thinning of thy-lakoid membranes (13–23%) (Murakami and Packer 1970).If this also occurs in vivo when plants are transferred fromthe dark to light, it implies truly enormous reversible changesin the structural organisation of core PSII and associatedLHCII within appressed granal thylakoids, as well as acrossthe narrowed partition gap. The extremely high ‘effectivetwo-dimensional concentration’ of surface charges at the
membrane surface may be equivalent to 10–15 M NaCl(Anderson 1975). Hence, alterations of surface chargebinding and screening during dynamic dark/light changesdue to the light-induced transport of protons and Ca++
(Ettinger et al. 1999) across thylakoids to the lumen, and thecounterbalancing transport of Mg++ from the lumen to thepartition gap and stroma will cause large conformationalchanges within grana stacks. Grana stacking at the structurallevel certainly influences both light harvesting and non-pho-tochemical quenching (see Garab and Mustárdy 1999;Horton 1999).
(i) Extent of grana membrane stacking is influenced by thevery different attenuation of light in terrestrial and aquaticenvironments
Given that grana stacking in terrestrial plant thylakoids israther a means to limit excessive spillover from PSII to PSI,it is necessary to consider how crucial differences in the lightqualities of terrestrial and aquatic habitats influence PSII/PSIstoichiometries, the light-harvesting antennae of both pho-toystems and the extent of membrane stacking. Grana stack-ing is especially relevant for shade plant chloroplasts as theindirect irradiance filtered through the plant canopy isheavily attenuated sunlight which is greatly enhanced in far-red light: shaded terrestrial habitats are highly enriched inPSI light. In these circumstances PSII needs to collect asmuch excitation energy as feasible without excessivespillover of excitation energy from PSII to PSI. However, thissegregation of most PSIIs into appressed domains is insuffi-cient to compensate for the enhancement of PSI light asshading increases steeply through the canopy, individualleaves and even chloroplasts themselves. Significantly, landplants always have more PSII than PSI. Relative to PSI, sunplants have more PSII complexes with smaller antennae thanshade plants which have fewer PSII complexes with muchlarger antennae, with PSII/PSI ratios of 1.1–1.6 for shade andlow-light plants and 1.8 or higher for most sun and high-lightplants (Anderson et al. 1988).
Conversely, the aquatic light environment is very differ-ent. In water with increasing depth, as the light intensity isdecreased, light quality is also changed: first far-red, then redand finally blue irradiance is progressively attenuated. Asintensity decreases, PSI irradiance is greatly attenuated, andPSII light is greatly enhanced (the reverse situation to landhabitats). To compensate for this imbalance in light qualitydistribution to PSII and PSI as water depth increases, aquaticalgae and cyanobacteria need more PSI than PSII complexes(Fujita 1997). Many more light-harvesting pigments are alsoassociated with each PSII, while the light-harvesting antennaof PSI is smaller (Glazer and Melis 1987). Given this photo-system stoichiometry, it is vital that the excitation energyabsorbed by PSII can be transferred to PSI.
Grana formation in vascular plants and some green algae,is but one way of achieving non-synchronous photon pro-
Insights into consequences of appressed grana thylakoids
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cessing by PSII compared to PSI. In Cyanophyta and thechloroplasts of red algae, both photosytems are homoge-nously located within the same unstacked membrane aidingenergy transfer. PSII has an enormous light-harvestingantenna system — the phycobilisomes attached to the outermembrane surface — that not only absorbs 80–90% of totalirradiance, but is usually coupled only to PSII (Glazer andMelis 1987). The absorption cross-section of PSI is rathersmall, but there are always more PSI than PSII complexes.Spillover from PSII to PSI is enhanced, and large differencesin the light-harvesting antenna of the photosystems arecounter-balanced by variable photosystem stoichiometrywith low PSII/PSI ratios of 0.3–0.8 (Fujita 1997)
The Chromophypta (brown algae, dinoflagellates,diatoms and cryptomonads) which possess light-harvestingchlorophyll a/c-proteins often have groups of three looselystacked thylakoid membranes that are separated by a 2–6-nmspace, larger than the separation between stacked thylakoidsin green algae (Staehelin 1986). Although not extensivelyinvestigated, there is no evidence of lateral segregation of thephotosystems in their stacked or unstacked thylakoids(Pyszniak and Gibbs 1992; cf. references in Trissl andWilhelm 1993).
