Review Chemical root to shoot signaling under drought · 2011-02-25 · Chemical root to shoot...

Chemical root to shoot signalingunder droughtDaniel P. Schachtman1 and Jason Q.D. Goodger2

1 Donald Danforth Plant Science Center, 975N. Warson Rd, St. Louis, MO 63132, USA2 School of Botany, The University of Melbourne, VIC 3010 Australia

Review

Chemical signals are important for plant adaptation towater stress. As soils become dry, root-sourced signalsare transported via the xylem to leaves and result inreduced water loss and decreased leaf growth. The pre-sence of chemical signals in xylem sap is accepted, butthe identity of these signals is controversial. Abscisicacid (ABA), pH, cytokinins, a precursor of ethylene,malate and other unidentified factors have all beenimplicated in root to shoot signaling under drought. Thisreview describes current knowledge of, and advances in,research on chemical signals that are sent from rootsunder drought. The contribution of these differentpotential signals is discussed within the context of theirrole in stress signaling.

Chemical and hydraulic signaling under droughtstressNumerous studies have shown that plant roots can sensechanges in abiotic factors such as soil water content [1,2],soil bulk density or compaction [3], soil oxygen content [4]and changes in the nutrient composition of soil (bothenrichment and depletion) [5]. Root sensing of water deficithas been widely studied, and this review focuses on theoutcomes of the early stage of water-deficit sensing: thetransport through the xylem of chemical signals that ulti-mately reduce leaf transpiration and leaf growth. Chemi-cal signals can be differentiated from hydraulic signals [6]but both are important because they reduce stomatalconductance and leaf growth under conditions of waterdeficit. Chemical signals most probably dominate duringearly stages of stress before hydraulic signals are produced[7], and become less important under severe drought whenleaf water potential declines and leaves wilt. It is likelythat the hydraulic signals that may trigger the productionof the hormone abscisic acid (ABA) in leaves dominate asplants becomemore water stressed [8]. This review focuseson chemical signals because of their importance during theearly stages of plant response to water deficit, and becausetheir manipulation has applications in increasing wateruse efficiency (WUE) in agriculture.

Agricultural practices involving chemical signals:deficit irrigation and partial root-zone dryingRoot to shoot signaling under conditions of both mild andsevere drought is an important area for research because ofits implications for agricultural production and theWUEofplants. Fresh water supplies are predominantly used for

Corresponding author: Schachtman, D.P. ([email protected]).

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agriculture, but this usage will need to be reduced in thefuture as supplies become limited. Where irrigation can bemanipulated, researchers have experimented with, andsuccessfully implemented, both deficit irrigation and par-tial root-zone drying (PRD) [9,10]. Both of these dryingregimes reduce leaf transpiration and limit vegetativegrowth, thereby increasing WUE, and have demonstratedefficacy in both woody perennials [9] and herbaceous cropplants [11,12] (Box 1). Although these treatments canincrease the WUE of certain crop species, knowledge ofthe role of chemical signals sent from root to shoot duringsuch practices is still developing [13]. Some studies haveshown that chemical signals such as ABA are generated bythe reductions in soil water content and act on the leaves toreduce transpiration and growth [14].

Where PRD and deficit irrigation have been used inagriculture, themost success has come from applications totree crops and to vines (in viticulture) [9,10]. With horti-cultural crops, the management of individual plants ismore intensive and economic return is often linked to fruitquality. In one study, the use of PRD or deficit irrigation inviticulture increased WUE by about 40% while onlydecreasing yield by 15% [10]. PRD improved the qualityof the grape berry by increasing the anthocyanin content[10] and the quality of wine produced [1]. In tomato (Lyco-persicon esculentum), PRD has been shown to increase cropquality. For example, one PRD study found a 21% increasein the Brix value of fruit (a measurement of the mass ratioof dissolved sugar to water), and a 50% increase in WUEwith only a 15% decrease in yield [15]. A physiologicalexplanation for such a small reduction in yield has beenproposed on the basis that the fruit receives water andminerals from the xylem early in development, but later,more than 90% of the water supplying the fruit is trans-ported via the phloem [15]. Particularly in arid climates,the use of precision irrigation methods such as PRD anddeficit irrigation puts into practice the principles developedfrom basic scientific studies on how root signals increaseWUE while maintaining crop quality and yields.

ABA is a key regulator of leaf stomatal conductanceThe production of ABA in roots and its transport to theleaves provides the plant with a mechanism for transmit-ting a chemical signal to report on the water status of thesoil. This system could have evolved specifically for thispurpose or the chemical signal could merely be a con-sequence of the increased production of ABA required tomaintain root growth under water deficit [16]. A dominant

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Box 1. Partial root-zone drying and deficit irrigation in

plants

Partial root-zone drying (PRD) and deficit irrigation (DI) are

examples of the manipulation of root signals to enhance the water

use efficiency (WUE) of agriculture. PRD is an irrigation method

where half the root zone is allowed to dry while the other half is kept

wet. After a period of time, the irrigation is alternated so that the

previously dry root zone receives water and the previously watered

side is allowed to dry. DI is an irrigation method in which the

amount of water supplied is less than that required to achieve

maximum crop evapotranspiration. Both methods can reduce

transpiration and leaf growth while increasing WUE with only small

decreases in yield.

Outstanding questions

Is the source of ABA that results in stomatal closure under drought

coming from the roots or the leaves?

How much variation in root to shoot signaling and xylem sap

composition is there within a species?

How does the nutrient status of a plant interact with chemical

signaling during water stress?

In which region of the root and in which particular root cells is ABA

synthesized?

What are the different forms of cytokinins that are transported in

xylem sap, and what roles do they play in root to shoot signaling?

What causes changes in pH in sap under drought and why do pH

changes appear inconsistent between certain species?

Review Trends in Plant Science Vol.13 No.6

role for ABA in root to shoot signaling under drought and inthe control of stomatal conductance was demonstrated inearly reports [17]. Several groups have also used a bioassayapproach to show that ABA that is fed to leaves decreasestranspiration [18,19]. It is also well known that ABA has adirect effect on guard cell closure [20]. However, the ABAthat acts on guard cells may not originate entirely in theroots. Recent reports suggest that the ABA that acts onguard cells could be produced in the leaves of some species,such as tomato and sunflower [21–23]. In tobacco (Nicoti-ana plumbaginifolia), grafting experiments showed thatABA came from the roots under conditions of drought [24].

ABA is synthesized in both roots [25] and leaves, but notmuch is known about the precise location of this synthesisin roots, which may influence how plants perceive andmonitor soil water content. ABA content in roots is wellcorrelated with both soil moisture and the relative watercontent of roots inmany plant species [26,27]. Some reportssuggest that ABA is synthesized in root tips [28], butsynthesis has been assessed mainly by measuring ABAcontent rather than synthetic activity, so conclusionsrelated to the site of synthesis need to be re-examinedmore carefully. One study using detached roots of pea(Pisum sativum) and Asiatic dayflower (Commelina com-munis) found that ABA synthesis occurred somewherebetween the root tip and a point 3 cm distal to the tip[29]. Recent studies have tried to locate enzymes that areinvolved in ABA biosynthesis, including aldehyde oxidase,which catalyzes the final steps in ABA biosynthesis; how-ever, these studies have focused on ABA production underdiffering nitrogen nutrition rather than drought stress[30]. More work will be required to clarify exactly wherethe ABA is produced in roots under drought stress.

Although the role of ABA in controlling stomatal con-ductance is strongly supported by many experimentsand experimental approaches [25], there may be other

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substances in the sap that act in concert with ABA tocontrol drought-induced stomatal closure. For example,recent findings have suggested an interaction betweenjasmonic acid and ABA in plants under drought stress[31]. In addition, the dominant role of ABA as a root toshoot signal has been challenged by experiments showingthat the ABA concentrations of xylem sap from drought-stressed plants were much lower than the concentrationsof exogenous ABA required to close stomata in detachedleaves [32]. It may be that the use of exogenous hormonesin bioassays may exclude important components of xylemsap that act synergistically with ABA in planta, thusaccounting for the higher levels of exogenous ABA thatare required to reduce transpiration. Grafting experimentshave also been used to determine the source of ABA indrought-induced stomatal closure. Some of these exper-iments suggest that leaf-sourced ABA is important forstomatal closure [22], but the influence of ABA precursorsthat might be transported from roots to leaves was notmeasured, and therefore the role of roots in chemicalsignaling was not completely resolved [25]. Other studiesshow that production of ABA in the rootstock has a stronginfluence on and is negatively correlated with stomatalconductance [33]. Overall, ABA is a dominate signal incontrolling growth and transpiration, but other factorscould also be important. The importance and role ofroot-sourced ABA is still controversial, but some of theconflicting findings may be due to differences in the inten-sity of stress imposed and the time-course of the devel-opment of water deficit.

Involvement of pH in signalingChanges in the pH of xylem sap commonly observed underdrought stress can be an important component of root toshoot signaling and may act synergistically with ABA. Inmany plant species (e.g. sunflower [Helianthus annuus][34],Phaseolus coccineus [35] andC. communis [36]), xylemsap pH becomes more alkaline when plants are waterstressed and this leads to enhanced stomatal closureand even reduced growth. The potential effects of pH havebeen outlined previously [37] and include: i) changes inABA metabolism resulting in increased leaf ABA concen-tration; ii) direct effects on leaf water status that couldalter guard cell turgor or sensitivity to leaf ABA concen-trations; iii) direct effects on ion fluxes through the guardcell plasma membrane; and iv) altered distribution of ABAin the leaf, specifically an increase the concentration ofABA in the apoplast outside of the guard cells (Figure 1). Itis this change in apoplast pH that probably has the great-est role in signaling.

As ABA is a weak acid it may become protonated ordeprotonated in the pH range found in the apoplast ofleaves. This was clearly shown in the early 1980s inexperiments on mesophyll protoplasts that documentedhigh rates of ABA uptake at acidic pH and almost nouptake at the more alkaline pH of 7.5 [38]. In certain plantspecies, the pH of the leaf apoplast increases as the soildries because of the delivery of xylem sap that is of a morealkaline pH. The ABA that arrives from roots via the xylemwill remain deprotonated under the more alkaline con-ditions and will not be taken up passively by mesophyll


cells. The result is that less ABA is transported into themesophyll cells and a build-up of ABA in the apoplast leadsto enhanced stomatal closure. Under well-watered con-ditions, when the apoplast is more acidic, ABA wouldpassively enter the symplast, and apoplastic concen-trations would not increase as rapidly as they do underwater stress. Therefore, the effect of pH is indirect in that itleads to the accumulation of ABA in the apoplast andenhances the effect of ABA on guard cells [35].

This mechanistic explanation is both attractive andintuitive. Nevertheless, the explanation sets up anotherhypothesis that ABA receptors are on the plasma mem-brane rather than inside the guard cells. Recently, threeABA receptors have been found [39–41]. Two of these ABAreceptors reside inside the cell but a third was found on thecell surface [39]. Therefore, plant cells could sense bothextracellular and intracellular ABA concentrations. Underconditions of drought, the increased stomatal closure thatoccurs because of the increased pH of sap suggests thatextracellular ABA is sensed by guard cells via receptors onthe plasma membrane.

Several studies have shown that pH does not act aloneon guard cells or in controlling leaf growth. For example,the use of tomato [42] and barley (Hordeum vulgare) [43]mutants that are deficient in ABA biosynthesis showedthat ABA was necessary for both stomatal closure and theinhibition of leaf growth. In the tomato mutant flacca,transpiration increased as the pH of artificial sapincreased, suggesting that ABA is also necessary to pre-vent stomatal opening andwater loss. In the barleymutantAz34, ABA was necessary for pH to act as a signal underconditions of drought stress [43]. Many studies that show arole for pH in stomatal closure have used tomato as amodelsystem. It is important to point out, however, that theremay be differences in response to ABA and pH betweenspecies and perhaps even between genotypes within thesame species. One study compared C. communis and Ara-bidopsis thaliana and found that the increased pH ofexternal solutions led to a 27% increase in stomatal open-ing in C. communis but had no effect on A. thaliana [44].Both species, however, responded similarly in the presenceof ABA and only changes in the kinetics of response wereobserved. In soybean (Glycine max) [45], xylem sap pH didnot change in response to mild drought and was notcorrelated with decreased stomatal conductance duringthe initial stages of drought. The xylem sap of soybeandid, however, become more alkaline during extendeddrought, but this alkalization occurred well after ameasured decrease in stomatal conductance.

The response of xylem sap pH to drought stress does notappear to be consistent in all species or even in differentexperiments using the same species. One study showedthat the sap pH of sunflower and C. communis did notchange significantly as soil dried, whereas the xylem sappH of tomato did increase as soil dried [46]. In maize (Zeamays) grown using ammonium as the nitrogen source,there was no change in sap pH during early [7] or evenduring later stages of drought (L. Ernst and D.P. Schacht-man, unpublished). Other experiments using maize havebeen conducted in the field and have found that the sap pHof drought-stressed plants growing on a loam soil wasmore

alkaline as the period of drought lengthened [47]. Suchinconsistencies may be explained by the fact that soluteconcentrations within the xylem sap vary with flow rate inintact plants [48,49]. The sap solute concentrations anddelivery rates that best represent those of xylem sap flow inintact plants are most accurately determined when sap iscollected at rates equivalent to whole-plant transpiration.Therefore the rate of whole plant transpiration must bedetermined prior to detopping plants to harvest sap. Exter-nal pressure must be applied to roots so that sap exudes atthe flow rate of whole plant transpiration, a fact oftenoverlooked by experiments in which only xylem sap iscollected [49].

The mechanism of pH change might involve nitrateavailability. As soils dry, nutrient availability is reducedbecause of physiochemical changes. Under conditions ofreduced nitrate availability, nitrate reductase activityshifts to roots, causing changes in the pH of sap [50].Changes in the activity of nitrate reductase that are causedby drought conditions lead to changes in organic acidproduction, which alter sap pH. In particular, malateconcentration increases in xylem sap under drought,resulting in sap that is less acidic than that inwell-wateredconditions when more nitrate is loaded into the xylem.When the effects of a nitrate reductase inhibitor (sodiumtungstate) were tested [51], the inhibitor was found toprevent the alkalization of the sap in tomato under droughtconditions. Similarly, the addition of nitrate to tomatounder water-stress conditions enhanced the alkalizationof the xylem sap [46]. A synergistic effect of xylem nitrateand ABA on stomatal conductance in detached leaves of C.communis has also been shown [46]. Moreover, the resultsof work on maize suggest that, under some circumstanceswhere neither compound is as effective alone, a combi-nation of ABA and nitrate is required to elicit an effect onshoot physiology [52].

The effects of changes in the internal pH of the guardcells have been very well characterized [20]. Less is knownabout the effects of apoplastic changes in pH on guard cells,but one study [53] showed that stomatal opening is inhib-ited by acidification and that the plasma membrane poten-tial is depolarized (which would facilitate the opening ofoutward-rectifying potassium channels) when the apoplastis acidified. More studies are needed to clarify the effects ofchanges in external pH on guard cells when the internalpH is held constant. If extracellular alkalinizationalso leads to an increased intracellular pH, it could bean important part of the signaling process for stomatalclosure.

Conjugates of ABA as potential signalsAlthough ABA seems to play a dominant role in root toshoot signaling under drought, it also seems likely, on thebasis of several studies, that other substances are alsoinvolved. ABA is present in xylem sap in conjugated forms,such as abscisic acid-glucose ester (ABA-GE). It has beensuggested that a conjugated form of ABA could serve as atransported form of the hormone and, moreover, as a stresssignal [32,54]. As many as six ABA conjugates have beenfound in xylem sap from well-watered and water-stressedsunflower [55], and a correlation was found in one study

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Figure 1. Changes in xylem sap composition under drought. Drought causes the alkalinization of xylem sap pH in certain plant species. (a) Well-watered plant with

apoplastic pH 6.0. (b) Plant under drought conditions with apoplastic pH 7.0. In plants in which xylem sap pH increases when the soil becomes dry, ABA-induced stomatal

closure is enhanced. This is thought to be due to increased apoplastic concentrations of ABA. Further changes in xylem sap composition under drought are also responsible


284


between increased ABA-GE concentrations and drought[56]. The importance of conjugated forms of ABA indrought responses and the mechanisms of hydrolysis[57] of such forms of ABA in the apoplast are, however,still poorly understood. A next step in establishing theimportance of conjugated forms of ABA might be to geneti-cally vary the hydrolysis of ABA conjugates. To do this, itwill be essential to identify the genes encoding the apo-plastic b-glucosidases that are responsible for hydrolysisbefore subsequent manipulation of these enzymes canproceed.

Cytokinins as signalsCytokinins could also be an important signal travelingfrom roots to the shoots. Root-produced cytokinins areclearly involved in responses to nutrient deprivation [5]and, as they are produced mainly in roots, could be import-ant in drought responses [58]. Despite this, there havebeen few reports that provide information on the cytokinincontent of xylem sap and how that content changes underdrought conditions. In grapevines, a 50% reduction inzeatin (Z) and zeatin riboside (ZR) was found in plantsthat had been subjected to PRD [59]. In amore recent studyin which Z, ZR and zeatin nucleotide (ZN) were measured,PRD of tomato reduced the ZN content of the xylem sap,but the magnitude of that change and the contribution ofZN to the total cytokinin content were not shown [60]. In atleast two studies on sunflower, xylem sap, combined Z andZR and combined isopentenyladenine and isopentenylade-nosine concentrations in xylem sap decreased underdrought-stressed conditions [61]. It is possible that theABA:cytokinin ratios in xylem sap are important for stresssignaling [55]. The effects of cytokinins are also supportedby a study showing that transpiration was higher in trans-genic plants that overexpressed the isopentenyltransfer-ase, causing the plants to produce more cytokinins after aheat shock treatment [62]. Although recent data showdecreased cytokinin concentrations in the xylem underdrought stress, it is still not clear that all plant speciesrespond in the same way to cytokinin at the concentrationsfound in the leaf and guard cells [63].

Strong conclusions about cytokinin content are prema-ture because the complexity of the cytokinin profiles hasnot been fully explored [64]. In a recent study onmaize [65],we found a decrease in Z and ZR concentrations in xylemsap coming from roots of drought stressed plants as com-pared to well watered controls. Surprisingly, we also foundhigh concentrations of the aromatic cytokinin 6-benzyla-minopurine (BAP) inmaize xylem sap, the concentration ofwhich increased significantly as a result of water stress[65]. With the availability of increasingly sensitive massspectrometry methods, it should be possible to explorecomprehensively the changes in concentrations of many

for reduced transpiration and inhibition of leaf growth. These changes include increases

in flow rates, and reductions in the concentrations of cytokinins, zeatin and zeatin ribosid

result in stomatal closure. (c) Stomatal closure under drought. The affects of ABA on

receptor, such as the recently identified GCR2 (G-PROTEIN COUPLED RECEPTOR2), b

magnesium protoporphyrin-IX chelatase that is localized in the chloroplast), cannot b

could also be important in transducing the increased ABA concentrations in the apoplast

(K+) and anions (A�) leads to stomatal closure. SLAC, slow anion channel; RAC, rapid

of the cytokinins, and to identify those that play a role inroot to shoot signaling.