Significantly, even with chlorophyll b-containing greenalgae which have stacked membranes, often extendingthroughout the cell, the lateral distribution of the photo-systems between stacked and unstacked membranes dependson the green algal species (Gunning et al. 1999). Usingimmunogold electron microscopy, Gibbs and colleagueshave shown that both PSII and PSI are located throughout theappressed and non-appressed thylakoid membranes inChlamydomonas reinhardtii and Tetraselmis subcordiformis(Song and Gibbs 1995; Bertos and Gibbs 1998). Gunning etal. (1999) have examined the distribution of PSII in greenalgal chloroplasts using confocal microscopy. The stackedmembrane regions of higher plants have much brighterchlorophyll fluorescence due to PSII which is mainly segre-gated to stacked granal membranes being much more fluo-rescent at room temperature than PSI. Conversely, severalmembers of the chlorophyll c-containing algae known not tohave lateral segregation of the photosystems although pos-sessing stacked membranes, as well as several red algaewhich have unstacked membranes with no segregation of thephotosystems, all showed uniform fluorescence (Gunning etal. 1999). Using this approach, some green algal chloroplasts(e.g. Nitella) show bright fluorescence due to PSII, similar tothe grana seen in higher plant chloroplasts (Fig. 2b) indicat-ing lateral heterogeneity in the distribution of the photo-systems, while others (e.g. Chlamydomonas, Codium,Ultrothrix) have uniform fluorescence indicating that thephotosystems are not laterally separated from each other(Gunning et al. 1999). This technique provides a novel, con-venient approach to determine lateral heterogeneity of thephotosystems in vivo amongst green algae. It also offers a
way to determine when grana appear in terms of plant evolu-tion, and when they become invariable components of somegreen aquatic algae and vascular plants.
Dynamic redox-controlled changes in extent of granamembrane stacking
Lateral heterogeneity in the distribution of the photosys-tems between appressed and non-appressed domains is anoperational definition. The static view of the molecularorganisation of thylakoid membranes inferred from electronmicrographs is really a snapshot in time, and cannot capturetheir highly dynamic nature. To survive and thrive underever-changing light conditions, which vary over two ordersof magnitude, plants have evolved both short and long termmolecular adaptation strategies which enable them to opti-mise photosynthetic efficiency and resource utilisation underall irradiances (Anderson et al. 1995). Dynamic, markedstructural rearrangements of membrane stacking occurduring both short term adaptations which involve lateralmovements of existing thylakoid components, while longterm acclimation modulates the composition, function andmembrane appression of thylakoids.
(i) Regulation of excitation energy distribution between thephotosystems
In the short-term, a dynamic redox-controlled molecularmechanism that regulates energy distribution between thephotosystems, via state 1–state 2 transitions (Allen 1992),also modulates the extent of grana stacking (cf. Staehelin1986). During a state 2 transition, over-excitation of PSIIrapidly results in over-reduction of the plastoquinone poolleading to activation of a LHC kinase that phosphorylates theperipheral 25 kDa protein of LHCIIb (a subset of up to 30%of total LHCIIb). The resultant increased negative chargecauses some grana destacking allowing phosphorylatedLHCIIb to migrate to non-appressed membrane domains.With overexcitation of PSI, the phosphorylated LHCIIb innon-appressed membrane domains are dephosphorylatedand laterally migrate to rejoin PSII-LHCII complexes ingrana-stacked membranes. This reversible change is accom-panied by lateral redistribution of some cytochrome bf com-plexes (Vallon et al. 1991). Activation of LHCII kinasedepends on plastoquinol binding at the quinol oxidation siteof reduced cytochrome bf complex (Vener et al. 1997). Thusthe redox state of both plastoquinone and cytochrome bfcomplex governs the distribution of absorbed light energybetween photosystems by controlling the phosphorylation ofmobile, peripheral LHCII which in turn modulates the extentof grana stacking, allowing rapid reorganisation in responseto sudden fluctuations of light quality and quantity.