Other chemical signalsChemical and protein-based factors other than ABA, cyto-kinins or pH could also be involved in root to shoot sig-naling. Although it has now been established clearly thatxylem sap from many plant species contains proteins[66,67], the presence of peptides in xylem sap has onlyrecently been demonstrated [68]. The addition of xylem sapfrom tomato induced rapid alkalinization in a tomato cell-suspension bioassay within minutes. This approach hasbeen used previously to identify peptides. In an experimentusing maize suspension cells, alkalinization was inducedbymaize xylem sap, but the activity could not be purified orabolished with proteases [65]. MicroRNAs have also beenimplicated as potential signal molecules that move sys-temically [69], and recent work on Arabidopsis found thatdrought treatment induced the synthesis of a specificmicroRNA: miR-169 g [70]. The functional significance ofpeptides, microRNAs and proteins in the xylem sapremains largely unknown, but peptides have been shownto play important signaling roles in plants and have alsobeen shown to move systemically via the phloem [71,72].

Many compounds, including hormones, inorganic ions,amino acids, sugars and organic acids have been identifiedin xylem sap [7,34], but among the organic acids found inabundance in the xylem sap, only malate has been impli-cated in the control of stomatal closure [73]. Stomatalopening in ash (Fraxinus excelsior) leaves could be pre-vented when these leaves were supplied with 0.5–3 mMmalate. Similarly, elevated extracellular concentrations ofmalate have also been shown to close stomates in Viciafaba [74]. Further work is needed to establish the role ofmalate in root to shoot signaling.

Ethylene could be another important factor underdrought conditions. It is known that a precursor of ethyl-ene, 1-aminocyclopropane-1-carboxylic acid (ACC), movesin the xylem from root to shoots [75]. Under drought stress,ACC transported in the xylem sap might result in themeasured increase of ethylene evolution in leaves [76]. Arole for ethylene under drought was demonstrated by theuse of an ACC oxidase (ACO) antisense in tomato. In theseantisense plants, ethylene evolution was much lower thannormal under both well-watered and drought conditions.Under soil-drying conditions, the stomatal response in theACO antisense plants was the same as wild type, but adecrease in leaf growth was measured in wild type but notthe ACO antisense plants in response to soil drying [76]. Inmaize, ethylene evolution in leaves could not be correlatedwith reductions in leaf elongation under drought [77]. Thisstudy also showed that ABA does not play a role in redu-cing leaf elongation in maize [77]. This suggests that

in malate and ACC (1-aminocyclopropane-1-carboxylate) concentrations, decreases

e. After guard cells perceive ABA, changes in potassium (K+) and anion (A�) fluxes

guard cells under drought suggest the importance of a plasma-membrane-bound

ut the importance of intracellular receptors, such as CHLH (the H subunit of the

e ruled out. Other unknown and yet-to-be-identified plasma membrane receptors

that cause stomatal closure. After guard cells perceive ABA, the efflux of potassium

anion channel.

285


ethylene may play a role in decreased leaf growth in oneplant species and that ACC may be one component of long-distance root-sourced signals under drought.

ConclusionsThe production of root-sourced chemical signals underconditions of water deficit has been associated withreduced transpiration and/or leaf growth. However, theidentity and relative contribution to signaling of these root-sourced chemicals remains controversial. This controversymay be due to differing responses between species, thedifferent intensities of stress treatments applied, the timeat which samples were collected during the imposition ofdrought, and/or the different methods used for xylem sapextraction [43]. Until recently [65], few studies have pro-vided comprehensive information on the composition ofxylem sap. Such comprehensive analyses will clarify thecontribution of different chemicals to root to shoot sig-naling, and the complexity of constituents and their inter-actions. As we increase our knowledge of the preciseidentity and biosynthesis of the primary chemical signalsthat are produced by roots, it will become feasible to alterplant sensitivity to soil water deficit. This will provide newmolecular breeding strategies for tailoring crops to main-tain or even increase yields under specific water stressconditions, such as intermittent and terminal drought.

AcknowledgementsDPS was supported in part by NSF-Plant Genome #0211842. JQDG is therecipient of an Australian Research Council Post-doctoral Fellowship(Inductry linkage project #LP0775362)

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40 Razem, F.A. et al. (2006) The RNA-binding protein FCA is an abscisicacid receptor. Nature 439, 290–294

41 Shen, Y.Y. et al. (2006) The Mg-chelatase H subunit is an abscisic acidreceptor. Nature 443, 823–826

42 Wilkinson, S. et al. (1998) Effects of xylem pH on transpiration fromwild-type and flacca tomato leaves. A vital role for abscisic acid inpreventing excessive water loss even from well-watered plants. PlantPhysiol. 117, 703–709

43 Bacon, M.A. et al. (1998) pH-regulated leaf cell expansion in droughtedplants is abscisic acid dependent. Plant Physiol. 118, 1507–1515

44 Prokic, L. et al. (2006) Species-dependent changes in stomatalsensitivity to abscisic acid mediated by external pH. J. Exp. Bot. 57,675–683

45 Liu, F.L. et al. (2003) Hydraulic and chemical signals in the control ofleaf expansion and stomatal conductance in soybean exposed todrought stress. Funct. Plant Biol. 30, 65–73

46 Jia, W. and Davies, W.J. (2007) Modification of leaf apoplast pH inrelation to stomatal sensitivity to root-sourced abscisic acid signals.Plant Physiol. 143, 68–77

47 Bahrun, A. et al. (2002) Drought-induced changes in xylem pH, ioniccomposition, and ABA concentration act as early signals in field-grownmaize (Zea mays L.). J. Exp. Bot. 53, 251–263

48 Else,M.A. et al. (1994) Concentrations of abscisic acid and other solutesin xylem sap from root systems of tomato and castor-oil plants aredistorted by wounding and variable sap flow rates. J. Exp. Bot. 45, 317–323

49 Jackson, M.B. (1997) Hormones from roots as signals for the shoots ofstressed plants. Trends Plant Sci. 2, 22–28

50 Liu, F.L. et al. (2005) A review of drought adaptation in crop plants:changes in vegetative and reproductive physiology induced by ABA-based chemical signals. Aust. J. Agric. Res. 56, 1245–1252

51 Wilkinson, S. (2004) Water use efficiency and chemical signalling. InWater Use Efficiency in Plant Biology (Bacon, M.A., ed.), pp. 75–112,Blackwell Publishing Ltd

52 Wilkinson, S. (2007) Nitrate signalling to stomata and growing leaves:interactions with soil drying, ABA, and xylem sap pH in maize. J. Exp.Bot. 58, 1705–1716

53 Roelfsema, M.R. and Prins, H.B. (1998) The membrane potential ofArabidopsis thaliana guard cells; depolarizations induced byapoplastic acidification. Planta 205, 100–112

54 Munns, R. and Sharp, R.E. (1993) Involvement of abscisic acid incontrolling plant growth in soils of low water potential. Aust. J.Plant Physiol. 20, 425–437

55 Hansen, H. and Dorffling, K. (1999) Changes of free and conjugatedabscisic acid and phaseic acid in xylem sap of drought-stressedsunflower plants. J. Exp. Bot. 50, 1599–1605

56 Sauter, A. et al. (2002) A possible stress physiological role of abscisic acidconjugates in root-to-shoot signalling. Plant Cell Environ. 25, 223–228

57 Dietz, K.J. et al. (2000) Extracellular beta-glucosidase activity in barleyinvolved in the hydrolysis of ABA glucose conjugate in leaves. J. Exp.Bot. 51, 937–944

58 Sakakibara, H. (2006) Cytokinins: activity, biosynthesis, andtranslocation. Annu. Rev. Plant Biol. 57, 431–449

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60 Kudoyarova, G. et al. (2007) Effect of partial rootzone drying on theconcentration of zeatin-type cytokinins in tomato (Solanumlycopersicum L.) xylem sap and leaves. J. Exp. Bot. 2, 161–168

61 Hansen, H. and Dorffling, K. (2003) Root-derived trans-zeatin ribosideand abscisic acid in drought-stressed and rewatered sunflower plants:interaction in the control of leaf diffusive resistance? Funct. Plant Biol.30, 365–375

62 Teplova, I. et al. (2000) Response of tobacco plants transformedwith the ipt gene to elevated temperature. Russ. J. Plant Physiol.47, 367–369

63 Dodd, I.C. (2003) Hormonal interactions and stomatal responses.J. Plant Growth Regul. 22, 32–46

64 Davies, W.J. et al. (2005) Long-distance ABA signaling and its relationto other signaling pathways in the detection of the soil drying and themediation of the plant’s response to drought. J. Plant GrowthRegul. 24,285–295

65 Alvarez, S. et al. (2008) Metabolomic and proteomic changes in thexylem sap of maize under drought. Plant Cell Environ. 31, 325–340

66 Alvarez, S. et al. (2006) Characterization of the maize xylem sapproteome. J. Proteome Res. 5, 963–972

67 Kehr, J. et al. (2005) Analysis of xylem sap proteins from Brassicanapus. BMC Plant Biol. 5, 11

68 Neumann, P.M. (2007) Evidence for long-distance xylem transport ofsignal peptide activity from tomato roots. J. Exp. Bot. 58, 2217–2223

69 Sunkar, R. et al. (2007) Small RNAs as big players in plant abioticstress responses and nutrient deprivation. Trends Plant Sci. 12, 301–309

70 Zhao, B. et al. (2007) Identification of drought-induced microRNAs inrice. Biochem. Biophys. Res. Commun. 354, 585–590

71 Lindsey, K. et al. (2002) Peptides: new signalling molecules in plants.Trends Plant Sci. 7, 78–83

72 Ryan, C.A. et al. (2002) Polypeptide hormones. Plant Cell 14 (Suppl),S251–S264

73 Patonnier, M.P. et al. (1999) Drought-induced increase in xylemmalateand mannitol concentrations and closure of Fraxinus excelsior L.stomata. J. Exp. Bot. 50, 1223–1229

74 Hedrich, R. et al. (1994) Malate-sensitive anion channels enable guard-cells to sense changes in the ambient CO2 concentration. Plant J. 6,741–748

75 Else, M.A. and Jackson, M.B. (1998) Transport of 1-aminocyclopropane-1-carboxylic acid (ACC) in the transpirationstream of tomato (Lycopersicon esculentum) in relation to foliarethylene production and petiole epinasty. Aust. J. Plant Physiol. 25,453–458

76 Sobeih, W.Y. et al. (2004) Long-distance signals regulating stomatalconductance and leaf growth in tomato (Lycopersicon esculentum)plants subjected to partial root-zone drying. J. Exp. Bot. 55, 2353–2363

77 Voisin, A.S. et al. (2006) Are ABA, ethylene or their interactioninvolved in the response of leaf growth to soil water deficit? Ananalysis using naturally occurring variation or genetictransformation of ABA production in maize. Plant Cell Environ. 29,1829–1840

287

Ecology of Water Relationsin PlantsYoseph Negusse Araya, The Open University, Milton Keynes, UK

Water is an important resource for plant growth. Availability of water in the soil determines

the niche, distribution and competitive interaction of plants in the environment.

Introduction

Importance of water for plants

Water typically constitutes 80–95%of themass of growingplant tissues and plays a crucial role for plant growth (Taizand Zeiger, 1998). Plants require water for a number ofphysiological processes (e.g. synthesis of carbohydrates)and for associated physical functions (e.g. keeping plantsturgid).

Water accomplishes its many functions because of itsunique characteristics: the polarity of the molecule H2O(which makes it an excellent solvent), viscosity (whichmakes it capable of moving through plant tissues bycapillary action) and thermal properties (which makes itcapable of cooling plant tissues).

Plants require water, soil nutrients, carbon dioxide, ox-ygen and solar radiation for growth.Of these,water ismostoften the most limiting: influencing productivity (Taiz andZeiger, 1998) as well as the diversity of species (Rodriguez-Iturbe and Porporato, 2004) in both natural and agricul-tural ecosystems. This is illustrated graphically in Figure 1.

How does water affect ecology of plants?

In order to understand the ecology of plant–water rela-tions it is important to understand from where and howplants acquire water in their environment (the latter is dis-cussed in the section on water uptake and movementthrough plants).

Unlike animals, which are capable of wandering aroundto forage for resources, plants are for the most part sta-tionary, depending on the availability of nutrients in theirsurrounding environment (soil and/or atmosphere). Ofthese two sources of resources, i.e. soil and atmosphere, thesoil is by far the major and more accessible reservoir. Con-sequently, the soil is the primary store and regulator in thewater flow of ecosystems, by intercepting precipitationinput and controlling its use by organisms (Rodriguez-It-urbe and Porporato, 2004). Figure 2 summarizes the soiland plant–water interrelationship.

Soil moisture availability is dependent on the soil particlesize distribution (also called soil texture) and arrangementof these particles (soil structure). The soil texture and struc-ture influence the size of soil pores where water is held by

Article Contents

Advanced article

. Introduction

. Water Uptake and Movement through Plants

. Water Stress and Plants

. Plant Sensing and Adaptation to Water Stress

. Distribution of Plants in Response to Water Regime

doi: 10.1002/9780470015902.a0003201

Moisture8

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selected vegetation communities, along an elevation gradient from SantaCatalina Mountains, Arizona (after Whittaker and Niering, 1975). The

elevation gradient ranges from 1000 to 3000m above sea level. Themoisture index relates to precipitation ranges of 190mm per annum

(moisture index 8) and 850mm per annum (moisture index 1).

1ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net

capillary forces. Soils with fine-sized particles, like clay holdmorewater than soils dominatedbycoarse grainparticles ofsand. However, this does not mean that all the water infine-particle sized soils is available for plant uptake. This isbecause the capillary forces holding water in the pores offine-textured soils are so powerful that the plants struggle toextract anywater. Figure3 shows soil water and soil aerationavailability for different soil texture classes.

Soil moisture availability primarily influences plants bytwo routes (see Figure 2), either by being directly limiting asa resource, or indirectly by filling pore spaces in soiland thereby excluding air, causing oxygen availability tobecome limiting for the activity of plant roots. This is ex-plained further in the section on water stress and plants.

Water Uptake and Movement throughPlants

Water is constantly moving from the soil, into plant roots,and through the xylem tissues of the stem to leaves where it

is ultimately lost to the atmosphere during transpiration.This cycle is referred to as the soil–plant–atmosphere con-tinuum (SPAC). See also: Plant–Water RelationsWhenwatermoves through the SPAC, it travels through

different mediums (including cell wall, cell membrane andair spaces) at different distances, which utilize differentmodes of transport.There are three principal modes of water transport:

diffusion, mass flow and osmosis. In diffusion, water mole-cules move spontaneously from regions of high concen-tration to regions of lower concentration, i.e. along aconcentration gradient. This movement is rapid over ashort distance and thus drives short-distance transport, forexample between cells and during the loss of water to theatmosphere from leaf stomata.In mass flow, groups of water molecules move under an

external force, such as a build-up of pressure that forms agradient. Mass flow of water is the predominant mode bywhich long-distance transport of water in stems is accom-plished. It also accounts for much of the water flow thoughthe soil and through the cell walls of plants.The third mode, called osmosis, is movement of water

molecules through a semipermeable membrane, an exam-ple of which is the cytoplasmic membrane. Osmosis occursspontaneously in both short- and long-distance transportas a response to driving forces of concentration (as indiffusion) and pressure gradient (as in mass flow).These driving forces of watermovement of both osmotic

(concentration) and mass flow (pressure) origin are collec-tively known as water potential. Water potential is meas-ured in units of pressure or suction, i.e. force per unit arearequired to move a specific amount of water. The mostcommon unit used for studying soil water potentials in thefield is kilopascals (kPa).The movement of water in the SPAC is thus dependent

on differences in water potential between surrounding soiland plant or atmosphere. Often, the water potential gra-dient is directed from the roots towards the shoot, as tran-spiring leaves exposed to the atmosphere have the lowestwater potential. However, under situations when the soil istoo dry this water potential gradient could be reversed,resulting in loss of water from plant roots to the soil. Alsoany environmental factors that influence the transpirationof the leaf stomata, e.g. wind or increase in temperaturemay further decrease the leaf water potential further,speeding up water loss.

Water Stress and Plants

In addition to an adequate level of water in their tissues,plants also require a continuous flux of water to performvital processes such as photosynthesis and nutrient uptake.Water for these is not always available in the right quan-tity and quality at the right time. This imbalance in water

Plants

Organic matterreturn

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Figure 2 Schematic summary of the processes that influence the

relationship between plants and soil water.

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representative sandy (solid line) and clayey (broken line) soils. Soil watercontentsonvolumebasis is shownagainst soilwaterpotential (suction) and

against air-filled pore space (volume of pore space not occupied by water).

Ecology of Water Relations in Plants

2

supply and plant requirements results in plants undergoingoccasional or, in some cases acute, water stress.

There are two types of water stresses that plants expe-rience. One is when water is not available in sufficientquantity – hence referred to as water-deficit, while thesecond one is that when water is available – but in excess,called waterlogging.

Water-deficit affects plants through decrease of leafwater potential, which in turn entails loss of cell turgor andstomatal closure. This results in decrease of transpirationand photosynthesis, which subsequently leads to reducedgrowth and if it persists, wilting. On the other hand, water-logging occurs when a large proportion of the pore spacesin the soil are occupied by water. This means the diffusionof oxygen and gas exchange between the soil, plants andatmosphere is limited. The result of this is decreased rootgrowth and functioning, which negatively affects plantgrowth and survival.

Plants start suffering the consequences of water stresswhen certain thresholds for water-deficit and waterloggingare breached. Physiological plant studies have shown thatsoil water potentials approaching 5 kPa are sufficient toinitiate plant stomatal closure, a classic response to waterdeficit (Hensen et al., 1989). On the other hand, water-logging which cause 5 10%air-filled pore space in the soil(0% is achievedat soil saturation), result in hampering rootactivity, and hence induce aeration stress (Wesseling andvan Wijk, 1957). Between these two thresholds of dryingandwaterlogging stress an optimal zone conducive to plantgrowth is achieved (Gowing et al., 2002).

However, to be even more meaningful stress thresholdsneed to take account of time duration, overwhich the plantis subjected to the stress, i.e. as short periods of stress areless damaging than gentler but longer-term ones. One in-dex thatmeasures and cumulates the level of stress over thetime duration it occurs, is called a sum exceedence value(SEV). SEVs are calculated separately for soil drying stressand for soil aeration stress, usually in unit of metre-weeks.SEVs were originally developed in the Netherlands by Sie-ben and colleagues in 1960s but later on successfully used inthe UK by Gowing and colleagues (e.g. Silvertown et al.,1999) to integrate temporal variation in soil moisture at ascale relevant to the physiological response of plants.Moreover, SEVs take into account differences in soil type(as thresholds are specifically developed for each soil typeunder consideration) and are hence transferable betweendifferent sites.

Plant Sensing and Adaptation to WaterStress

Water stress is damaging to plants, so they have evolved anumber of short-term responses as well as life historystrategies that help them to cope. For this, a mechanism

that senses water stress is crucial to the initiation ofdefensive processes. See also: Plant Stress Physiology

Plant sensing of soil drying

Water-deficit is the most common form of water stressstudied in relation to sensing of impending soil drying byplant roots and the subsequent communication to shoots.In this connection, signals of a chemical nature havereceived a lot of attention, as they are suited for rapidcommunication between plant tissues.A well-known chemical signal of impending water stress

originating from exposed roots is abscisic acid (ABA).ABA is synthesized by dehydrating roots in nongrowingtissues as well as in apices, and in the cortex (Hartungand Davies, 1991). An increase in ABA concentration inresponse to an increase in soil drying is known to initiatewater-saving measures like reduction in transpiration rateand conductance (e.g. Hensen et al., 1989).A consequence of this sensing is that it determines

the response of the plant and its competitive ability. Forexample, a plant which responds to the tiniest sign ofstress will trade-off productivity for safety (‘pessimiststrategy’), while a plant that waits longer will trade safetyfor productivity (‘optimist strategy’). Depending on theextent and duration of the actual stress, either of thesetwo types of plants will emerge as the one having a bettercompetitive advantage (Davies and Gowing, 1999). Thiswill then influence, within the limits of physiologicalplasticity, their success in the plant community (see thesection on Distribution of plants in response to waterregime).

Plant adaptations to water stress

Plants respond towater stress in twoways: by avoidance ofthe stress or by tolerating it. Stress avoidance is accom-plished when plants alter their growth schedule to escapethe exposure to damaging stress. Well-known examples inthis category include completing the life cycle while con-ditions are optimal, or using strategies to maximize wateruptake from the environment and or conservation. On theother hand, the tolerance response to water stress occurswhen plants develop certain characteristics, often of bio-chemical and or morphological nature, to minimize thepotential damage from stress. An example of the lattercould be additions to photosynthetic pathways such ascrassulacean acid metabolism (Scott, 2000) for dryingstress. Somemorphological adaptations for flooding stressinclude development of air-space tissue (aerenchyma)within tissues and ventilation roots (pneumatophores).The development of the ability to metabolize productsof anaerobic respiration and/or tolerate an accumulationof anaerobic metabolites is also another biochemical


3

adaptation utilized by wetland plants. See also: PlantResponse To Water-deficit Stress

As a closing remark to this section, it is worth mention-ing, an extreme form of adaptation to water-deficit by agroup of plants known as poikilohydrics or resurrectionplants. Poikilohydric plants show an ability of maturetissues such as the shoot, stem and leaves to tolerate almostcomplete dehydration of the tissues and then return asfunctional units very rapidly on rehydration, sometimesin as short as 24 h (Norwood et al., 2003). Obviously,such plants are native to and inhabit ecological niches thatare subjected to lengthy periods of drought with briefperiods of rain during the year, e.g. deserts of SouthernAfrica, Southern America and Western Australia (Scott,2000).

Distribution of Plants in Response toWater Regime

Differences in water regime have been known to be behindexistence of different vegetation types and ecosystems.Some widely known examples include global and regionaldistribution of plant communities. At global level examplesinclude the major world biomes, such as tropical rainforest,deserts and tundra. At this level, precipitation differences asa result of latitude and incoming solar radiation define cer-tain plants to prevail. A regional example is where plantcommunities are defined by precipitation differences asso-ciated with topographic features, such as elevation. A wellknown such example being a study as given in Figure 1. Thedistribution of plant species in relation to water regime atregional level, had also been examined using the subjective

Ellenberg values, developed from field observations by theeminent German botanist, Heinz Ellenberg.However, only recently has the potent role of fine-scale

heterogeneities in hydrology on a plot scale identified asprincipal driver for the defining structures in plant com-munities (Silvertown et al., 1999). These fine-scale differ-ences in hydrological regime accomplish this structuringby creating realized niches, which are capable of beingexploited by specific species. This is illustrated in Figure 4,withhydrological niches of eight species of sedge inUKwetmeadows.The ecology of plant–water relations thus can be eluci-

dated by examination of the species’ hydrological nicheswithin a community. These niches are a result of distinctplants’ differing physiological response towater stress, andthe presence of other neighbouring plants.

References

Davies WJ and Gowing DJG (1999) Plant responses to small perturbat-

ions in soil water status. In: Press MC, Scholes JD and Barker MG

(eds) Plant Physiological Ecology, Vol. 39, pp. 67–89. Oxford: Black-

well Science.

Gowing D, Lawson C, Youngs E et al. (2002) The Water Regime Re-

quirements and the Response to Hydrological Change of Grassland

Plant Communities: DEFRA commissioned project BD1310, Final

report to the Department for Environment, Food and Rural Affairs,

Cranfield University, Silsoe.

Hartung W and Davies WJ (1991) Drought-induced changes in phys-

iology and ABA. In: Davies WJ and Jones HG (eds) Abscisic Acid

Physiology and Biochemistry, pp. 63–79. Oxford: BIOS Science Pub-

lications Ltd.

Hensen IE, Jensen CR and Turner NC (1989) Leaf gas exchange and

water relations of lupins and wheat. I Shoot responses to soil water

deficits. Australian Journal of Plant Physiology 16: 401–413.

8

6

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0

6

4

2

00 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 12

SEV drought stress

SEV

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ress

Carex panicea Carex flacca Carex acutiformis Carex acuta

Carex distichaCarex hirtaCarex ripariaCarex nigra

Figure 4 Distributions of eight sedge species showing differentiation in niche space defined by hydrological axes on a fine-scale gradient. The

x-axis depicts increasing soil drying stress, while the y-axis shows increasing flooding (i.e. aeration) stress (see the section on water stress andplants for explanation). The vertically hatched area in each graph shows the range of possible hydrological regimes and the solid area indicates the

zone in which the species occurs significantly more frequently than by chance. Data are cumulated across 18 different meadow sites (after Gowing et al.,2002). Reproduced with permission from Her Majesty’s Stationery Office.


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Norwood M, Toldi O, Richter A and Scott P (2003) Investigation into

the ability of roots of the poikilohydric plant Craterostigma plant-

agineum to survive dehydration stress. Journal of Experimental Botany

54(391): 2313–2321.

Rodriguez-Iturbe I and Porporato A (2004) Ecohydrology of

Water-Controlled Ecosystems: Soil Moisture and Plant Dynamics.

Cambridge, UK: Cambridge University Press.

Scott P (2000) Resurrection plants and the secrets of eternal leaf. Annals

of Botany 85: 159–166.

Silvertown J, Dodd ME, Gowing DJG and Mountford JO (1999)

Hydrologically defined niches reveal a basis for species richness in

plant communities. Nature 400: 61–63.

Taiz L and Zeiger E (1998)Plant Physiology, 2nd edn. Sunderland,MA,

USA: Sinauer Associates, Inc.

Wesseling J and van Wijk WR (1957) In: Luthin JN (ed.) Drainage

in Agricultural Lands, pp. 461–504. Madison, Wisconsin: American

Society for Agronomy.

Whittaker RH andNieringWA (1975) Vegetation of the Santa Catalina

Mountains, Arizona. V. Biomass, production, and diversity along the

elevation gradient. Ecology 56: 771–790.

Further Reading

Archibold OW (1995) Ecology of World Vegetation. London, UK:

Chapman & Hall.

Ellenberg H (1988) Vegetation ecology of Central Europe, 4th edn.

Cambridge, NY: Cambridge University Press.

Etherington JR and William A (1976) Environment and Plant Ecology.

London, UK: Wiley.

Grime JP, Hodgson JG and Hunt R (1988) Comparative Plant Ecology:

A Functional Approach to Common British Species. London, UK:

Unwin Hyman.

Lambers H, Stuart Chapin III F and Pons TL (1998)Plant Physiological

Ecology. New York, USA: Springer New York, Inc.

Proctor MCF and Tuba Z (2002) Poikilohydry and homoihydry:

antithesis or spectrum of possibilities? New Phytologist 156: 327–349.

Smith JAC and Griffiths H (1993)Water Deficits: Plant Responses from

Cell to Community. Oxford, UK: BIOS Scientific.


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ANRV342-PP59-13 ARI 2 April 2008 8:55

Flooding Stress:Acclimations andGenetic DiversityJ. Bailey-Serres1,∗ and L.A.C.J. Voesenek2,∗

1Center for Plant Cell Biology, University of California, Riverside, California 92521;email: [email protected] Ecophysiology, Institute of Environmental Biology, Utrecht University,NL-3584 CA Utrecht, The Netherlands

Annu. Rev. Plant Biol. 2008. 59:313–39

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev.arplant.59.032607.092752

Copyright c© 2008 by Annual Reviews.All rights reserved

1543-5008/08/0602-0313$20.00

∗Both authors contributed equally to this paper.

Key Words

aerenchyma, anoxia, response strategy, hypoxia, reactive oxygenspecies, submergence

AbstractFlooding is an environmental stress for many natural and man-madeecosystems worldwide. Genetic diversity in the plant response toflooding includes alterations in architecture, metabolism, and elon-gation growth associated with a low O2 escape strategy and an anti-thetical quiescence scheme that allows endurance of prolonged sub-mergence. Flooding is frequently accompanied with a reduction ofcellular O2 content that is particularly severe when photosynthesisis limited or absent. This necessitates the production of ATP andregeneration of NAD+ through anaerobic respiration. The exami-nation of gene regulation and function in model systems providesinsight into low-O2-sensing mechanisms and metabolic adjustmentsassociated with controlled use of carbohydrate and ATP. At the de-velopmental level, plants can escape the low-O2 stress caused byflooding through multifaceted alterations in cellular and organ struc-ture that promote access to and diffusion of O2. These processes aredriven by phytohormones, including ethylene, gibberellin, and ab-scisic acid. This exploration of natural variation in strategies thatimprove O2 and carbohydrate status during flooding provides valu-able resources for the improvement of crop endurance of an envi-ronmental adversity that is enhanced by global warming.

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Hypoxia (e.g.,<20.9% and >0%O2 at 20◦C):characterized byincreased anaerobicmetabolism,increased ATPproduction viaglycolysis owing tolimited availability ofO2 for oxidativephosphorylation, andincreased NAD+regeneration vialactate and ethanolicfermentation.Cellular ATPcontent is reduced

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 314GENETIC DIVERSITY OF

STRATEGIES TO SURVIVEFLOODING . . . . . . . . . . . . . . . . . . . . 315

ACCLIMATION TO FLOODINGAT THE CELLULAR LEVEL. . . 316Overview of Cellular Adjustments

to Oxygen Deprivation . . . . . . . . 316Low-Oxygen Sensing . . . . . . . . . . . . . 318Management of the

Energy Crisis . . . . . . . . . . . . . . . . . 318THE LOW-OXYGEN ESCAPE

SYNDROME . . . . . . . . . . . . . . . . . . . . 323Enhanced Growth Leading to

the Emergence of Shoots . . . . . . 323Improvement of the Oxygen and

Carbohydrate Status inSubmerged Plants . . . . . . . . . . . . . 326

Improvement of Internal GasDiffusion: Aerenchyma . . . . . . . . 328

CONCLUSIONS AND FUTUREPERSPECTIVES . . . . . . . . . . . . . . . . 329

INTRODUCTION

Partial to complete flooding is detrimentalfor most terrestrial plants because it hampersgrowth and can result in premature death.Some plant species have a remarkable capacityto endure these conditions, and certain speciescan even grow vigorously in response to flood-ing. This interspecific variation has a strongimpact on species abundance and distributionin flood-prone ecosystems worldwide (12, 31,122, 138, 150, 151). Furthermore, floodinghas a severe negative influence on the produc-tivity of arable farmland because most cropsare not selected to cope with flooding stress(121). The Intergovernmental Panel on Cli-mate Change (IPCC) (http://www.ipcc.ch)reported that the anthropogenically inducedchange of world climate increases the fre-quency of heavy precipitation and tropical cy-clone activity. This is likely to engender more

frequent flooding events in river flood plainsand arable farmland, particularly affecting theworld’s poorest farmers (1).

The observation that some plant speciescan cope with flooding stress and others can-not imposes the question of why a floodedenvironment is detrimental. The adversity islargely due to the dramatically reduced gasexchange between plants and their aerial en-vironment during partial to complete submer-gence. Gases such as O2, CO2, and ethylenediffuse very slowly in water (46). Because ofthis tremendous barrier for gas diffusion, thecellular O2 level can decline to concentrationsthat restrict aerobic respiration (39, 46). De-pending on the tissue and light conditions, thecellular CO2 level either increases in shoots inthe dark and roots (47) or decreases in shootsin the light (83). The endogenous concen-tration of the gaseous plant hormone ethy-lene increases in tissues surrounded by water(59, 148). This accumulation activates adap-tive signal transduction pathways, whereassimilar concentrations hamper normal growthin many terrestrial plants (93). Furthermore,complete submergence decreases light inten-sity, dampening photosynthesis (141). A thirdmajor change in the flooded environment isthe reduction of oxidized soil components totoxic concentrations (12). In summary, flood-ing is a compound stress in which the de-cline in molecular O2 and thus the restrictionof ATP synthesis and carbohydrate resourceshave major consequences for growth and sur-vival. However, O2 depletion is not the onlyactive stress component, and often its impactis restricted to nonphotosynthesizing organs(84).

O2 shortage (hypoxia/anoxia) is not re-stricted to flooding stress. It is a frequentmetabolic status of cells during normal devel-opment, particularly in tissues with high celldensity, a high O2 demand, and/or restrictedO2 entry, such as meristems, seeds, fruits, andstorage organs (43). Fundamental insight intothe low O2–sensing mechanism, downstreamsignal transduction, and metabolic alterationsthat promote survival is key to increased crop

314 Bailey-Serres · Voesenek

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production in flood-prone environments andhas wider implications for biologists (3, 43).Most studies on flooding stress have focusedon relatively flood-tolerant species from gen-era such as Oryza, Rumex, and Echinochloa.Single species studies are valuable for an un-derstanding of the regulation of various accli-mations but less meaningful in an ecologicalperspective. Here genetic diversity in accli-mations to flooding stress is discussed side byside with the molecular regulation of low-O2

responses and flooding tolerance. Ultimatelywe aim to shed light on the genes, proteins,and processes controlling these phenotypes.

GENETIC DIVERSITY OFSTRATEGIES TO SURVIVEFLOODING

Not all species in flood-prone environmentsare flood tolerant. Some species avoid flood-ing by completing their life cycle between

Anoxia (e.g., 0% O2at 20◦C):characterized byanaerobicmetabolism, NAD+regeneration vialactate and ethanolicfermentation, andATP productionsolely via glycolysis(2–4 mol ATP permole hexose).Cellular ATPcontent is low, andADP content iselevated

LOES: low-oxygenescape syndrome

two subsequent flood events, whereas flood-ing periods are survived by dormant lifestages [e.g., Chenopodium rubrum thrives infrequently flooded environments by timingits growth between floods and producingseeds that survive flooding (134)]. Estab-lished plants also use avoidance strategiesthrough the development of anatomicaland morphological traits. This ameliora-tion response, here called the low oxy-gen escape syndrome (LOES) (Figure 1),facilitates the survival of submerged organs.Upon complete submergence several speciesfrom flood-prone environments have the ca-pacity to stimulate the elongation rate of peti-oles, stems, or leaves. This fast elongation canrestore contact between leaves and air but canalso result in plant death if energy reservesare depleted before emergence. Concomitantwith high elongation rates, the leaves also de-velop a thinner overall morphology, developthinner cell walls and cuticles, and reorient

Drained Submerged

Low Shoot elongation

Low High

High

Aerenchyma

High Low Leaf thickness

Around intercellular spaces Toward epidermis Chloroplast position

Trait

aaa

bb

Figure 1Various species display the low-oxygen escape syndrome (LOES) when submerged. The syndromeincludes enhanced elongation of internodes and petioles, the formation of aerenchyma in these organs (airspaces indicated by arrows labeled a), and increased gas exchange with the water layer through reducedleaf thickness and chloroplasts that lie directed toward the epidermis (indicated by arrows labeled b).Photographs are courtesy of Ronald Pierik, Liesje Mommer, Mieke Wolters-Arts, and Ankie Ammerlaan.

www.annualreviews.org • Acclimation to Flooding Stress 315

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Sub1: Submergence1polygenic locus ofrice; determinessubmergencetolerance

Oxygendeficiency/deprivation: thenatural andexperimentalconditions in whichcellular oxygencontent is reducedbut metabolic statusis not determined

mtETC:mitochondrialelectron transportchain

ROS: reactiveoxygen species

chloroplasts toward the leaf surface. Thesetraits reduce the resistance for diffusion ofCO2 and O2, facilitating inward diffusion andthereby improving underwater photosynthe-sis and aerobic metabolism (82, 83). Thus,the LOES improves the aeration of the plant,which is further enhanced by the relativelylow resistance for internal gas diffusion ow-ing to a system of interconnected gas conduitscalled aerenchyma, a property typical of manywetland plants (24, 33). These conduits areconstitutive, induced in existing tissues (roots,petioles, stems) (33) or formed during thedevelopment of adventitious roots that arisefrom the root shoot junction or stem nodes(115, 142). In specialized cases the longitudi-nal diffusion of O2 to the root apex is furtherenhanced by the development of a barrier toradial oxygen loss to minimize escape of O2

to the surrounding environment (24, 25).LOES is costly and will only be selected for

in environments where the cost is outweighedby benefits such as improved O2 and carbo-hydrate status, both contributing to a higherfitness (120). The flooding regime is an im-portant determinant for selection in favor ofor against LOES. A study on the distribu-tion of species in the Rhine floodplains con-firmed this hypothesis. Here LOES occurspredominantly in species from habitats char-acterized by prolonged, but relatively shallow,flooding events (150). However, the benefitsof LOES do not outweigh the costs whenthe floods are too deep or ephemeral. Theseregimes favor a quiescence strategy character-ized by limited underwater growth and con-servation of energy and carbohydrates (39,91). This strategy is a true tolerance mech-anism, driven by adjustment of metabolism.With respect to low-O2 stress, this includesthe downregulation of respiration and limitedstimulation of fermentation to create a posi-tive energy budget when organ hypoxia starts(43, 148). The SUB1A gene of the polygenicrice (Oryza sativa L.) Submergence1 (Sub1) lo-cus was shown to confer submergence toler-ance through a ‘quiescence’ strategy in whichcell elongation and carbohydrate metabolism

is repressed (41, 91, 159) (Figure 2).SUB1A, encodes an ethylene-responsive ele-ment (ERF) domain–containing transcriptionfactor (41). The lack of SUB1A-1 or the pres-ence of a slightly modified allele is associatedwith reduced submergence tolerance and theinduction of the LOES. This example demon-strates that environment-driven selection ona single locus can significantly alter survivalstrategy.

ACCLIMATION TO FLOODINGAT THE CELLULAR LEVEL

Overview of Cellular Adjustmentsto Oxygen Deprivation

During flooding, the onset of O2 deprivationis rapid in the dark and in nonphotosyntheticcells. The reduced availability of O2 as thefinal electron acceptor in the mitochondrialelectron transport chain (mtETC) mediates arapid reduction of the cellular ATP:ADP ratioand adenylate energy charge (AEC) ([ATP +0.5 ADP]/[ATP+ADP+AMP]) (46). Cellscope with this energy crisis by relying primar-ily on glycolysis and fermentation to gener-ate ATP and regenerate NAD+, respectively.Whether a LOES or a quiescence response toflooding is activated, cellular acclimation totransient O2 deprivation requires tight regu-lation of ATP production and consumption,limited acidification of the cytosol, and ame-lioration of reactive oxygen species (ROS)produced either as O2 levels fall during flood-ing or upon reoxygenation after withdrawal ofthe flood water.