A striking example of changes in the redox control ofLHCII-PSII organisation that initiates a marked redistribu-tion of complexes between stacked and unstacked mem-branes via state 1–state 2 transitions has been demonstrated
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in vitro by Weis and colleagues. Isolated thylakoids weredriven to state 1 (incubation under oxidising conditionswithout ATP) or state 2 (incubation with 1 mM ATP and 10mM NaF in low light) and then fragmented into granal,margins and stromal fractions (Spittel et al. 1995). Understate 1, defined as the state with minimal LHCII phosphory-lation, grana are assumed to be tightly stacked: 47% of totalchlorophyll and almost all PSII complexes were located instacked membranes, with 10% of total chlorophyll in granamargins (Spittel et al. 1995). Under state 2, the total amountof chlorophyll in the grana margins increased at the expenseof stacked membranes, and there was exchange of pigmentcomplexes between stroma and margins, indicating thatgradual destacking of grana thylakoids led to a largerexposed margin area (Andrée et al. 1995). Concomitantly,there was a marked redistribution of cytochrome bf com-plexes with state 1–state 2 transitions, with the fraction oftotal cytochrome bf complexes decreasing from about 70% instate 1 to 30%, as the stacked membrane area is diminished(Spittel et al. 1995). This dynamic redox-controlled redistri-bution of cytochrome bf complex between non-appressedand appressed membrane domains in vivo regulates linearand cyclic electron transport.
(ii) Acclimation of composition leads to changes in struc-ture and function
During long term acclimation, there are marked decreasesin grana stacking in sun and high-light plants compared withshade and low-light plants, resulting from changes in thecomposition of thylakoid components (Anderson et al.1988). The adjustment of photosystem stoichiometry whichaccompanies modulation of grana stacking is striking:marked adjustments of both the amounts of core PSII and PSIcomplexes and LHCII and LHCI are required to optimisequantum efficiency at limiting light of diverse quality (Chowet al. 1990). Sun and high-light plant chloroplasts (higherChl a/Chl b ratios) have many more PSII complexes eachwith smaller light-harvesting antennae, relative to PSI, thanshade and low-light thylakoids with fewer PSII complexeseach with larger light-harvesting antennae (Anderson et al.1988). Shade and low-light chloroplasts have more chloro-phyll and more stacked vs unstacked domains for maximallight capture, but less electron transport, photophosphoryla-tion and carbon fixation components resulting in lowerphotosynthetic rates which saturate at lower intensities.Conversely, sun and high- light chloroplasts are limited inelectron transport rather than light capture: they have greateramounts of cytochrome bf complex, ATP synthase, plasto-quinone, plastocyanin, ferredoxin and carbon fixationenzymes to support high photosynthetic rates which saturateat high irradiance (Anderson et al. 1988).
These acclimative changes of composition, function andstructure are so beautifully orchestrated that even the
Chl a/Chl b ratios of leaves are a simple index of light accli-mation. Chl a/Chl b ratios are linearly related to the contentand activity of cytochrome f, ATP synthase, Rubisco, andinversely correlated with amounts of LHCII and LHCI(Anderson et al. 1988) and extent of grana stacking(Anderson and Aro 1994). Such dynamic adjustments of thephotosynthetic apparatus to varying environmental cues arefully reversible. Shade and low-light plants transferred tohigh light, or vice versa, will acclimate and both compositionof the photosynthetic apparatus and amounts of stackedmembranes are dynamically altered (Melis 1991; Andersonet al. 1988, 1995).