O2 concentration is 20.95% at 20◦C in airbut ranges from 1 to 7% in the core of well-aerated roots, stems, tubers, and developingseeds (14, 44, 46, 107, 136, 137). Within aroot, O2 levels and consumption vary zonally;the highly metabolically active meristematiccells are in a continuous state of deficiency.Upon flooding, the ∼10,000-fold-slower dif-fusion of O2 in water rapidly limits its avail-ability for mitochondrial respiration. Thisdeprivation is progressively more severe as


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Lowland Rice Deepwater Rice

Tolerant Intolerant

Strategy Quiescence LOES LOES

High

N.D.

High

Sub1 haplotype SUB1A-1, SUB1B,SUB1C

SUB1B, SUB1C orSUB1A-2, SUB1B, SUB1C

SUB1B, SUB1C orSUB1A-2, SUB1B, SUB1C

Carbohydrateconsumption Limited by SUB1A-1 High

GA response Inhibited by SUB1A-1 Promoted by SUB1C

Fermentationcapacity High Moderate

Figure 2Rice responds via different strategies to submergence. Flood-tolerant rice varieties invoke a quiescencestrategy that is governed by the polygenic Submergence1 (Sub1) locus that encodes two or threeethylene-responsive factor proteins (41, 159). SUB1A is induced by ethylene under submergence andnegatively regulates SUB1C mRNA levels. Flood-intolerant varieties avoid submergence via the lowoxygen escape syndrome (LOES). To this end SUB1C expression is promoted by gibberellic acid (GA)and is associated with rapid depletion of carbohydrate reserves and enhanced elongation of leaves andinternodes. The LOES is unsuccessful when flooding is ephemeral and deep. Deepwater rice varietiessurvive flooding via a LOES, as long as the rise in depth is sufficiently gradual to allow aerial tissue toescape submergence (61). N.D., not determined.

distance from the source increases and tissueporosity decreases. For example, the cortex ofnonaerenchymatous maize (Zea mays L.) rootsexposed to 10% O2 becomes hypoxic, whereasthe internal stele becomes anoxic. Even theapex of aerenchymatous roots encounters se-vere O2 deprivation (46). In dense storage or-gans such as potato (Solanum tuberosum L.) tu-bers and developing plant seeds, exposure to8% O2 significantly reduces the endogenousO2 level. However, the decrease in cellularO2 is strikingly nonlinear from the exterior

to the interior of the organ; cells at the in-terior of the tuber or endosperm maintain ahypoxic state (44, 136). This has led to thesuggestion that an active mechanism may al-low cells to avoid anoxia (43). Such a mecha-nism may include proactive limitation in theconsumption of both ATP and O2. The lowKm for cytochrome c oxidase (COX) [140 nM(∼0.013%) O2] should ensure that the activityof COX continues as long as O2 is available(31, 46). However, a mechanism that inhibitsthe mtETC at or upstream of COX or inhibits


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Normoxia (e.g.,20.9% O2 at 20◦C):characterized byaerobic metabolism,NAD+ regenerationprimarily via themitochondrialelectron transportchain, and ATPproduction viamitochondrialoxidativephsophorylation(30–36 mol ATP permol hexoseconsumed); cellularATP content isnormal

O2 consumption by other enzymes may allowcells to sustain hypoxia and avoid death.

Low-Oxygen Sensing

In animals the perception of O2 deficit in-volves O2-binding proteins, ROS, and mi-tochondria. The O2-consuming prolyl hy-droxylases (PHDs) are direct sensors of O2

availability. Under normoxia, PHDs targetthe proteosomal degradation of hypoxia in-ducible factor 1α (HIF1α), a subunit of a het-erodimeric transcription factor that regulatesacclimation to hypoxia (51). The concomitantdrop in PHD activity stimulates an elevationin HIF1α as O2 declines. A paradox is thatthe production of ROS at the mitochondrialubiquinone:cytochrome c reductase complex(Complex III) is necessary to initiate O2 deficitresponses (7, 51).

There is limited understanding of themechanisms by which plant cells sense andinitiate signaling in response to O2 deficit (3,39, 43). Plants lack a HIF1α ortholog, al-though PHD mRNAs are strongly induced byO2 deficit in Arabidopsis thaliana and rice (67,146). Furthermore, significant increases inmRNAs encoding enzymes involved in ROSsignaling and amelioration (16, 63, 67, 70, 71)and evidence of ROS production have beenreported in several species upon transfer tolow O2 conditions. A challenge in monitoringROS production during O2 deficit is that ROSare produced readily upon reoxygenation.However, ethane, a product of membrane per-oxidation by ROS, evolves from submergedrice seedlings in a closed system as levels of O2

fall to as low as 1% (112), providing evidencethat ROS form as O2 levels decline. Blokhinaand colleagues (11) demonstrated that in re-sponse to anoxia, H2O2 accumulates to higherlevels in the apoplast of root meristems of hy-poxic wheat (Triticum aestivum) than in themore anoxia-tolerant rhizomes of Iris pseu-dacorus. In Arabidopsis seedlings, H2O2 lev-els increase in response to O2 deprivationin a ROP GTPase–dependent manner (6).Genotypes that limit ROP signaling under hy-

poxia display lower levels of H2O2 accumula-tion and altered gene regulation in stressedseedlings. Indications that mitochondria arecrucial to low-O2 sensing in plants comesfrom the release of Ca2+ from mitochondria ofcultured maize cells within minutes of trans-fer to anoxia (127). This release may be ac-tivated by mitochondrial ROS production atComplex III of the mtETC (99). A rapidspike in cytosolic Ca2+ was also observed inthe cotyledons of Arabidopsis seedlings upontransfer to anoxia and again at higher ampli-tude upon reoxygenation (119). These Ca2+

transients are required for alterations in geneexpression that enhance ethanolic fermenta-tion and ATP management during the stress(3, 66, 88, 119, 126, 128). Further studies areneeded to confirm whether mitochondrion-to-nucleus signaling, mediated by ROS pro-duction and Ca2+ release from mitochondria,contributes to reconfiguration of metabolismunder low O2. Additional players in the ac-climation response may be the reduction ofATP content and decline in cytosolic pH aswell as change in levels of metabolites such assucrose and pyruvate (3, 39). mRNAs encod-ing mitochondrial alternative oxidase, (AOX)are strongly induced by low-O2 stress (63, 67,70, 71). AOX diverts ubiquinone from Com-plex III; if active as O2 levels decrease, AOXwould paradoxically reduce oxygen availabil-ity for COX and decrease ATP production.However, if active as O2 levels rise upon re-oxygenation, AOX may limit mitochondrialROS production (99).

Management of the Energy Crisis

Within minutes of transfer to an O2-depletedenvironment, cells reliant on external O2 limitprocesses that are highly energy consumptiveand alter metabolism to increase anaerobicgeneration of ATP by cytosolic glycolysis (31).This shift is followed by fermentation of pyru-vate to the major end products, ethanol orlactate, yielding NAD+ to sustain anaerobicmetabolism (Figure 3). A crisis in ATP avail-ability ensues because glycolysis is inefficient,


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yielding 2 to 4 mol ATP per mol hexose ascompared with 30 to 36 mol ATP by themtETC. Evaluation of gene transcripts, en-zymes, and metabolites in a variety of speciesand genotypes demonstrated the productionof minor metabolic end products that are alsoimportant for NAD+ and NAD(P)+ regen-eration. Although mutant analyses with sev-eral species have demonstrated that glycol-ysis and fermentation are necessary for cellsurvival under O2 deprivation, the enhance-ment of these processes is not well corre-lated with prolonged endurance of this stress(31, 46).

The anaerobic energy crisis necessitates ablend of optimized ATP production with lim-ited energy consumption. ATP-demandingprocesses such as DNA synthesis and celldivision are curtailed (46), and the produc-tion of rRNA is dramatically reduced (36).In Arabidopsis and other plants, low-O2 stressmarkedly limits protein synthesis but main-tains the initiation of translation of a subset ofcellular mRNAs, many of which encode en-zymes involved in anaerobic metabolism andthe amelioration of ROS (16, 36). Therefore,under O2 deprivation, a mechanism oper-ates that sequesters untranslated mRNAs andlessens ATP expenditure, thereby allowing forthe recovery of protein synthesis within min-utes of reoxygenation.

Carbohydrate mobilization and sucrosecatabolism. The metabolic response to O2

deprivation is orchestrated by the availabilityand mobilization of carbohydrates (31, 137).In some plants and tissues, the induction ofamylases by low O2 or flooding promotes theconversion of starch to glucose (Figure 3).However, the mobilization of starch dur-ing O2 deprivation is not universal. Boththe tubers of potatoes and rhizomes of theflood-tolerant marsh plant Acorus calamus L.have considerable carbohydrate reserves, butAcorus rhizomes are more capable of mo-bilizing starch into respirable sugars underanoxia (2). This slow consumption of starchallows the rhizomes to sustain a low level of

metabolism that affords survival of long peri-ods of submergence. Seeds of rice, rice weeds(e.g., some Echinochloa species), and tubers ofPotamogeton pectinatus also mobilize starch un-der anoxia (29, 40, 50). In rice seeds, this starchmobilization requires the depletion of solublecarbohydrates, suggesting regulation by sugarsensing (50, 72). In organs lacking starch re-serves or effective starch mobilization, the ex-haustion of soluble sugars prior to reoxygena-tion is likely to result in cell death.

Plants possess two independent routes forthe catabolism of sucrose, the bidirectionalUDP-dependent sucrose synthase (SUS) andthe unidirectional invertase (INV) pathways(Figure 3). The net cost for entry into gly-colysis is one mol pyrophosphate (PPi) permol sucrose via the SUS route, if the UTPproduced by UDP-glucose pyrophosphory-lase (UGPPase) is utilized by fructokinase(FK) in the subsequent conversion of UDP-glucose to glucose-6P or the ATP consumedby FK is recycled by nucleoside diphosphate(NDP) kinase. By contrast, the cost via theINV pathway is two mol ATP per mol su-crose. The SUS route is positively regulatedunder O2 deprivation through opposing in-creases in SUS and the repression of INVgene expression and enzymatic activity (10,14, 43, 44, 64, 67). The energetic disadvantageof the INV route was confirmed by the inabil-ity of transgenic potato tubers with elevatedINV activity to maintain ATP levels under8% O2 (14). The SUS pathway is enhancedin a variety of species by rapid increases intranscription of SUS mRNAs, which is mostlikely driven by sucrose starvation (64, 71).Other glycolytic reactions may utilize avail-able PPi during O2 deprivation, thereby im-proving the net yield of ATP per mol sucrosecatabolized. The phosphorylation of fructose-6P to fructose-1,6P2 by the bidirectionalPPi-dependent phosphofructokinase (PFP)is favored over the unidirectional ATP-dependent phosphofructokinase (PFK), anda pyruvate Pi dikinase (PPDK) may substi-tute for cytosolic pyruvate kinase (PK) in O2-deprived rice seedlings (95).


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Glycolysis

Ethanol

ADH

NADH

NADH

NADH

NADH

NADH

Sucrose

SUSInvertase

H2O

H2O

Invertaseinhibitor

2-oxyglutarateGlutamate

Aspartate

HCO3

PDC

PEPC

AspAT

FFumarase

MDMDH

ICDCDH

AconitaAconitase

SuSuccccinyl CoA syn n

2-O2-OGDH

CS

Malatete

Citratetrate

2-oxyglutarate

Succiccinyl CoA

SucciSuccinate

FumararateSDH

IsocitratIsocitrate

Fumarase

MDH

ICDH

Aconitase

Succinyl CoA syn

2-OGDH

CS

PFK PFP

PPDK

CoASH

x4

Fructose + Glucose UDP-glucose + Fructose

Glucose-1P

Glucose-6P

PGM

HXK

FK FK

UGPPase

PGI

ALDH

Fructose-1,6P2

Malate

OxalaloacetacetateOxaloacetate

Citrate

2-oxyglutarate

Succinyl CoA

Succinate

FumarateSDH

+CoASHH+

Starch

amylases

PCK

Glucose

Glucose-1P

Starch Pase

Glutamate

GABA

GlutamineGDC

NH4

+

NH4

+

CO2

CO2

CO2

NiR

NR

2-oxyglutarate

GOGAT

GS

GDH

GABASuccinate

semialdehyde

GHBDHGABA-T

2-oxyglutarateGlutamate

PyruvateAlanine

SSADH

PyruvateLactate

Acetaldehyde

LDH

Fructose-6P

Alanine

Pi

CO2

CO2

PK

NAD+

NAD+

NAD+

NAD+

UDP

UDP

Pi

Phosphoenolpyruvate

Isocitrate

Pi

NO2

–

NO3

–

γ-hydroxybutyrate

ATP

FAD+

FADH2

NAD(P)+

TCA cycle

PDH

PPi

PPi

UTP

ADP

NDP kinase

ATPUDP

ADP

ATP

ATP

ADP

ATP+Pi

AMP+PPi

NADH

ATP

ADP

NADH

NAD+

AlaAT

NAD(P)+

NAD(P)H

NAD(P)+

NAD(P)H

NAD(P)+

NAD(P)H

Acetyl CoAAcetyl CoA

NAD+

ADP

NAD+

NAD(P)H

NADH

NADH NAD+

ATP

NAD+ADP

UTPADP

ATPUTP


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Metabolic end products. During O2 depri-vation, pyruvate decarboxylase (PDC) con-verts pyruvate to acetaldehyde, which is me-tabolized by alcohol dehydrogenase (ADH) toethanol, with the regeneration of NAD+ tosustain glycolysis. PDC- and ADH-deficientgenotypes confirm the essentiality of ethano-lic fermentation in the acclimation to floodingand low-O2 stress (6, 31, 46, 65). In Arabidop-sis seedlings, the level of induction of ADH iscontrolled by the activation of a ROP GTP-ase (5). O2 deprivation promotes an increasein active ROP, which leads to the elevation oftranscripts that encode ADH and ROPGAP4,a GTPase that inactivates ROP. In a ropgap4null mutant, ADH mRNA and ROS are signif-icantly elevated under hypoxia, and seedlingsurvival is reduced. This led to the proposalthat a ROP rheostat controls the temporalregulation of ADH expression under low O2

(3, 39).The production of ethanol is benign ow-

ing to its rapid diffusion out of cells, whereasthe intermediate acetaldehyde is toxic. Ac-etaldehyde dehydrogenase (ALDH) catalyzesthe conversion of acetaldehyde to acetate,with the concomitant reduction of NAD+ toNADH. A mitochondrial ALDH is signifi-cantly induced by anoxia in coleoptiles of rice(67, 87), but not in seedlings of Arabidopsis(65). ALDH activity correlates with anaerobicgermination capability of Echinochloa crus-galli

under strict anoxia (40). Under O2-limitingconditions, ALDH consumes NAD+ and maythereby limit glycolysis, whereas upon reoxy-genation acetaldehyde converted to acetate bymitochondrial ALDH enters the tricarboxylicacid (TCA) cycle (Figure 3).

In addition to ethanol, lactate is producedin plant cells under O2 deprivation. The ac-cumulation of lactate under low-O2 stress hasgarnered considerable interest (31, 35, 48, 98)ever since the demonstration that its transientappearance precedes that of ethanol in theroot tips of maize seedlings (105). The pHof the cytosol of maize root tips declines from7.5 to a new equilibrium at pH 6.8 following-transfer to anoxia. It is posited that the tran-sition from lactic to ethanolic fermentation iscontrolled by a pH-stat. The ∼0.6 unit de-crease in cytosolic pH favors the catalytic op-timum of PDC and thereby limits lactate andpromotes ethanol production. Anoxic ADH-deficient root tips continue to produce lac-tate and fail to stabilize the cytosolic pH, re-sulting in rapid cytosolic acidification and celldeath (106). Thus, the switch from lactic toethanolic fermentation is critical for the main-tenance of cytosolic pH. An alternative pro-posal is that this switch, under conditions ofO2 deprivation and in aerobic cells in whichethanol is produced, is driven by a rise inpyruvate rather than the increase in lactateor reduction of cytosolic pH (130). When

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Metabolic acclimations under O2 deprivation. Plants have multiple routes of sucrose catabolism, ATPproduction, and NAD+ and NAD(P)+ regeneration. Blue arrows indicate reactions that are promotedduring the stress. Metabolites indicated in bold font are major or minor end products of metabolismunder hypoxia. Abbreviations are as follows: 2-OGDH, 2-oxyglutarate dehydrogenase; ADH, alcoholdehydrogenase; AlaAT, alanine aminotransferase; ALDH, acetaldehyde dehydrogenase; AspAT, aspartateaminotransferase; CoASH, coenzyme A; CS, citrate synthase; FK, fructokinase; GABA-T, GABAtransaminase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; GHBDH,γ-aminobutyrase dehydrogenase; GOGAT, NADPH-dependent glutamine: 2-oxoglutarateaminotransferase; GS, glutamine synthase; HXK, hexokinase; ICDH, isocitrate dehydrogenase; LDH,lactate dehydrogenase; MDH, malate dehydrogenase; NDP kinase, nucleoside diphosphate kinase; NiR,nitrite reductase; NR, nitrate reductase; PCK, phosphenolpyruvate carboxylase kinase; PDC, pyruvatedecarboxylase; PDH, pyruvate dehydrogenase; PEPC, phosphenolpyruvate carboxylase; PFK,ATP-dependent phosphofructokinase; PFP, PPi-dependent phosphofructokinase; PGI,phosphoglucoisomerase; PGM, phosphoglucomutase; PK, pyruvate kinase; PPDK, pyruvate Pi dikinase;SDH, succinate dehydrogenase; SSADH, succinate semialdehyde dehydrogenase; Starch Pase, starchphosphorylase; SUS, sucrose synthase; UGPPase, UDP-glucose pyrophosphorylase.


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pyruvate levels increase, the low Km of mi-tochondrial pyruvate dehydrogenase (PDH)and high Km of PDC serve to limit carbon en-try into the TCA cycle and promote ethanolicfermentation.

Flooding stress is likely to involve a gradualtransition from normoxia to hypoxia, allow-ing cells to initiate processes that favor sur-vival. Plants exposed to a period of hypoxiafor 2 to 4 h prior to transfer to an anoxicenvironment are more capable of avoidingcell death than those that undergo an abruptanoxic shock (31). The preexposure to 3%or 4% O2 reduces the severity of ATP de-pletion, allows the synthesis of stress-inducedand normal cellular proteins (19), and acti-vates a lactate efflux mechanism (158). Lac-tate removal from the cytoplasm may be ac-complished by the hypoxia-induced nodulinintrinsic protein (NIP2;1), which was iden-tified in Arabidopsis as a plasma membrane–associated protein capable of driving lactatetransport in Xenopus oocytes (23). Most likely,a decline in cytosolic pH of 0.2 to 0.5 units un-der O2 shortfall establishes a new pH set pointthat influences multiple aspects of metabolism(35, 48, 95). The management of this pH de-cline involves ethanolic fermentation and isbenefited by the availability of a lactate ef-flux mechanism and proton ATPase activity.However, some species or organ systems, suchas the tuber shoots of Potamogeton pectinatus,do not show an adjustment in cytosolic pHduring O2 deprivation. The stem elongationin these shoots under anoxia results from cellexpansion that occurs in the absence of an ad-justment in cytosolic pH and appears to bemaintained by tight constraints on ATP pro-duction and consumption (29).