(iii) Dynamic changes in grana stacking are regulated byredox-controlled signal transduction elicited by the bindingof plastoquinol to cytochrome bf complex
Significantly, the signal transduction mechanism gov-erned by the redox state of plastoquinone and cytochrome bfcomplex that regulates the short-term distribution of lightenergy between the photosystems by controlling the phos-phorylation of mobile peripheral LHCIIb as outlined above,is also involved in regulating gene transcription.Pfannschmidt et al. (1999) have elegantly demonstrated thatthe rate of transcription of chloroplast genes encoding thereaction centre apoproteins of PSII and PSI also depends onthe redox state of plastoquinone: this chloroplast geneexpression is direct and rapid, permitting transcriptionalresponses within minutes of perturbation of the redox state ofelectron carriers between the photosystems. Thus this signaltransduction mechanism of redox control of transcription ofchloroplast genes regulates the stoichiometry between thetwo photosystems, and adjusts the PSII/PSI ratio in responseto changes in illumination that favour either of the two pho-tosystems. In eukaroytes, these PSII and PSI reaction centreproteins are encoded universally within the chloroplast,while other genes have been removed to the cell nucleus:Allen (1993) suggested that direct and rapid chloroplast tran-scriptional responses may depend upon the genes concernedbeing in the same organelle as the electron transport chainthat regulates their expression (Pfannschmidt et al. 1999).
The rate of transcription of nuclear genes encodingLHCIIb is also controlled by the redox state of plastoquinonein Dunaliella species (Escoubas et al. 1995; Maxwell et al.1995), but this has not yet been demonstrated in plants. Intransgenic tobacco, generated using an antisense constructagainst the Rieske Fe-S protein, that contained very lowlevels of cytochrome bf complex, there was no light acclima-tion to irradiance, despite marked photochemical reductionof the plastoquinone pool: this demonstrates that both plasto-quinol and cytochrome bf complex must act together toregulate gene transcription (Anderson et al. 1997b). Thisrequirement for the interaction of plastoquinol withcytochrome bf has been directly shown to occur in the signal
Insights into consequences of appressed grana thylakoids
Jan M. Anderson 634
transduction between light and protein phosphorylation,where functional LHCII kinase-cytochrome bf complexinteraction is analogous to a signal transduction systemwhere the receptor is cytochrome bf complex, and the ligandis plastoquinol attached to its QO site (Vener et al. 1997).
Other cellular pathways, including carbon fixation, carbo-hydrate and nitrogen metabolism, exert feedback influenceson photosynthesis which will also be sensed by the redoxstate of the intermediate carriers (Anderson et al. 1995;Huner et al. 1996; Durnford and Falkowski 1997) (Fig. 7).Since the oxidation of plastoquinol by cytochrome bfcomplex is the rate-limiting step of photosynthesis, it is theperfect point for redox control to regulate not only light har-vesting between the photosystems, but also transcription ofchloroplast and nuclear genes. The grand design of photo-synthesis is wondrously woven into the tapestry of plant cellfunctionality under normal and stressed conditions(Anderson et al. 1995), since the redox poise of plasto-quinol/cytochrome bf complex is also central to the function-ality of PSII in the complementary processes of light usagebeing dependent on the redox state of QA (Öquist et al. 1992)and energy dissipation by non-photochemical quenching, asalready discussed.
Grana protect non-functional PSII complexes stillcontaining D1 protein from degradation under
sustained high irradiance
Paradoxically, as a dynamic molecular machine, PSIIcomplex has a limited life. The extremely high oxidisingpotential of P680+ required to oxidise water means that PSIIfunctionality is always at risk, despite many photoprotectivestrategies which have evolved to regulate photosystem IIlight absorption. These include physiological responseswhich decrease incident light, such as leaf and chloroplastmovement, waxy cuticles, anthocyanin content, and so on. Aspointed out, dynamic acclimation of the photosyntheticapparatus modulates both energy utilisation (via state transi-tions, cyclic electron transport around each photosystem andacclimation) and energy dissipation including modulation ofthe Chl a/b-proteins of LHCII and LHCI, regulation of thetotal pool of xanthophyll cycle carotenoids (violaxanthin,antheraxanthin and zeaxanthin) and amount of photocon-vertable violaxanthin. These photoprotective responses,together with D1 protein turnover to restore PSII function,and enhancement of oxygen scavenging systems, are all inte-grated in a dynamic time continuum. Nevertheless, an
Fig. 7. Chloroplasts not only transduce light, but also sense environmental and metabolic cues. Light absorbed by PSII and PSI is consumed in many bio-chemical reactions of photosynthesis and the contiguous metabolic processes of carbon, nitrogen, sulphur and lipid metabolism or dissipated as heat mainlyin PSII. Under stress, be it high light or other factors, the capacity to utilise this light is greatly diminished due to feedback inhibition of photosynthesis orassociated metabolic processes. The photosynthetic apparatus responds to energy imbalance between light utilisation and consumption elicited by environ-mental stress, by dynamic redox sensing/signalling mechanisms in the photosynthetic electron transport chain: sensing between the photosystems by reducedplastoquinol bound to cytochrome bf complex, and beyond PSI by ferredoxin/thioredoxin. Ensuing signal transduction cascades modulate the rate of chloro-plast and nuclear gene transcription of the photosynthetic apparatus to allow acclimation to stress, and once again to balance excitation energy supply to thephotosystems and its consumption via utilisation and dissipation.
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inevitable consequence of PSII function is its photoinactiva-tion in vivo, which is a probability and light dosage event thatdepends on the number of photons absorbed rather than therate of absorption (Anderson et al. 1997a). This means thatPSII photoinactivation occurs at all light levels from limitingto saturating and super-saturating light. It is no trivial eventfor a leaf: even in weak light (100 µmol photons m–2 s–1),some million PSIIs per mm2 of leaf surface are inactivatedevery second (Lee et al. 1999).
While the molecular mechanism of the initial steps of PSIIphotoinactivation in vivo is unknown, it appears to depend onthe generation and maintenance of increased concentrationsof the primary radical pair, P680+ Pheo–, and the variousways charge recombination is regulated under different envi-ronmental conditions (Anderson et al. 1998). We suggest thatP680+ is the dominant oxidative agent that photoinactivatesPSII in vivo rather than triplet P680 generating singletoxygen (Anderson et al. 1998). We hypothesise that P680 isshielded from oxygen in functional PSII, and any tripletP680 formed is rapidly quenched and unable to act with O2:it is only in non-functional PSIIs which undergo a markedconformational change upon inactivation that triplet P680formed by charge combinations reacts with O2 to singlet O2.
The decline in functional PSIIs is greatly accelerated bythe chloroplast-encoded protein inhibitor, lincomycinshowing that the rate of photoinactivation reflects a balancebetween damage to D1 protein and repair (Anderson et al.1997a). To restore PSII function, the targeted D1 protein ofthe D1/D2 protein heterodimer that binds the redox compo-nents of the PSII reaction centre must be degraded and syn-thesised de novo (Aro et al. 1993). In higher plant thylakoids,photoinactivation of PSII occurs in appressed granaldomains, while the coordinated processes of D1 proteindegradation and de novo synthesis occur in non-appresseddomains, the region accessible to chloroplast ribosomes(Melis 1991; Aro et al. 1993).
Under prolonged high-light illumination, non-functionalphotoinactivated PSIIs still possessing D1 protein accumu-late in stacked granal domains where they are protected fromdisassembly. Hence grana stacking in higher plants is ameans whereby the rate of D1 protein degradation and D1protein turnover is highly regulated (Anderson and Aro 1994and references therein). In peas acclimated to growth in dif-ferent light intensities the extent of membrane stacking islinearly related to the extent of photoinactivation of PSII, butinversely related to the rate of D1 protein degradation(Fig. 8). Hence low-light plants with greater amounts ofappressed membrane are more susceptible to photoinhibitionbut less D1 protein is degraded than in high-light plants.Furthermore, in leaves, unlike in many algae, D1 proteindegradation and turnover increases up to light saturation,with D1 protein turnover being fast enough to prevent netphotoinactivation of PSII in vivo. However, D1 proteindegradation and turnover decline under sustained photoin-
hibitory light, when non-functional PSIIs still containing D1protein accumulate, especially in the huge granal stacks ofdeep shade plant chloroplasts (Fig. 9). Light-dependentphosphorylation of D1 protein of non-functional PSIIs inappressed thylakoids prevents its degradation (Rintamäki etal. 1995). These non-functional PSIIs continue to dissipateexcess excitation as heat and may help protect functionalPSII-LHCII neighbours in grana stacks (Öquist et al. 1992).Recovery of PSII function occurs under low light because D1protein synthesis saturates at low irradiance (Anderson et al.1997a). This is vital because much of crop and tree photo-synthesis takes place at low light due to attenuation throughthe canopy.