Besides the major fermentation end prod-ucts, lactate and pyruvate, O2 deficiency isassociated with the elevation of alanine, γ-aminobutyric acid (GABA), succinate, andoccasionally malate (29, 31, 46, 113, 137,139). Strong induction of cytosolic and mito-chondrial alanine aminotransferase (AlaAT),aspartate aminotransferase, mitochondrialglutamate dehydrogenase (GDH), and mi-

tochondrial Ca2+/calmodulin-regulated glu-tamate decarboxylase (GDH) mRNA and/orenzymatic activity is consistent with pyruvateconversion to alanine or GABA (Figure 3)(63, 67, 70, 71, 100, 139). GABA may be fur-ther metabolized via the mitochondrial GABAshunt to γ-hydroxylbutyrate with the regen-eration of NAD(P)+ (17). Upon reoxygena-tion, alanine can be recycled back to pyru-vate, and GABA can be converted to succinate.Amino acid oxidation may thereby minimizethe decline in cytosolic pH and reduce carbonloss via ethanol or lactate. An appreciation ofthe relative significance of the major and mi-nor pathways of anaerobic metabolism will re-quire metabolite profiling and flux studies thatresolve organ specific and temporal aspects ofproduction in relationship to changes in redoxand energy status.

Nitrite, nitric oxide, mitochondria, andhemoglobin. Nitrate and nitrite are also im-plicated in cellular adjustment to O2 depriva-tion. Nitrate is assimilated and reduced to am-monia via nitrate reductase (NR) and nitritereductase (NiR) (Figure 3). NR but not NiRmRNAs increase significantly in response tohypoxia/anoxia in Arabidopsis and rice (67, 70,71). Even without an increase in NR levels, areduction of cytosolic pH may increase nitriteproduction because of the low pH optimumof this enzyme (57). Roots of tobacco plantsengineered to have reduced NR levels displayseveral metabolic anomalies under anoxia, in-cluding higher levels of soluble hexoses andATP, enhanced ethanol and lactate produc-tion, and increased acidification of the cytosol(125). By contrast, maize seedling roots sup-plied with nitrate during anoxia maintain aslightly higher cytosolic pH than do controlseedlings (69). Notably, the provision of mi-cromolar levels of nitrite to seedling roots hada similar effect on the adjustment of cytosolicpH. This unexpected benefit of low levels ofnitrite is unlikely to be due to a direct effecton NAD(P)+ regeneration and may indicate arole of nitrite in a regulatory mechanism thataugments homeostasis under low O2.


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A plant-specific association has surfacedbetween nitrate/nitrite metabolism, mito-chondrial ATP synthesis, and a low-O2-induced nonsymbiotic Class 1 hemoglobin(HB). Plant mitochondria provided with mi-cromolar levels of nitrite under anoxia havethe capacity to coordinate the oxidationof NADH and NAD(P)H with low lev-els of ATP production (124). This nitrite-promoted process involves the evolution ofnitric oxide (NO) via a pathway that re-quires the activity of rotenone-insensitiveNAD(P)H dehydrogenases, mtETC Com-plex III (ubiquinone:cytochrome c reduc-tase), and Complex IV (COX). In the pro-posed pathway (124), NAD(P)H producedduring O2 deficit is oxidized by Ca2+-sensitiveNAD(P)H dehydrogenases on the inner mito-chondrial membrane surface, providing elec-trons to the ubiquinone pool. In the absenceof O2, nitrite may serve as an electron accep-tor at Complex III or IV, yielding NO, whichmay activate signal transduction by promot-ing mitochondrial ROS production and Ca2+

release. The cytosolic HB that accumulatesunder O2 deprivation, however, scavenges anddetoxifies NO in planta by converting it intonitrate in an NAD(P)H-consuming reactionover a broad pH optimum (30, 57). The cou-pled activities of HB and cytosolic NR re-generate nitrite that may enter the mitochon-drion, where it continues the cycle of NOand ATP production (94, 124). A major chal-lenge is to confirm in planta that nitrite con-version to NO functions as a surrogate finalelectron acceptor. Nonetheless, the scenariois consistent with reports that overexpressionof HB in several species decreases rates ofethanolic fermentation, augments ATP main-tenance, and fosters NO production underhypoxia. By contrast, the inhibition of HBexpression increases NAD(P)H:NAD(P)+ ra-tios and reduces cytosolic pH (30, 56, 57,123). Notably, NO inhibits COX activity andthereby reduces ATP production under nor-moxia. Might NO formed during the transi-tion from normoxia to hypoxia be the factorthat dampens O2 consumption to avoid cellu-

lar anoxia (43)? If so, the production of NOprior to the synthesis of HB may allow thecell to transition slowly from normoxia to hy-poxia, providing a segue that augments energymanagement.

THE LOW-OXYGEN ESCAPESYNDROME

Enhanced Growth Leadingto the Emergence of Shoots

Plants forage for limiting resources by adjust-ing carbon allocation and overall plant archi-tecture such that the capture of resources isconsolidated (93, 96). As O2 and CO2 be-come limiting for plants in flood-prone en-vironments, species from widely dispersedfamilies that share the capacity to survive inflood-intense environments initiate signalingpathways that lead to fast extension growthof shoot organs (101, 147). These leaves,when reaching the water surface, functionas snorkels to facilitate the entrance of O2

and the outward ventilation of gases such asethylene and methane trapped in roots (24,145). Another benefit of the emergence of leafblades is a higher rate of carbon gain fromaerial photosynthesis (82).

Fast shoot elongation under water isnot restricted only to species occurring inenvironments with periodic floods (e.g.,deepwater rice, Rumex palustris, Ranunculussceleratus) (61, 148, 150). It persists in trueaquatics that develop floating leaves orflowers [e.g., Nymphoides peltata (82)] andin species that germinate in anaerobic mudfollowed by an extension growth phase toreach better-aerated water/air layers (e.g.,seedlings of Oryza sativa, Potamogeton pectina-tus, P. distinctus) (58, 113, 129). The exploredmechanism of shoot elongation in Marshdock (R. palustris) and deepwater rice (92,115, 149) can be used to shed light on themechanistic backbone of genetic diversity inflooding-induced shoot elongation.

The shoot elongation response can occurin petioles or internodes, depending on the


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developmental stage or predominant growthform of the plant. Interestingly, petiole elon-gation in rosette plants is accompanied by hy-ponastic growth that changes the orientationof the petiole from prostrate to erect. Thisdirectional growth brings the leaf in such aposition that enhanced petiole elongation willresult in leaf blade emergence in the shortestpossible time. Accordingly, petiole elongationlagged behind hyponastic growth in R. palus-tris rosettes (26).

It is generally accepted that the submer-gence signal for enhanced shoot elongation isthe gaseous phytohormone ethylene (76, 101,147). Ethylene is biosynthesized via an O2-dependent pathway, and the endogenous con-centration of this hormone is determined pre-dominantly by production rate and outwarddiffusion. Both aspects are affected by sub-mergence. Several biosynthetic genes [e.g.,those encoding ACC synthase (ACS) andACC oxidase (ACO)] are upregulated by sub-mergence (102, 135, 154), whereas diffusionof ethylene to the outside environment isstrongly hampered. As a result, the endoge-nous concentration rises to a new, higher equi-librium. Ethylene production persists in sub-merged shoots as O2 continues to diffuse fromthe water into the shoot, guaranteeing rela-tively high endogenous O2 concentrations inshoot cells even in the dark (80). Submergenceor low oxygen also upregulates the expressionof ethylene receptor genes, including RpERS1in R. palustris (155), OsERL1 in deepwater rice(156), and ETR2 in Arabidopsis (16, 63, 70, 72).An elevation of ethylene receptor levels fol-lowing submergence is counterintuitive be-cause these molecules are negative regulatorsof ethylene signaling. However, this increasewould allow rapid cessation of ethylene sig-naling as the plants emerge from the waterand vent off the accumulated ethylene.

Ethylene is the input signal for severalparallel pathways required for fast elonga-tion under water (Figure 4). Under fully sub-merged conditions the accumulated ethylenedownregulates abscisic acid (ABA) levels viaan inhibition of 9-cis-epoxycarotenoid dioxy-

genase (NCED) expression, a family of rate-limiting enzymes in ABA biosynthesis that be-longs to the carotenoid cleavage dioxygenases,(CCDs) and via an activation of ABA break-down to phaseic acid (9, 61, 110). The de-cline of the endogenous ABA concentrationin R. palustris is required to stimulate the ex-pression of gibberellin (GA) 3-oxidase, an en-zyme that catalyzes the conversion to bioac-tive gibberellin (GA1) (8), and in deepwaterrice to sensitize internodes to GA (61). Down-stream of GA, three sets of genes play a role insubmergence-induced shoot elongation. Thefirst group encodes proteins involved in cellwall loosening; the second, those involved inthe cell cycle; and the third, those involvedin starch breakdown. Additional genes withputative regulatory roles in enhanced intern-ode elongation have been identified in floodeddeepwater rice (22, 108, 117, 132, 133).

The rigid cell wall constrains the rate anddirection of turgor-driven cell growth. Sig-nificant increases in acid-induced cell wall ex-tension upon submergence were observed inrice (20), R. palustris (152), and Regnellidiumdiphyllum (62). This could be reversed evenwhen R. palustris petioles were desubmerged,emphasizing the correlation between exten-sibility and submergence-induced elongation(152). Cell wall extensibility is thought to beassociated with cell-wall-loosening proteins,such as expansins (EXPs) and xyloglucan en-dotransglycosylase/hydrolases (XTHs) (27).Submergence-induced elongation is stronglycorrelated with increases in mRNAs encod-ing expansins A (EXPA) and B (EXPB), alongwith EXP protein abundance and activity(21, 62, 68, 89, 152, 153). Interestingly, insome species ethylene directly regulates EXPexpression (62, 152, 153) (Figure 4). In sub-merged R. palustris petioles, ethylene not onlyenhances EXP expression but also stimulatesproton efflux into the apoplast (153), which isessential for EXP action.

The second group of GA-regulated genesis involved in cell cycle regulation. Invery young petioles of the fringed wa-terlily (Nymphoides peltata) and the youngest


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Cell elongation and/or division

ABA

GA

RpNCED1-4RpNCED6-10

RpGA3OX1

OsEXPA2OsEXPA4OSEXPB3OsEXPB4OsEXPB6OsEXPB11OsXTR1OsXTR3

cyc2Os1cyc2Os2cdc2Os2HistoneH3OsRPA1

OsTMKOsGRF1-3OsGRF7,8,10,12OsDD3-4OsSBF1OsGRF9

RpEXPA1RdEXPA1

OsUSP1

Apoplasticacidification

OsSUB1A

OsSUB1COsAMY3D

OsABA8ox1

Submergence EthyleneOsACS1OsACS2OsACS5RpACS1

OsACO1RpACO1

RpERS1

Figure 4Schematic model of the plant processes, hormones, and genes involved in submergence-induced shootelongation (blue signifies upregulated genes and red signifies downregulated genes). Gene abbreviationsare as follows: CYC2Os, cyclin; CDC2Os, cyclin-dependent kinase; OsACO and RpACO, ACC oxidase;OsACS and RpACS, ACC synthase; OsDD, differentially displayed (61); OsAMY, amylase (41); OsEXP,RdEXP, and RpEXP, expansins; OsGRF, growth-regulating factor (22); OsRPA, replication protein A1;OsSBF, sodium/bile acid symporter family (108); OsSUB1, submergence1; OsTMK, transmembraneprotein kinase (133); OsUSP, universal stress protein (117); RpERS1, ethylene receptor (155); RpNCED,9-cis-epoxycarotenoid dioxygenase; RpGA3ox, gibberellin 3-oxidase (8); OsXTR, xyloglucanendotransglucosylase-related (27); OsABA8ox, ABA 8′-hydroxylase (110). Os indicates Oryza sativa, Rdindicates Regnellidium diphyllum, and Rp indicates Rumex palustris.

internode of deepwater rice, ethylene pro-motes not only cell elongation but also celldivision. Consistent with this increase in celldivision is the observed upregulation of cy-clin (CYC2Os1, CYC2Os2), cyclin-dependentkinase (CDC2Os2), HistoneH3, and replicationprotein A1 (OsRPA1) (114, 115, 131).

The third group of GA-regulated genesis involved in starch breakdown. R. palustrisplants depleted of soluble sugars and starch

show a very restricted underwater elongationresponse (49). Carbohydrates are required todeliver energy and the building blocks for newcell wall synthesis (115, 148). The require-ment of carbohydrates can be fulfilled by thetranslocation of photosynthates and by thedegradation of starch reserves via an increasein α-amylase activity (115). Fukao and col-leagues (41) reported that α-amylase gene ex-pression (OsAmy3D) in leaves of submerged


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rice is regulated by SUB1C, an ethylene-responsive factor (ERF)-domain-containingprotein of the polygenic Sub1 locus. This geneis regulated positively by GA and negativelyby a related ERF in the Sub1 locus, SUB1A-1, which is present in some rice accessions.These results imply that carbohydrate levelsin submerged plants are also under hormonalcontrol.

There is considerable genetic variation be-tween and within species in submergence-induced elongation capacity. The closely re-lated species Rumex acetosa and R. palustrisshow inhibition and stimulation of petioleelongation upon exposure to ethylene, respec-tively. Both accumulate significant amountsof ethylene when submerged (4), but R. ace-tosa lacks ABA downregulation (9), GA upreg-ulation (104), and increased EXP expression(153). However, when R. acetosa is exposed toelevated GA levels without enhanced ethyleneor when ABA levels are reduced with fluri-done in submerged plants, petiole elongationis strongly stimulated (9, 104). This demon-strates that signal transduction componentsrequired for elongation growth downstreamof ABA and GA are present in this species andcan be activated. It also shows that in R. acetosa,contrary to R. palustris, ethylene cannot switchon this cascade. Most likely, elements down-stream of ethylene but upstream of ABA/GAexplain differences in ethylene-induced elon-gation between Rumex species.

Rice cultivars also show variation in elon-gation capacity during submergence (28, 41,120). The Sub1 locus controls underwaterelongation through genetic distinctions in thetwo to three ERF proteins it encodes (41,159) (Figure 2). SUB1A-1 is present in theSub1 locus only in submergence-tolerant linesand is induced by ethylene. SUB1C is presentin all rice lines and is induced by GA. Thebetween-cultivar variation in elongation cor-relates with genotypic variation and expres-sion of ERFs of the Sub1 locus. The slowlyelongating rice varieties of indica rapidly andstrongly induce SUB1A-1 upon submergence,whereas all elongating indica and japonica

varieties lack either the SUB1A gene or theSUB1A-1 allele (159). Transformation of anelongating japonica variety with a SUB1A-1full-length cDNA under the control of themaize Ubiquitin1 promoter resulted in a sig-nificant repression of underwater elongation(159). The expression of SUB1A-1 coincideswith repressed accumulation of transcripts forEXPs and reduced expression of SUB1C (41),suggesting that SUB1A acts upstream of GAregulation of EXPs and SUB1C.

Improvement of the Oxygenand Carbohydrate Statusin Submerged Plants

At the whole-plant level, complete submer-gence leads to a dramatic shift in the carbonbudget and energy status, potentially result-ing in death. Some relief of this problem,with the leaves still submerged, is underwa-ter photosynthesis (83). The significance ofthis was exposed by studies showing that lightavailability enhances survival under water inboth flood-tolerant and intolerant species (55,81, 86, 141) and that O2 levels in submergedplants are affected by light intensity (90). Im-proved survival of submergence in the lightis correlated with a higher carbohydrate sta-tus (97) and internal O2 concentrations (80,84, 103). However, underwater photosynthe-sis can be limited by low light and CO2

availability. Consistent with these findings arestudies showing that illumination can main-tain sugar transport and leaf ATP content atnear-normoxic levels under strict O2 depriva-tion in rice and wheat leaves (85).

True aquatics develop specialized leavescharacterized by an overall thin leaf andcuticle, a high degree of dissection, and epi-dermal cell chloroplasts. These traits reducethe diffusion barriers and shorten the dif-fusion pathways, thus enhancing carbon in-put per leaf area and unit time (111). Otherstrategies, developed by true aquatics toenhance carbon gain, are the utilizationof HCO3 as carbon source, C4 or CAMmetabolism, or hydrosoil CO2 consumption


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(75, 83). Very little information is availableabout the occurrence of these last strate-gies in terrestrial plants from flood-proneenvironments.

Leaf acclimations to submergence havebeen characterized for R. palustris (82) andother amphibious species (18, 37, 157). Leavesdeveloped under water are 20% thinner withan increased specific leaf area (SLA) (m2 g−1),indicating a large surface area relative to mass.The higher SLA is related not only to thelower leaf thickness of aquatic leaves but alsoto their tenfold-lower starch content. Fur-thermore, aquatic leaves have thinner epider-mal cell walls and cuticles, and their chloro-plasts lie close to the epidermis rather thantoward the intercellular spaces as is typical foraerial leaves (82). These acclimations are con-sistent with the view that CO2 directly entersthe mesophyll cells of these leaves via diffusionthrough the epidermis and not via stomataand intercellular gas diffusion. This diffusionpathway under water has a much higher dif-fusion resistance for gases than does intercel-lular diffusion. Calculations for R. palustris in-dicate a 15,000-fold-higher resistance to CO2

diffusion in leaves under submergence thanwhen in air (81). However, the morphologicaland anatomical changes decrease gas diffusionresistance for CO2 (38). In R. palustris theseacclimations result in a dramatic reduction ofthe diffusion resistance between submergedleaves and leaves in air to a factor of less than400 (81). Functional consequences of these ac-climations in R. palustris include higher ratesof net underwater assimilation and lower CO2

compensation points (81). Similar effects arealso described for amphibious species (13, 55,140). The relatively low diffusion resistance inaquatic leaves also permits increased inwarddiffusion of O2 from the water layer into theshoot. This results, in the dark, in an inter-nal O2 concentration of 17% in acclimatedpetioles of R. palustris when submerged in air-saturated water, whereas nonacclimated peti-oles reach only 9% (80).

These observations demonstrate that thewater column can function as an important

source of O2 for terrestrial plants when theyare exposed to submergence and that O2 lev-els in leaves, stems, and petioles below thecritical O2 pressure (0.8%; 31) are rare andprobably restricted to densely packed tissuesor to aquatic environments that are extremelystagnant or have low O2 levels. Although rootsystems will likely benefit from these shoot ac-climations, O2 pressures in the roots will stillbe much lower than the values mentioned herefor shoots, especially at night, when there is nophotosynthesis (90). It is therefore expectedthat even with LOES acclimations, roots willalso rely on the metabolic cellular adjustmentsto O2 deprivation for survival.

Plants in frequently flooded environmentsare expected to display these traits at a higherfrequency than do those in rarely flooded ar-eas. Consistently, Ranunculus repens popula-tions in temporary lakes are characterized byconstitutively dissected leaves. This morphol-ogy allows for a relatively large leaf surfaceand an improved gas exchange and results inrelatively high rates of underwater photosyn-thesis. Plants from more terrestrial popula-tions have less-dissected leaves and relativelylow rates of underwater photosynthesis (73,74). However, a comparative study of ninespecies, both flooding tolerant and intolerant,showed that gas exchange acclimations un-der water are not restricted to flood-tolerantspecies (84). In this study all but one speciesdeveloped aquatic leaves that were thinnerand had thinner outer cell walls and cuticlesand a higher SLA. These responses were in-dependent of the species’ flooding tolerances.Furthermore, leaf plasticity upon submer-gence resulted in increased O2 levels in allspecies. Therefore, between-species variationin inducible leaf acclimations in terrestrialplants, to optimize gas exchange when sub-merged, is not related to the variation inflooding tolerance of the species investigated(84). This conclusion hints toward a lim-ited role of submergence signals, such aselevated ethylene, in inducing leaf acclima-tions that enhance gas exchange under wa-ter. Plants that are not exposed to flooding


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throughout their life are not expected to usethese signals to switch on signaling cascadesthat lead to altered leaf anatomy and mor-phology. More likely, signals associated withchanged rates of photosynthesis and/or re-duced levels of carbohydrates induce theseleaf acclimations. This hypothesis is consis-tent with observations that shade-acclimatedplants with reduced rates of photosynthe-sis develop thinner leaves with higher SLA(78). Consistently, transgenic tobacco plantswith substantially reduced Rubisco levels havereduced photosynthesis and increased SLA(34).