Again, this photoprotective regulatory mechanism locatedin appressed granal domains in higher plants that preventsD1 protein degradation and turnover in non-functional PSIIs,does not occur in lower plants like mosses whose membranesare not structurally differentiated (Rintamäki et al. 1995). Inmany Chlorophyta, such as Chlamydomonas, two or threethylakoid membranes are loosely stacked over very longregions within the cell, yet there may be no lateral hetero-geneity in the distribution of the photosystems (Bertos andGibbs 1998; Gunning et al. 1999). Not suprisingly, the D1protein of Chlamydomonas is not phosphorylated but isreadily degraded under sustained high light (de Vitry et al.1991). Similarly in the Cyanophyta and red algae, where PSIIand PSI complexes as well as chloroplast ribosomes arehomogeneously distributed through unstacked membranes,there is neither accumulation of non-functional PSII still
Insights into consequences of appressed grana thylakoids
Hal
f-lif
etim
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Granal cross-sectional area (%)
10 15 20 25
140
120
100
80
60
20
40
60
80
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/Fm
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Fig. 8. Relationships between both the susceptibility of PSII to photoinac-tivation (measured as the decline in Fv/Fm ratios (●●) and the half-life time ofD1 protein following photoinhibitory light treatment of lincomycin-treatedleaves (●) with the extent of granal stacking of pea leaves acclimated to dif-ferent growth irradiances. More PSII is photoinactivated and less D1 proteinis degraded in low-light peas with more appressed thylakoids than high-lightplants. The extent of granal appressed membranes which was measured bytheir cross-sectional area per chloroplast is inversely proportional toChl a/Chl b ratios (Anderson and Aro 1994). (Redrawn from Anderson andAro 1994).
Jan M. Anderson 636
containing undegraded D1 protein nor reversible D1 proteinphosphorylation (Kanervo et al. 1993). It appears that inaquatic environments when light intensity is rapidly attenu-ated with depth, many algae and cyanobacteria can moverapidly away from high light, and with short life cycles thereis little need to store non-functional PSIIs, in contrast to thevery long-lived leaves of extreme shade plants.
Membrane compartmentation aids regulatory pathwaysand separates function
More generally, it seems that grana stacking is an exampleof specialised extension of cellular compartmentation whichhas occurred during evolution as a general strategy for theregulation of increasingly complicated pathways, as well asthe avoidance of uncontrolled interactions between catalyticcentres (Anderson and Andersson 1988). As pointed out,lateral heterogeneity in the distribution of the photosystemsin grana-containing chloroplasts increases both the ability of
PSII to regulate light energy effectively by balancing photo-chemical utilisation with non-photochemical dissipation, toenhance maximal linear electron transport, and to protectnon-functional photosytem II parked in stacked granaldomains under sustained high irradiance. The need for theimportant photoprotective mechanism to prevent high D1protein turnover under high photon exposure in higher plants,especially those grown in the shade, is due to conflictingdemands between efficient use of low irradiance and protec-tion of PSII under high light.
However, there may be other advantages with membranecompartmentation concerning the more fluid, non-appressedmembrane domains. Since the area occupied by proteincomplexes is very much greater in appressed granal mem-branes (Staehelin 1986), unstacked membrane domains,grana margins and inter-linking stroma thylakoids, havemore fluid lipid bilayers. More fluid non-appressed mem-brane domains allow free lateral diffusion of componentsalong the membrane required for light energy regulationbetween the photosystems, a redistribution of componentsduring membrane stacking processes, and permit the inser-tion of proteins and lipids during membrane biogenesis andacclimation. Like the large head group of ATP synthase,bulky chloroplast ribosomes cannot penetrate between granapartitions. Thus, both chloroplast-encoded and nuclear-encoded peptides are inserted into the more fluid non-appressed thylakoids facilitating assembly into thylakoidcomplexes or transport to the chloroplast lumen.