Improvement of Internal GasDiffusion: Aerenchyma

Important traits for survival in flooded en-vironments are those that reduce the resis-tance for diffusion of O2 and CO2 from theenvironment to the plant. Equally signifi-cant, however, is the resistance that hampersgas diffusion within organs. Fast gas diffu-sion can be accomplished only in a gaseousdiffusion medium, over short distances, bylimited loss of the gas along the diffusionpath, and by restricted tortuosity of the dif-fusion route. These requirements are met inaerenchymatous tissue, characterized by lon-gitudinally interconnected gas spaces in rootsand shoots. Aerenchyma is either constitu-tively present and/or induced upon flooding(116, 144) and develops in existing tissues orconcomitant with the development of newroots (32). Distinct physiological processesare at the basis of aerenchyma formation. Thisled to the discrimination of two aerenchymatypes: (a) lysigenous aerenchyma formed bycell death and (b) schizogenous aerenchymain which gas spaces develop through theseparation of previously connected cells (23,33, 60). A third type, termed expansigenousaerenchyma, is characterized by intercellu-lar gas spaces that develop through cell divi-sion and cell enlargement, without cell sepa-ration or collapse/death (118). Combinationsof these aerenchyma types also exist (118), and

within one plant species different types can bepresent in different organs (32, 33).

The mechanism of schizogenous aeren-chyma formation is largely unknown as com-pared with that of lysigenous aerenchyma.Low O2 and elevated ethylene can inducelysigenous aerenchyma development in rootsof maize in a manner that is phenotypicallysimilar to the process promoted by flooding(32). Under flooded conditions, subambientO2 concentrations stimulate the productionof ethylene, which accumulates in roots sur-rounded by water and induces programmedcell death (PCD) in the cortex tissue (53).Accordingly, hypoxic roots, exposed toinhibitors of ethylene biosynthesis or ac-tion, form no gas spaces (53). Downstreamcomponents of this regulatory route includeprotein kinases, protein phosphatases, Gproteins, Ca2+, and inositol phospholipids(54). The targets of these signaling routesinclude proteins associated with cell wallbreakdown. The activity of cellulase increasesin roots upon exposure to low O2 or ethylene(52). Furthermore, increases in pectinaseand xylanase activity (15) and the inductionof XTH mRNAs occur in diverse species inresponse to flooding or hypoxia (67, 70, 109).

Large data collections are available on ge-netic diversity in traits that contribute to thedelivery of O2 to root tips. Justin & Armstrong(60) compared 91 species from wetland, in-termediate, and nonwetland habitats. Nearlyall the species from nonwetland environmentshad low root porosities, whereas high consti-tutive and increased porosities upon floodingwere associated with species from wetland en-vironments. Also, other studies confirmed thestrong correlation between high root porosi-ties and occurrence in wet environments (45,77, 143). Interestingly, a comparative studyon 35 wild Hordeum accessions from environ-ments that differ in flooding intensity showedthat this correlation does not always exist andthat aerenchyma development can be con-strained by phylogeny (42).

Aerenchyma is also formed in shoot or-gans, providing a system of interconnected


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channels from leaf to root tip. In a study with14 species divided over seven families, theaerenchyma content of petioles strongly cor-related with plant survival during completesubmergence. This robust correlation per-sisted in environmental conditions with (light)and without (dark) underwater photosynthe-sis (79, 84). These observations suggest thataerenchyma is important not only for survivalduring partial flooding but also during com-plete submergence. Petiole aerenchyma likelyfacilitates the diffusion of O2 from shoot or-gans to the roots. The O2 involved can be pho-tosynthetically derived during the light periodor obtained from the water layer by the shootduring the dark.

CONCLUSIONS ANDFUTURE PERSPECTIVES

The growing understanding of the molec-ular basis and genetic diversity in submer-gence and flooding acclimations provides

opportunities to breed and engineer crops tol-erant of these conditions that would benefitthe world’s farmers. The evaluation of diver-sity exposes plasticity in metabolic and de-velopmental acclimations that enable distinctstrategies that increase fitness in a floodedenvironment. Natural variation in acclima-tion schemes provides opportunities for de-velopment of crops with combinations of sub-mergence tolerance traits that are optimal atspecific developmental stages and under par-ticular flooding regimes, which vary substan-tially worldwide. The first example of this isthe use of marker-assisted breeding to intro-duce the submergence-tolerance conferringSub1 genotype to selected rice cultivars (159),which may appreciably benefit rice produc-tion in flood-prone lands in the Third World.The further exploration of the molecular ba-sis of genetic diversity in flooding tolerancesis critical given the global climate change sce-narios that predict heavy precipitation in re-gions of our planet.

SUMMARY POINTS

1. Evaluation of diversity exposes the remarkable plasticity in metabolic and develop-mental acclimations that enable increased fitness in a flooded environment.

2. Plants employing an escape strategy develop a suite of traits collectively called thelow-oxygen escape syndrome (LOES).

3. A consequence of low-O2 stress is a requirement for energy conservation that isinvoked through adjustments in gene expression, carbohydrate catabolism, NAD(P)+

regeneration, and ATP production.

4. Energy conservation is influenced by a low-O2-induced nonsymbiotic hemoglobinthat regulates cytosolic and mitochondrial processes, including rates of fermentation,NO, and ATP production.

5. Enhanced shoot elongation upon submergence requires the action of at least threehormones (ethylene, ABA, and GA) that regulate processes such as apoplastic acidifi-cation, cell wall loosening, cell division, and starch breakdown.

6. Anatomical and biochemical leaf acclimations upon submergence facilitate underwa-ter photosynthesis as well as the inward diffusion of O2 from the floodwater.

7. Aerenchyma in root and shoot tissue not only is important for survival during partialsubmergence but also facilitates O2 diffusion from shoot to root while the plant iscompletely submerged.


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FUTURE ISSUES

1. Plant species differ in their growth response to ethylene during submergence. This isfar from understood but probably involves signal transduction components upstreamof ABA and GA. The characterization of SUB1A is an important finding in thisrespect. More work is needed because this is probably an important selection pointto differentiate survival strategies.

2. A quiescence strategy (carbohydrate conservation) in rice is associated with submer-gence tolerance. However, not all plants that fail to elongate under water are tolerant.The question arises as to whether cells of these plants are metabolically inactiveor simply lack other aspects also needed for tolerance (e.g., the ability to manageATP, cytosolic pH, or cellular O2 content; protection against ROS; or aerenchymadevelopment).

3. Characterization of the Sub1 locus provides an opportunity to breed or engineersubmergence-tolerant rice that could benefit farmers in flood-prone areas. Studiesare needed to determine if submergence and salt tolerance can be combined becausefloodwaters can be saline.

4. The development of rice cultivars with improved underwater germination and low-O2

escape capabilities may reduce herbicide use.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTS

The authors thank Ronald Pierik for his critical comments and his efforts to condense thisreview. Furthermore, we thank Tim Colmer, Takeshi Fukao, Robert Hill, Liesje Mommer,Angelika Mustroph, Pierdomenico Perata, and Eric Visser for comments and discussion. Weregret that many publications were not cited owing to space limitations. Submergence and low-O2 stress research in the Bailey-Serres lab is currently supported by the NSF (IBN-0420152)and USDA (06-35100-17288). Flooding research in the Voesenek lab has been continuouslysupported by the Netherlands Organization for Scientific Research.

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Annual Review ofPlant Biology

Volume 59, 2008Contents

Our Work with Cyanogenic PlantsEric E. Conn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

New Insights into Nitric Oxide Signaling in PlantsAngelique Besson-Bard, Alain Pugin, and David Wendehenne � � � � � � � � � � � � � � � � � � � � � � � � � 21

Plant Immunity to Insect HerbivoresGregg A. Howe and Georg Jander � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 41

Patterning and Polarity in Seed Plant ShootsJohn L. Bowman and Sandra K. Floyd � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 67

Chlorophyll Fluorescence: A Probe of Photosynthesis In VivoNeil R. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 89

Seed Storage Oil MobilizationIan A. Graham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �115

The Role of Glutathione in Photosynthetic Organisms:Emerging Functions for Glutaredoxins and GlutathionylationNicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot � � � � � � � � � � � � � � � � � � � � �143

Algal Sensory PhotoreceptorsPeter Hegemann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �167

Plant Proteases: From Phenotypes to Molecular MechanismsRenier A.L. van der Hoorn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �191

Gibberellin Metabolism and its RegulationShinjiro Yamaguchi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �225

Molecular Basis of Plant ArchitectureYonghong Wang and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �253

Decoding of Light Signals by Plant Phytochromesand Their Interacting ProteinsGabyong Bae and Giltsu Choi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �281

Flooding Stress: Acclimations and Genetic DiversityJ. Bailey-Serres and L.A.C.J. Voesenek � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

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Roots, Nitrogen Transformations, and Ecosystem ServicesLouise E. Jackson, Martin Burger, and Timothy R. Cavagnaro � � � � � � � � � � � � � � � � � � � � � � �341

A Genetic Regulatory Network in the Development of Trichomesand Root HairsTetsuya Ishida, Tetsuya Kurata, Kiyotaka Okada, and Takuji Wada � � � � � � � � � � � � � � � � � �365

Molecular Aspects of Seed DormancyRuth Finkelstein, Wendy Reeves, Tohru Ariizumi, and Camille Steber � � � � � � � � � � � � � � �387

Trehalose Metabolism and SignalingMatthew J. Paul, Lucia F. Primavesi, Deveraj Jhurreea, and Yuhua Zhang � � � � � � � �417

Auxin: The Looping Star in Plant DevelopmentRene Benjamins and Ben Scheres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �443

Regulation of Cullin RING LigasesSara K. Hotton and Judy Callis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �467

Plastid EvolutionSven B. Gould, Ross F. Waller, and Geoffrey I. McFadden � � � � � � � � � � � � � � � � � � � � � � � � � � � � �491

Coordinating Nodule Morphogenesis with Rhizobial Infectionin LegumesGiles E.D. Oldroyd and J. Allan Downie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �519

Structural and Signaling Networks for the Polar Cell GrowthMachinery in Pollen TubesAlice Y. Cheung and Hen-ming Wu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �547

Regulation and Identity of Florigen: FLOWERING LOCUS T MovesCenter StageFranziska Turck, Fabio Fornara, and George Coupland � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �573

Plant Aquaporins: Membrane Channels with Multiple IntegratedFunctionsChristophe Maurel, Lionel Verdoucq, Doan-Trung Luu, and Veronique Santoni � � � �595

Metabolic Flux Analysis in Plants: From Intelligent Designto Rational EngineeringIgor G.L. Libourel and Yair Shachar-Hill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �625

Mechanisms of Salinity ToleranceRana Munns and Mark Tester � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �651

Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal CellsLacey Samuels, Ljerka Kunst, and Reinhard Jetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �683

Ionomics and the Study of the Plant IonomeDavid E. Salt, Ivan Baxter, and Brett Lahner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �709

vi Contents

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Alkaloid Biosynthesis: Metabolism and TraffickingJorg Ziegler and Peter J. Facchini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �735

Genetically Engineered Plants and Foods: A Scientist’s Analysisof the Issues (Part I)Peggy G. Lemaux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �771

Indexes

Cumulative Index of Contributing Authors, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � �813

Cumulative Index of Chapter Titles, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �818

Errata

An online log of corrections to Annual Review of Plant Biology articles may be foundat http://plant.annualreviews.org/

Contents vii

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Flooding-Stress in PlantsMartin M Sachs, Agricultural Research Service, United States Department of Agriculture,

Urbana, Illinois, USA

Flooding is a stress that crop plants frequently encounter and results in huge economic

losses. Studies on the effects of flooding at the molecular level may lead to

improvements that will result in flood-tolerant crop plants.

Introduction

When heavy rains occur shortly after planting a crop,leaving standing water in the field, it can lead to unaccept-able losses and may necessitate replanting. Waterloggedsoils are rapidly depleted of oxygen by microorganisms,leaving the germinating seedlings in an anoxic environ-ment. During the 1993 flooding in the midwestern UnitedStates about 20 million acres of corn and soybean wereinundated, leading to heavy economic losses. Measures toenhance a crop plant’s tolerance to this type of floodingwould be very beneficial to farmers. The response of plants(especially maize; Zea mays ssp. mays) to anoxia has beenthe subject of extensive genetic, physiological, biochemicaland molecular analyses that may eventually lead to suchimprovements. See also: Plant Stress Physiology

The Anaerobic Response

Anaerobic treatment of maize seedlings drastically altersthe profile of total protein synthesis. Initially during an-oxia, there is an immediate and complete repression ofpreexisting (aerobic) protein synthesis along with a rapidincrease in the synthesis of a small class of polypeptides(� 33 kDa, the transition polypeptides; TPs). After 90minof anoxia, the synthesis of a second class of 20 ‘anaerobic’proteins (ANPs) is induced. After 5 h these 20 ANPs areselectively synthesized, and account for more than 70% ofthe total translation, whereas TP synthesis becomes neg-ligible (Figure 1).

Regulation

Regulation of both transcription and translation is in-volved in this response. At the translation level, anaerobic

treatment of maize seedlings disrupts most polysomes andleads to a redirection of protein synthesis. After 72 h, pro-tein synthesis decreases concurrently with the start of celldeath. At the transcription level, genes encoding the ANPsare activated by anoxia, whereas other genes are repressed.Putative regulatory sequences have been found in the pro-motors of a number of anaerobically induced genes. Ca2+

appears to be a signal used to transfer information that aplant is being flooded from themitochondria to the nucleusand starts the cascade of events leading to the anaerobicresponse. See also: Calcium Signalling and Regulation ofCell Function

Other tissues

In the presence of air, each maize organ examined syn-thesizes a tissue-specific spectrum of polypeptides. Mostorgans (e.g. the scutellum and endosperm of the devel-oping kernel) actually synthesize many or all of theANPs constitutively, along with many other proteinsunder aerobic conditions. Only the root tissue ofmature plants and seedlings and other pre-emergentseedling organs (e.g. the coleoptile) show a dramatic in-crease in the level of synthesis of the ANPs by anaerobicinduction. However, under anoxia, most other organsexamined also selectively synthesize the ANPs. More-over, except for a few characteristic qualitative andquantitative differences, the patterns of ANP synthesis indiverse organs are remarkably similar. This response hasalso been observed in maize tissue culture. A similar shiftin the pattern of protein synthesis due to anaerobiosishas been observed in root tissue of many other plantspecies. A notable exception is maize leaves, which can-not synthesize protein under anoxia, and do not surviveeven a brief exposure to anaerobic conditions.

Hypoxia versus anoxia

Hypoxia (low oxygen) has also been found to inducethe synthesis of the ANPs in maize primary roots, but doesnot cause the complete repression of preexistingprotein synthesis observed during anoxia. Significantlevels of ‘aerobic’ protein synthesis are still observed atoxygen concentrations as low as 0.2%. In addition, anovel set of polypeptides, not normally observed under

Advanced article

Article Contents

. Introduction

. The Anaerobic Response

. Role of the ANPs in Anaerobic Metabolism

. Other Responses to Anoxia and Flooding Tolerance

Mechanisms in Plants

. Future Research

. Biography Information

Online posting date: 15th December 2008

ELS subject area: Plant Science

How to cite:Sachs, Martin M (December 2008) Flooding-Stress in Plants. In:Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0001317.pub2

This is a US Government work and is in the public domain in the United States of America.

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aerobic or anaerobic conditions, is synthesized underhypoxic conditions. Additionally, a set of ethylene re-sponses is induced by hypoxia, but not anoxia.See also: Ethylene

As with leaves, it has been found that the maize primaryroot tip is also very sensitive to anoxic treatment, survivingonly a few hours in the absence of oxygen. However, it hasbeen shown that a hypoxic pretreatment allows survival ofthe root tip for a few days under subsequent anoxia. Incontrast, the more proximal part of the primary root andthe pre-emergent shoot (i.e. with an intact coleoptile) cansurvive anoxia for a few days without a hypoxicpretreatment.

Besides this reprogramming of gene expression in a co-ordinated fashion, metabolic (e.g. switch to a fermentativepathway) and structural (e.g. aerenchyma formation)changes also occur during flooding. See also: AnaerobicRespiration

Hypoxia also occurs in plant organs that are bulky and/or have fast metabolic rates, under otherwise well aeratedconditions.

Role of the ANPs in AnaerobicMetabolism

Most of the ANPs identified were found to be enzymes ofglycolysis or sugar-phosphate metabolism such as aldolaseenolase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, sucrose synthase and alcoholdehydrogenase (ADH). In addition, the induction of tran-scription and enzyme activity of pyruvate decarboxylase(PDC) in maize, and lactate dehydrogenase (LDH) in bar-ley, have been reported during anaerobic stress, indicatingthat they may represent other ANPs. Synthesis of theseenzymes appears to be an adaptation allowing someadenosine triphosphate (ATP) production when oxygenis limited.

Alcohol dehydrogenase

Molecular studies of the maize anaerobic responsestemmed from the extensive analysis of maize ADH ge-netics. ADH activity is essential for the survival of maize

Figure 1 Protein synthesis in a maize primary root during anaerobic treatment. Fluorographs of native–sodium dodecyl sulfate (SDS) 2-D polyacrylamide gels

that were loadedwith extracts frommaize primary roots after the following treatments. (a) One hour pulse labellingwith [3H]-leucine under aerobic conditions.

(b–e) Pulse labelling with [3H]-leucine during the specified times under anaerobic conditions. The arrow labelled TPs indicates the position of the transition

polypeptides. The unlabelled arrow indicates the position of alcohol dehydrogenase-1 (ADH1). MW, molecular mass.

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seeds and seedlings during flooding. Normal seedlings sur-vive � 3 days of anoxia, while mutants that lack ADHsurvive only a few hours of flooding. ADH is the majorterminal enzyme of fermentation in plants and is respon-sible for recycling NAD+ during anoxia.

Most animal cells are very sensitive to anoxia. One pos-sible reason for this is that animals rely solely on lactic acidfermentation, since they lack the enzymePDC that catalysesa necessary intermediate step in ethanolic fermentation.Amodel proposedbyDavies (1980) postulates that there is atight regulation of pH to prevent cytoplasmic acidosis dur-ing anoxia in plants. Initially upon anaerobiosis, LDH isactive, causing a drop in cytoplasmic pH with the accumu-lation of lactic acid. However, as the pHdrops LDH (with arelatively high pH optimum) activity decreases while PDC(with a relatively low pHoptimum) activity increases. Thus,it is proposed that after a short period of oxygen depriva-tion, ethanolic fermentation would predominate in plants.Thus, as inanimal cells,maizeADH-nullmutantsmight relyonly on LDH for recycling NAD+ and succumb to cyto-plasmic acidosis. Alternatively, a plant that is ADH-nullmay accumulate toxic levels of acetaldehyde, the substratefor ADH produced by the enzymatic reaction catalysed byPDC. A mutation (atn1), which allows ADH-null seedlingsto survive 24h of anoxia, may allow further understandingof the role ofADH inflooding tolerance.See also: OxidativePhosphorylation

Although some minimum level of ADH activity appearsto be required for any flooding tolerance, natural variationobserved in long-term seedling survival of anaerobic stressdoes not appear to be correlated with variation in the levelsof ADH activity. Variation in flooding tolerance has beenfound among maize lines. When maize lines are compared,survival during anoxia ranges from 2 to 6 days, and thistolerance appears to be inherited as a simple dominant trait.The lines showing variation in flooding tolerance also varyin induced ADH activity levels over a fivefold range, butthere is no apparent relationship between these two traits.

Other Responses to Anoxia andFlooding Tolerance Mechanisms inPlants

Most genes characterized in plants that are induced by ox-ygen deprivation (anoxia or hypoxia) encode enzymes ofglucose-phosphate metabolism (mostly glycolysis and fer-mentation), and thus function to allow some energy pro-duction in the face of limited oxygen supply. In fact, mostresearch on molecular aspects of oxygen deprivation re-sponses in plants has focused on the enzymology of energyproduction that aids in short-term survival of flooding.However, flooding also induces structural modifications,such as the formation of cortical intercellular air spaces(aerenchyma). These modifications can facilitate oxygendiffusion to submerged tissues and, as such, are generallyproposed to aid in prolonging survival during flooding.

Other anoxia-induced maize genes have been identifiedthat are not involved in glucose-phosphate metabolism.These genes, with unique kinetics of mRNA (messenger ri-bonucleic acid) induction under anaerobic conditions com-pared to other anoxia-induced genes, were not induced byheat, cold or salt stress, or by seedling death. Thus, theirinduction appears to be specific to oxygen deprivation. Thefunction of one of these (xet1) has been identified as en-coding a xyloglucan endotransglycosylase, a putative cellwall loosening and degradation enzyme that is proposed toplay a role in wall metabolism during germination, cell ex-pansion and fruit ripening. This enzymemay be involved inaerenchyma formation during flooding. The mechanism ofaerenchyma formation is through selective degradation ofsome cells in the cortex and is termed, lysigeny, i.e. pro-grammed cell death (PCD) and it is mainly induced by lowoxygen concentrations in the soil, coordinatedwith inducedethylene accumulation, during such events as excessive rain,irrigation or flooding. Specific cellulase and protease iso-forms are also induced, in maize roots, by oxygen depriva-tion. These may also be involved in morphological changesthat occur in plants during flooding, such as root tip death.See also: Plant Salt Stress; Plant Temperature StressAnother mechanism of aerenchyma formation called

schizogeny prevails in various plant species where gas-filledspaces are formed by controlled cell division and expan-sion. This is more characteristic of plants inhabiting ex-cessively wet and flooded soils and under suchcircumstances the process is predominantly a constitutiveevent.Adventitious root formation at the soil surface is another

adaptation to flooding conditions. This characteristic al-lows the root system to obtain oxygen directly from the airbecause the adventitious roots develop at or immediatelybelow thewaterlogged soil surface.Othermechanisms suchas use of alternative electron acceptors, changes inmitochondrial untrastructure and enhanced tolerance tooxidative stress during recovery from anoxia are also beinginvestigated in roles that confer increased flooding toler-ance to plants.

Future Research

In addition to variation found in maize, some plants havebeen found to be extremely tolerant to long-term flooding.Natural tolerance to flooding is found in vegetation grow-ing in flood plains, swamps and other areas that are fre-quently waterlogged. Z. mays ssp. huehuetenangensis,Z. luxurians and especially Z. nicaraguensis, close relativesof maize, all show excellent tolerance to flooding. Theseteosintes differ from cultivatedmaize in having constitutiveschizogenous aerenchyma, adventitious root formation atthe soil surface and show tolerance to Fe2+, H2S in wa-terlogged soils (Mano and Omori, 2007). Other floodingtolerant grass species include rice (Oryza sativa) and barn-yard grass (Echinochloa phyllopogon) whose seeds can ger-minate under anaerobic conditions. A variant allele



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(Sub1A-1) in rice enhances submergence tolerance (Xuet al., 2006). Other grasses, such as reed mannagrass(Glyceria maxima), common reed (Phragmites australis)and plants such as American cranberry (Vaccinium mac-rocarpon), sweet flag (Acorus calamus) and pondweeds (inthe genus Potamogeton), have also developed mechanismsfor long-term survival in standing water. Studies of themechanisms that flooding tolerant plants possess are un-derway in order to gain an understanding as to how theseplants cope with extended periods of oxygen deprivation.Additional studies are underway to understand how someplants can alter theirmetabolism so thatATP consumptionis minimized at oxygen concentrations well above thoseactually limiting for respiration (either during flooding orin cases of localized hypoxia). Coupledwith further knowl-edge of how other more susceptible plants respond to an-oxia, understanding mechanisms involved in long-termsurvival during anaerobiosis will ultimately allow molec-ular breeders to produce flooding tolerant varieties of cropssuch as maize. See also: Plant Breeding and CropImprovement

Biography Information

Martin M. Sachs, PhD Genetics 1981, University ofCalifornia, Berkeley. Discovered the anaerobic response(selective translation of ANPs) in plants. Research onmaize flooding-stress has focused on understanding thegenes involved in this response and signal transductionevents allowing plants to perceive oxygen deprivation andleading to the redirection in transcription and translationthat results and related adaptive morphological changesthat occur.

References

Davies DD (1980) Anaerobic metabolism and production of or-

ganic acids. In: StumpfPKandConnEE (eds)TheBiochemistry

of Plants: A Comprehensive Treatise, vol. 2, pp. 581–611. New

York: Academic Press.

Mano Y and Omori F (2007) Breeding for flooding tolerant

maize using ‘teosinte’ as a germplasm resource. Plant Root

1: 17–21.

Xu K, Xu K, Fukao T et al. (2006) Sub1A is an ethylene respon-

sive-factor-like gene that confers submergence tolerance to rice.

Nature 442: 705–708.

Further Reading

Bailey-Serres J and Chang R (2005) Sensing and signalling in

response to oxygen deprivation in plants and other organisms.

Annals of Botany 96: 507–518.

Bailey-Serres J and Voesenek LACJ (2008) Flooding stress: accli-

mations and genetic diversity. Annual Review of Plant Biology

59: 313–339.

Geigenberger P (2003) Response of plant metabolism to too little

oxygen. Current Opinion in Plant Biology 6: 247–256.

Jackson MB (2008) Ethylene-promoted elongation: an adapta-

tion to submergence stress. Annals of Botany 101: 229–248.

Sachs MM and Vartapetian BB (2007) Plant anaerobic stress I.

Metabolic adaptation to oxygen deficiency. Plant Stress

1: 123–135.

Subbaiah CC and Sachs MM (2008) Responses to oxygen

deprivation and potential for enhanced flooding tolerance in

maize. In: Bennetzen JL and Hake SC (eds) Handbook of

Maize: Its Biology. New York: Springer (in press), http://

www.springer.com/life+sci/plant+sciences/book/978-0-387-

79417-4

Vartapetian BB and Crawford RMM (2007) The international

society for plant anaerobiosis: history, functions and activity.

Plant Stress 1: 1–3.

Vartapetian BB, Sachs MM and Fagerstedt KV (2008) Plant an-

aerobic stress II. Strategy of avoidance of anaerobiosis and

other aspects of plant life under hypoxia and anoxia. Plant

Stress 2: 1–19.

Voesenek LACJ, Colmer TD, Pierik R,Millenaar FF and Peeters

AJM (2006) How plants cope with complete submergence.New

Phytologist 170: 213–226.



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ANRV342-PP59-24 ARI 2 April 2008 9:6

Plant Aquaporins:Membrane Channelswith MultipleIntegrated FunctionsChristophe Maurel, Lionel Verdoucq,Doan-Trung Luu, and Veronique SantoniBiochimie et Physiologie Moleculaire des Plantes, SupAgro/INRA/CNRS/UM2UMR 5004, F-34060 Montpellier Cedex 1, France; email: [email protected],[email protected], [email protected], [email protected]

Annu. Rev. Plant Biol. 2008. 59:595–624

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev.arplant.59.032607.092734

Copyright c© 2008 by Annual Reviews.All rights reserved

1543-5008/08/0602-0595$20.00

Key Words

environmental stress, gating, major intrinsic protein, nutrient,transport selectivity, water relations

AbstractAquaporins are channel proteins present in the plasma and intracel-lular membranes of plant cells, where they facilitate the transportof water and/or small neutral solutes (urea, boric acid, silicic acid)or gases (ammonia, carbon dioxide). Recent progress was made inunderstanding the molecular bases of aquaporin transport selectiv-ity and gating. The present review examines how a wide range ofselectivity profiles and regulation properties allows aquaporins to beintegrated in numerous functions, throughout plant development,and during adaptations to variable living conditions. Although theyplay a central role in water relations of roots, leaves, seeds, and flow-ers, aquaporins have also been linked to plant mineral nutrition andcarbon and nitrogen fixation.

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MIP: majorintrinsic protein

PIP: plasmamembrane intrinsicprotein

TIP: tonoplastintrinsic protein

NIP: nodulin-26–like intrinsicprotein

SIP: small basicintrinsic protein

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 596MOLECULAR AND CELLULAR

PROPERTIES . . . . . . . . . . . . . . . . . . . 596The Plant Aquaporin Family . . . . . . 596Mechanisms of Transport . . . . . . . . . 597Molecular Mechanisms of

Regulation . . . . . . . . . . . . . . . . . . . . 599AQUAPORIN FUNCTIONS

THROUGHOUT PLANTGROWTH ANDDEVELOPMENT. . . . . . . . . . . . . . . 603Water Transport . . . . . . . . . . . . . . . . . 603Nitrogen, Carbon, and

Micronutrient Acquisition . . . . . 606AQUAPORINS IN A VARIABLE

ENVIRONMENT. . . . . . . . . . . . . . . 607Changes in Irradiance . . . . . . . . . . . . 607Water, Salt, and Nutrient Stresses . 608Cold Stress . . . . . . . . . . . . . . . . . . . . . . . 611Anoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . 612Biotic Interactions . . . . . . . . . . . . . . . . 612

CONCLUSIONS. . . . . . . . . . . . . . . . . . . 613

INTRODUCTION

Aquaporins are small integral membrane pro-teins that belong to the ancient family ofmajor intrinsic proteins (MIPs), with mem-bers in animals, microbes, and plants. Fifteenyears after their discovery in plants, it nowappears that studies on aquaporins have pro-vided unique perspectives into multiple in-tegrated aspects of plant biology. Aquapor-ins first raised considerable interest becauseof their water channel activity. This findingwas unexpected in plants and, although it maynot have induced a real paradigm shift inunderstanding of membrane water transport(124, 140), it led researchers to revisit manyaspects of plant water relations and to linkthese aspects to novel physiological contexts.More recently, MIPs were proved to be morethan water channels (141), and other transportsubstrates of great physiological significancehave been identified.

Although the term aquaporin was ini-tially restricted to water-transporting MIPs,we now use this term in a broader sense, re-ferring to all plant MIPs as aquaporins. Themolecular and cellular properties of aquapor-ins were recently reviewed in detail (18, 91).The aim of the present review is to examinehow a wide range of selectivity profiles andregulation properties allows aquaporins to beintegrated in numerous functions throughoutplant development and during adaptations tovariable environmental conditions.

MOLECULAR AND CELLULARPROPERTIES

The Plant Aquaporin Family

Subfamilies. Plant aquaporins show a highmultiplicity of isoforms, with 35 and 33 ho-mologs in Arabidopsis and rice, respectively(62, 113, 120). On the basis of sequence ho-mology, aquaporins in most plant species canbe divided into four subgroups. The plasmamembrane intrinsic proteins (PIP) (with twophylogenic subgroups, PIP1 and PIP2, and13 isoforms in Arabidopsis) and the tono-plast intrinsic proteins (TIP) (10 homologsin Arabidopsis) are the most abundant aqua-porins in the plasma membrane and vacuolarmembrane (tonoplast), respectively (62, 113).The third subfamily comprises the nodulin-26–like intrinsic membrane proteins (NIPs),which were named after soybean (Glycine max)nodulin-26 (GmNOD26), an abundant aqua-porin expressed in the peribacteroid mem-brane of N2-fixing symbiotic root nodules.NIPs are also present in nonlegume plantspecies (9 homologs in Arabidopsis) (149). Afourth class comprises small basic intrinsicproteins (SIPs) (3 homologs in Arabidopsis)(56, 62, 113). Although these four classesare conserved among all plant species, theaquaporin gene family shows signs of rapidand recent evolution and orthologs cannotnecessarily be distinguished between species(120). In addition, some plant species have ac-quired additional, novel types of aquaporins.

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For instance, a homolog of the bacterial glyc-erol facilitator GlpF has been acquired by themoss Physcomitrella patens by horizontal genetransfer (45), and the genome of this organ-ism and some higher plants (such as poplar)encodes a fifth class of aquaporins, which areclosely related to but yet clearly distinct fromPIPs (139; U. Johanson, personal communi-cation).

Subcellular localization. Plant aquaporinslocalize in all subcellular compartments form-ing or derived from the secretory pathway.This broad localization pattern reflects thehigh degree of compartmentation of the plantcell and the need for the cell to control wa-ter and solute transport not only across theplasma membrane but also across intracellu-lar membranes. Similar to PIPs, some NIPslocalize in the plasma membrane (82, 134). Bycontrast, the three Arabidopsis SIP homologsreside mainly in the endoplasmic reticulum(56).

However, aquaporins cannot simply beassigned to homogeneous subcellular com-partments. For instance, immunocytochemi-cal studies using isoform-specific anti-TIP an-tibodies revealed that distinct types of vacuolethat can coexist in the same cell are equippedwith specific combinations of TIP isoforms;TIP1 and TIP2 isoforms are preferentiallyassociated with the large lytic vacuoles andvacuoles accumulating vegetative storage pro-teins, respectively (59). More recently, Ara-bidopsis thaliana AtTIP1;1 was shown to accu-mulate in spherical structures named bulbs,tentatively identified as intravacuolar invagi-nations made of a double tonoplast membrane(118). Preferential expression of PIPs in plas-malemmasomes (convoluted plasma mem-brane invaginations that dip into the vacuole)has also been observed in Arabidopsis leaves(116). Finally, preferential expression of a PIPand a NIP homolog on the distal side of rootexo- and endodermal cells has been describedin maize and rice, respectively (46, 82). Suchcell polarization is consistent with the uptakeand centripetal transport of water and solute

Plasmalemmasome:convoluted plasmamembraneinvagination

in roots (see below). A future challenge isto understand how aquaporins can be specif-ically targeted to membrane subdomains inthe plant cell and how targeting contributesto their functional specialization.

Mechanisms of Transport

Pore structure and transport mecha-nisms. X-ray crystallography determinationof atomic structures of microbial, animal,and plant homologs points to highly con-served structural features in the aquaporinfamily (38, 137). Aquaporins are 23–31 kDaproteins comprising six membrane-spanningdomains tilted along the plane of the mem-brane and linked by five loops (A to E ) lo-cated on the intra- (B, D) or extracytoplas-mic (A, C, E ) side of the membrane. TheN- and C-terminal extremities are both ex-posed to the cytosol (Figure 1). A centralaqueous pore is delineated by the transmem-brane domains and loops B and E, which bothcarry a conserved Asn-Pro-Ala (NPA) motifand dip from either side of the membraneinto the center of the molecule. Projectionstructures determined by cryo-electron mi-croscopy indicate that, similar to their animaland microbial counterparts, PIPs and TIPsoccur as tetramers in their native membranes(24, 34). X-ray structures have confirmed thistype of assembly (38, 137) and in combi-nation with molecular dynamics simulationshave provided critical insights into the funda-mental principles of aquaporin transport se-lectivity (38, 133) (Figure 1). In brief, thesubstrate specificity of aquaporins can be ex-plained by several mechanisms, including sizeexclusion at two main pore constrictions [aro-matic/Arg (Ar/R) and NPA] and stereospe-cific recognition of the substrate mediated byspatially defined H-bonding and hydropho-bic interactions within the pore. The remark-able impermeability of aquaporins to protonsis explained by electrostatic repulsion, dipoleorientation, and transient isolation of thewater molecule as it passes within a single

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a

Phe81

Asn101

Arg225

His210

Asn222

b

Figure 1Representative atomic structure of a plant aquaporin (a) and general molecular mechanisms of transportselectivity (b). (a) Structure of the open conformation of Spinacia oleracea plasma membrane intrinsicprotein 2;1 (SoPIP2;1) [Protein Data Bank (PDB) ID 2B5F] (137) showing a typical tetramericarrangement. Each monomer is composed of six tilted transmembrane helices; the N-terminal (red ) andC-terminal ( green) helices of the top left monomer are shown. The pores of individual monomers areemphasized by the space-filling representation of the three other monomers. (b) The two highlyconserved Asn-Pro-Ala (NPA) motifs (represented by Asn101 and Asn222, green) are in close proximityto form one of the main pore constrictions. Another constriction called Ar/R (red ) is formed on theextracytoplasmic side of the membrane by a spatial arrangement of aromatic (Ar) residues, such as Phe81and His210, facing an Arg (R) residue, here Arg225. Proton transport is blocked by electrostaticrepulsion in the Ar/R constriction and the dipole orientation of the water molecule by the two Asnresidues of the NPA motifs. This results in a transient isolation of the water molecule within the singlefile of water molecules that fills the pore (orange spheres).

file of water molecules through the center ofthe pore (11, 38, 133) (Figure 1).

The molecular basis of plant aqua-porin selectivity has been investigated morespecifically by homology modeling of porestructures at the Ar/R constriction (8, 150).Analysis of all 35 Arabidopsis homologs yieldedup to nine pore types (150) and additionaltypes exist in maize and rice (8). Whereasall PIPs exhibit a narrow pore structure typ-ical of orthodox, water-selective aquaporins,larger substrate specificity was predicted forother plant homologs. According to this anal-ysis, AtNIP6;1 belongs to one of two NIPsubgroups and as such exhibits a low andhigh permeability to water and urea, respec-tively (151). An Ala119Trp substitution, madeto mimic the pore configuration of mem-bers of the other NIP subgroup, also con-

fers novel permeability properties, i.e., higherpermeability to water and failure to trans-port urea. This result and other examples inanimal aquaporins (11) show that point mu-tations can drastically alter transport speci-ficity and that these proteins may be engi-neered to accommodate novel substrates ofinterest.

Transport assays and aquaporin sub-strates. Functional expression in Xenopusoocytes or yeast was essential to show thatplant MIP homologs of all four subclassescan function as water channels (56, 66, 94,115). Enhanced water permeability of pro-teoliposomes containing a purified aquaporinprovides the ultimate proof of water channelactivity. Such functional reconstitution hasbeen performed with GmNOD26 purified

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from native peribacteroid membranes (27) orafter production of Spinacia oleracea SoPIP2;1in Pichia pastoris (67). Although strict compar-ative measurements have not been performedin plants, plant aquaporins may, similar totheir animal homologs, exhibit marked differ-ences (up to 30-fold) in intrinsic water trans-port activity (154).