Unstacked thylakoids also house most of the auxiliaryenzymes associated with stress regulation, regulatory pro-teases, thylakoid kinases and phosphatases associated withsignal transduction mechanisms and gene expression, as wellas enzymes associated with the final stages of chlorophylland carotenoid biosynthesis, lipid desaturases, and more.Many of the ‘house-keeping’ functions of the photosyntheticapparatus such as the proteases are located in stroma thy-lakoids (Andersson and Aro 1997). It may also be advanta-geous to locate these many auxiliary proteins well away frompotential damage from highly oxidative species such assinglet O2 and other reactive species associated with water-splitting processes of PSIIα in grana stacks, particularlybecause the auxiliary proteins all occur at very much lowerconcentrations than the light-harvesting and electron/protontransport proteins.
Finally, since the signal transduction mechanism mediatedby the redox state of intermediate carriers that controlsenergy imbalance between the photosystems as well as genetranscription involves both plastoquinol and cytochrome bfcomplex, it may be significant that some cytochrome bfcomplex migrates from appressed to non-appressed domainsunder the influence of plastoquinone reduction (Vallon et al.1991; Andrée et al. 1995). The kinetics of chloroplast geneexpression of reaction centre proteins being rapid and directare reminiscent of transcriptional control in prokaryotes
Fig. 9. Comparison between sun and shade thylakoids showing long-termacclimation of PSII and extent of appressed vs. non-appressed membranedomains under high photon exposure; (based on Anderson and Aro 1994).
637
(Pfannschmidt et al. 1999). Since prokaryotic membranesare unstacked, plastoquinol/cytochrome bf complex mayneed to migrate to non-appressed membranes before initiat-ing transcription of chloroplast genes. The redox state of theferredoxin/thioredoxin which always occurs in non-appressed domains, also regulates feeback regulation ofenzymes involved in carbon fixation (Fig. 7).
Conclusions
To summarise, some consequences of grana stacking are:(i) enhancement of PSII function from limiting, sub-limitingto saturating light; (ii) protection of PSII at sustained highirradiance; (iii) regulation of dynamic grana stacking whichis driven by signal transduction dependent of the redox stateof plastoquinol and cytochrome bf complex; and (iv) alinkage to more fluid non-appressed domains to aid in regu-lation of auxiliary enzymes, proteases, and redox transcrip-tional control of chloroplast- and nuclear-encoded genes.Fascinating challenges for further exploration include ques-tions such as when did grana evolve, and why? Does the dra-matic contraction of grana stacks and concomitant thinningof thylakoids in light compared to dark observed with iso-lated thylakoids occur in vivo, with resultant enhancement inlight excitation energy utilisation and dissipation?
Epilogue
‘We shall not cease from explorationAnd the end of all our exploringWill be to arrive where we startedAnd know the place for the first time’
T.S. Elliot, The Waste Land (1922)
Acknowledgments
‘No man is an island’: my ‘why grana?’ exploration owesmuch to those colleagues who have accompanied me,lengthily or briefly during this particular journey. I amextremely grateful for the help and support of HeatherAdamson, Bertil Andersson, Eva-Mari Aro, Jack Barrett,Keith Boardman, Olle Björkman, Fred Chow, John Evans,Robin Hill, David Goodchild, Tony Larkum, Ta-Yan Leong,Li-Xia Liu, Dick Malkin, Tasso Melis, Stephanie McCaffery,Barry Osmond, Gunnar Öquist, Youn-Il Park, John Sinclair,Cecilia Sundby Emanuelsson, Bill Thomson, and JohnThorne. I appreciate helpful discussions with Fred Chow,Paul Kriedemann and Barry Osmond, and access tomanuscripts in press. This paper is dedicated to my friends,Barry and Cornelia Osmond, who hosted a splendidFestschrift at Schloss Arnsberg.
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Manuscript received 11 May 1999, accepted 27 July 1999
Insights into consequences of appressed grana thylakoids