Expression studies in Xenopus oocytes alsoshow that, similar to animal and bacterialaquaglyceroporins, some plant aquaporin iso-forms can transport small neutral solutes suchas glycerol (12), urea (42), formamide, ac-etamide (115), methylammonium (53), boricacid (134), silicic acid (82), or lactic acid (20).Ammonia (NH3) and CO2 transport is de-tected using substrate-induced extra- and in-tracellular acidification, respectively, whereasammonium (NH4

+) transport by Triticum aes-tivum TaTIP2;1 results in inward currents(53, 144). Finally, expression in yeast cells de-ficient in endogenous systems responsible forurea or hydrogen peroxide uptake has provedefficient to screen, on the basis of a survivalassay, aquaporin isoforms that possibly trans-port these molecules; these properties are sub-sequently confirmed by true transport assays(13, 77).

Several approaches have established thataquaporins contribute significantly to the per-meability of plant membranes to water andsmall neutral solutes. In most studies, mercuryderivatives, which act through oxidation andbinding to Cys residues, were used as com-mon aquaporin blockers. Plant aquaporins donot have Cys residues at conserved positionsand various residues may be involved in plantaquaporin inhibition (23). We also note thatmercury-resistant PIPs have been described inArabidopsis and tobacco (12, 25). In some stud-ies, the permeability profiles of the vacuolar,peribacteroid, and plasma membranes werecharacterized by stopped-flow spectropho-tometry on purified membrane vesicles, andmercury induced a marked (50%–90%) inhi-bition of water transport in the first two typesof membranes (42, 95, 104, 105, 115). In addi-tion, a good parallel was established between

the high permeability of the tobacco tonoplastand soybean peribacteroid membrane to ureaand formamide, respectively, and the capacityof Nicotiana tabacum NtTIPa and GmNOD26to transport these solutes (42, 115). In otherstudies, the respective water permeabilitiesof the plasma membrane and the tonoplastand their sensitivity to mercury were in-ferred from independent osmotic swellingassays on protoplasts and isolated vacuolesand calculations using a three-compartmentmodel (92, 99, 102). Figure 2 summarizesthe contribution of plant aquaporins to wa-ter and solute transport in multiple subcellularcompartments.

Molecular Mechanisms of Regulation

Cotranslational and posttranslationalmodifications. Because of their high abun-dance in plant membranes, and despitetheir high hydrophobicity, some aquaporinshave proved to be particularly amenable tobiochemical analysis, in comparison withother membrane proteins (34, 48, 63). Pro-teomics, and mass spectrometry techniques inparticular, have recently been added to moreclassical techniques to produce a thoroughdescription of aquaporin co- and posttrans-lational modifications (26, 121, 122). Forinstance, N-terminal maturation of PIP1sand PIP2s occurs through N-α-acetylation orcleavage of the initiating residue, respectively(121). In vivo and in vitro labeling studies,experiments with antiphosphopeptide anti-bodies, and mass spectrometry analyses haveprovided direct evidence for phosphorylationof Ser residues in the N-terminal and C-terminal tails of Phaseolus vulgaris PvTIP3;1,GmNOD26, and SoPIP2;1 (26, 43, 63, 64,96). PIPs show a conserved phosphorylationsite in loop B and multiple (up to three)and interdependent phosphorylations occurin adjacent sites of their C-terminal tail(63, 64; S. Prak, S. Hem, J. Boudet, N.Sommerer, G. Viennois, M. Rossignol, C.Maurel & V. Santoni, unpublished results).Purification of calcium-dependent protein


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kinases acting on aquaporins has beenundertaken by several laboratories (48, 129).Although most plant aquaporins do not ex-hibit glycosylation, this type of modification

has been observed in GmNOD26 and in an iceplant TIP (96, 146). In the latter case, glycosy-lation was required for subcellular redistribu-tion (described below). Aquaporins were also

Silicicacid

Hydrogen peroxideWater

Ammonia?Water

Boricacid

Water

CO2

AmmoniaWater

GlycerolUreaWater

Protein storagevacuole

Vegetative proteinstorage vacuole

Lacticacid

Water

PIP1s

PIP2s

TIP1s

TIP2s

TIP3s

AtNIP2;1

Lsi1/OsNIP2;1

AtNIP5;1

NOD26

SIPs

Nucleusand ER

ChloroplastChloroplast

Golgiapparatus

Lytic/central vacuole

Earlyendosome

Plasmalemmasome

Vacuolarbulb

PlasmamembraneCell wall

Peribacteroidmembrane

Multivesicular body / lateendosome / prevacuolar

compartment

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the first plant membrane proteins found to bemethylated (121). For instance, AtPIP2;1 cancarry one or two methyl groups on its Lys3 andGlu6 residues, respectively. These data showthat, in addition to a high isoform multiplic-ity, plant aquaporins occur in a large variety ofmodified forms, which suggests intricate co-and posttranslational regulation mechanisms.

Gating. The gating of aquaporins, i.e.,the opening and closing of the pore, canbe regulated by multiple factors. A rolefor phosphorylation in gating PvTIP3;1,GmNOD26, and SoPIP2;1 was first deducedfrom functional expression in oocytes ofthese aquaporins, either wild-type or withpoint mutations at their phosphorylation sites(43, 63, 93), and by using pharmacological al-terations of endogenous protein phosphatasesand kinases. A role for phosphorylation inGmNOD26 gating has been unambiguouslyestablished by stopped-flow measurements inpurified peribacteroid membranes, showingthat alkaline phosphatase-mediated dephos-phorylation leads to reduced water perme-ability (43). Water transport measurementsin plasma membrane vesicles purified fromArabidopsis suspension cells or Beta vulgarisroots also suggest that PIPs can be gatedfrom the cytosolic side by protons and diva-lent cations (4, 41). A half-inhibition of water

Gating: openingand closing of amembrane channelpore

transport is observed at ∼pH 7.5 and for freeCa2+ concentrations in the 100 μM range(4, 41). Beet plasma membranes exhibit anadditional affinity component in the 10 nMrange (4).

The molecular bases of aquaporin gatinghave been elucidated from structure-functionanalyses in Xenopus oocytes and more re-cently from the atomic structures of SoPIP2;1in its open and closed conformations (137,138). These studies established that protonsare sensed by a His residue that is perfectlyconserved in loop D of all PIPs (138). Themolecular mechanisms that lead to a confor-mational change of loop D and occlusion ofthe pore upon protonation of the His residueor binding of divalent cations are detailed inFigure 3. The atomic structure of SoPIP2;1also indicates how phosphorylation of loop Bwould unlock loop D to allow the open confor-mation. By contrast, phosphorylation of theC-terminal tail would act in trans to preventloop D of an adjacent monomer from adopt-ing a closed-pore conformation (137).

A role for solutes in gating aquaporinshas been proposed, based mainly on pressureprobe measurements in Chara cells (155). In-hibition of cell water permeability is linked tothe presence of the solute on either side of themembrane and is strongly dependent on so-lute molecular size. A tension/cohesion model

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2The multiple cellular functions of plant aquaporins. The figure illustrates the variety of transportfunctions achieved by aquaporins in various subcellular compartments. The different aquaporinsubclasses or isoforms are identified below the illustration in distinct colors. Isoforms of the plasmamembrane intrinsic protein 1 (PIP1) and PIP2 subfamilies are thought to follow the secretory pathway,which carries cargo from the endoplasmic reticulum (ER) toward the plasma membrane through theGolgi apparatus. PIPs also undergo repeated cycles of endocytosis and recycling through endosomalcompartments before being eventually targeted to the lytic vacuole through the multivesicular body. InArabidopsis leaves, PIP1s label plasmalemmasomes (116). Tonoplast intrinsic protein 1s (TIP1s) are foundin the lytic vacuole membrane. AtTIP1;1 localizes in spherical structures named bulbs in epidermal cellsof young cotyledons or salt-treated roots (15, 118). TIP2s and TIP3s are preferentially associated withvacuoles that accumulate vegetative storage proteins and seed protein storage vacuoles, respectively.Nodulin-26–like intrinsic membrane proteins (NIPs) show a broad range of subcellular localizationpatterns. AtNIP2;1 is localized in the endoplasmic reticulum and the plasma membrane (20, 97), theOryza sativa silicon influx transporter low silicon rice 1 (Lsi1, also namedOsNIP2;1) and the Arabidopsisthaliana boric acid channel AtNIP5;1 are localized in the plasma membrane, whereas Glycine maxnodulin-26 (GmNOD26) is exclusively expressed in the peribacteroid membrane.


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Loop D

Loop BLoop D

a

b

His193

His193

Leu197

Leu197

Glu31

Arg118

Glu31

Asp28

Ser115

Asp28

Ser115Arg118

Figure 3Molecular mechanisms of plasma membrane intrinsic protein (PIP)gating. The Spinacia oleracea SoPIP2;1 structure was solved in an open(a) [Protein Data Bank (PDB) ID 2B5F] and in a closed (b) (PDB ID1Z98) conformation (137). The His193 residue ( green) is perfectlyconserved in loop D of all PIPs. In the open conformation (a), His193 isnot protonated and loop D is distal from the other cytoplasmic loop B.By contrast, the protonation of His193 (b) allows interaction with anacidic residue of the N terminus, Asp28 ( purple). This in turn drives aconformational change of loop D and occlusion of the pore bydisplacement of the hydrophobic side chain of Leu197 ( yellow) into thecytosolic pore mouth. Binding of divalent cations [Cd2+ in the atomicstructure, ( purple sphere)] would also involve Asp28 and an adjacent acidicresidue (Glu31). Loop D would then be stabilized in the closed poreconformation through a network of H-bond and hydrostatic interactions,involving Arg118. In this model, phosphorylation of loop B, at Ser115( pink), would disrupt this network of interactions and unlock loop D toallow the open conformation.

was proposed in which exclusion of the solutefrom the narrow vestibule of the pore wouldresult in osmotic forces and tensions, which inturn would collapse the pore (155). Hydroxylradicals also induce a marked (≥90%) and re-versible inhibition of water transport in Characells, which was interpreted in terms of directoxidative gating of aquaporins (50). By con-trast, the inhibition of aquaporins by reactiveoxygen species (ROS) in the Arabidopsis rootseems to involve cell signaling mechanisms(Y. Boursiac, J. Boudet, O. Postaire, D.-T.Luu, C. Tournaire-Roux & C. Maurel, un-published results).

Tetramer assembly and cellular traffick-ing of PIPs. Recent studies have pointed toaquaporin trafficking as a critical point forregulating aquaporin expression and function.The inability of some PIP1 isoforms to befunctionally expressed in Xenopus oocytes hasbeen reported by several laboratories. Fet-ter and coworkers (31) explained this inabil-ity by a failure of these aquaporins to trafficto the oocyte plasma membrane, and showedthat coexpression of maize PIP1s in Xeno-pus oocytes with reduced amounts of PIP2isoforms could alleviate this defect. Affin-ity copurification and coimmunopurificationstudies provided the first biochemical evi-dence that PIP1s and PIP2s physically in-teract, both in oocytes and plants (31, 158).The results of fluorescence resonance energytransfer (FRET) imaging in living maize pro-toplasts coexpressing PIP1s and PIP2s fur-ther support a model in which aquaporins ofthe two classes directly interact, very likely byheterotetramerization, to facilitate PIP1 traf-ficking (158). A possible role for PIP1 phos-phorylation was recently added to this model(135). Phosphorylation on loop B of a MimosaPIP1 is not necessary for aquaporin interac-tion but enhances the overall water transportactivity of PIP1-PIP2 complexes in oocytes(135). Whereas interaction-dependent traf-ficking of PIP1s and PIP2s offers a broadrange of combinatorial regulations, a futurechallenge is to determine to what extent this

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process can dominate the expression of PIP1or PIP2 homotetramers. Antisense inhibitionexperiments in Arabidopsis of PIP1s and PIP2s,alone or in combination, have suggested thatthe two classes of aquaporins contribute tothe same functional water transport units(90).

Similar to other membrane proteins,PIP2 aquaporins are subjected to constitu-tive cycling. Their endocytosis is clathrin-dependent (28) and reduced by auxin (108).Export of PIP2 aquaporins from the endo-plasmic reticulum is also critically controlledand the role of a di-acidic motif containedin the N-terminal tail of PIP2s was recentlyuncovered in maize and Arabidopsis (F. Chau-mont, personal communication; M. Sorieul,D.-T. Luu, V. Santoni & C. Maurel, un-published results). The cellular mechanismsthat determine aquaporin trafficking and theirsubcellular relocalization in response to stim-uli will surely fuel intense investigations in thecoming years.

AQUAPORIN FUNCTIONSTHROUGHOUT PLANTGROWTH AND DEVELOPMENT

Water Transport

Principles of plant water transport. A widerange of cell water permeabilities can beobserved between distinct cell types andthroughout plant development. For instance,cell pressure probe measurements indicatethat, in growing epicotyls of pea, cortical cellshave a ∼30-fold higher hydraulic conductiv-ity than epidermal cells (130). Also, swellingassays on isolated protoplasts from rape rootsindicate that their osmotic water permeabilitycoefficient increases from 10 to 500 μm sec−1

within less than two days (114). Although thecontribution of the lipid membranes shouldbe taken into account, one challenge will beto determine how the aquaporin equipment ofeach individual cell can determine such strik-ingly different water transport properties. Inthese respects, attempts have been made to re-

Hydraulicconductivity: waterpermeability (i.e.,intrinsic capacity totransport water) of amembrane, cell, ortissue

late the cell-specific expression of aquaporinisoforms in radish taproots and maize primaryroots and the water permeability of proto-plasts derived from the various cell types ofthese materials (46, 132).

In the whole plant, long-distance transportof fluids occurs mostly through vascular tis-sues, which do not present significant mem-brane barriers. Yet, living tissues can be thesite of intense flows of water during transpi-ration or expansion growth. For this, watercan flow along various paths: (i ) the apoplas-tic path, i.e., within the cell wall continuum,(ii ) the symplastic route through cytoplas-mic continuities and plasmodesmata, and (iii )the transcellular path across cell membranes(mainly plasma membranes), which in manytissues is mostly mediated by aquaporins. Al-though it is not very specific and is inactiveon certain aquaporins (see above), mercuryrepresents one of the very few tools availableto evaluate the contribution of aquaporins towater transport in plant tissues. The generaltoxicity of this compound in vivo must becarefully evaluated (reviewed in 61), and re-searchers checked that mercury does not per-turb xylem solute transport and respiration inaspen roots. By contrast, mercury depolarizeswheat root cells in parallel to inhibition ofwater transport (61). A reversibility of mer-cury effects by reducing agents is also requiredto show that the blocking effects are due tooxidation mechanisms and not to irreversibledamage of the cells. Despite all these restric-tions, the effects of mercury on water trans-port have been characterized in a large va-riety of physiological contexts. Overall, thesestudies provide a consistent picture of the roleof aquaporins, in particular during root wa-ter transport (61). Yet, new specific aquaporinblockers are critically needed. Gold and silverions have been described as potent aquaporinblockers in vitro but the use of these com-pounds in vivo seems to be problematic andtheir mode of action is as yet unknown (106).

Transpiration. Because it induces an in-tense renewal of water throughout the plant,


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ZmPIP2;1/2ZmPIP2;5

Lsi1/OsNIP2;1Lsi2

PIP1sTIP1s

Maize

a

Rice

ExodermisEpidermis

Endodermis

i

ii

b

Movement of water

Movement of CO2

Bundle sheath

b

Spongymesophyll

Palisademesophyll

Phloem sieve tube

Xylem vessel

Epidermis

Cuticle

Guard cell

Movement of waterUptake of silicic acid

Figure 4Aquaporin-mediated transport of water and solutes in roots (a) and leaves (b). Schematic cross sectionswith representations of the tissue-specific expression patterns of aquaporins and paths of transport areshown. Aquaporin expression and water transport in maize roots is summarized according to Reference46, whereas uptake of silicic acid in rice roots by Oryza sativa Lsi1 (OsNIP2;1) in combination with theefflux transporter Low silicon rice 2 (Lsi2) is drawn according to References 82 and 83. Expression ofplasma membrane intrinsic protein 1s (PIP1s) and tonoplast intrinsic protein 1s (TIP1s) in Brassica napusleaves was summarized according to Reference 35. The movement of water can follow the cell-to-cell(symplastic and transcellular) (i ) or apoplastic (ii ) path.

Lpr: root hydraulicconductivity

transpiration represents an obvious contextin which to investigate aquaporin function inroots and leaves. A thorough description ofcell-specific expression of aquaporins in rootswas recently performed in maize using in situand quantitative RT-PCR and immunolocal-ization (46). Strong expression of ZmPIP2;1and ZmPIP2;5 is observed in both the exo-dermis and endodermis of the mature zone,suggesting that because of the presence ofCasparian strips a bypass of the apoplasticpath may be necessary in these cell layers (46)(Figure 4a). Also, a strong expression in thestele and vascular tissues is consistently ob-served in roots of several plant species (46,124, 132). Several lines of functional evidencealso show that aquaporins significantly con-tribute to water uptake by roots. Firstly, mer-

cury inhibits the root hydraulic conductivity(Lpr) by 30%–90% in more than ten plantspecies (61). In addition, antisense inhibitionof aquaporins of the PIP1 and/or PIP2 sub-classes reduced Lpr by approximately 50%in tobacco or Arabidopsis (90, 128). Finally,two allelic Arabidopsis pip2;2 knockout mu-tants show, with respect to wild-type plants,a reduction of 25%–30% in hydraulic con-ductivity of root cortex cells (60). In addition,the osmotic hydraulic conductivity of entireroots, as derived from free exudation measure-ments, is decreased by 14% in the mutants,pointing to PIP2;2 as an aquaporin special-ized in osmotic fluid transport in the Arabidop-sis root (60). Further analysis of mutants cor-responding to aquaporin genes with distinctcell-specific expression patterns may help

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dissect the contribution of the various celllayers to distinct modes (i.e., osmotic orhydrostatic) of water transport within theroot.

The preferential expression of some aqua-porin isoforms in vascular tissues and celltypes such as tracheary elements, xylemparenchyma cells, and phloem-associatedcells suggests a general role of aquaporinsin sap transport throughout the plant body(reviewed in 92). In the leaf, the water sup-ply at the evaporating sites is sustained by aflow of liquid water from the vascular systemthrough the extravascular compartment, in-cluding the vascular bundles and the meso-phyll (Figure 4b). A role for aquaporins inmediating water transfer from the veins tothe stomatal chamber has been proposed onthe basis of two lines of evidence. Firstly,mercury can inhibit leaf hydraulic conduc-tance (Kleaf) in sunflower and in six temper-ate deciduous trees (1, 103). Secondly, light-dependent changes in Kleaf in walnut occurwithin one hour, are associated with changesin expression of PIP2 aquaporin transcripts,and both are inhibited by 100 μM cyclohex-imide, indicating a role for protein synthesis inKleaf regulation (22). In a recent study, meso-phyll protoplasts were isolated from Arabidop-sis genotypes differing in stomatal apertureor from plants grown at varying relative airhumidity (98). Surprisingly, researchers ob-served an inverse relationship between therate of transpiration in the plant and the wa-ter permeability of the isolated protoplasts.These variations occur without any alterationin the leaf PIP content, suggesting that aqua-porin function is controlled at the posttrans-lational level. The physiological significanceof this control and its occurrence in otherplant species are as yet unknown. Expressionof aquaporins has also been reported in stom-atal guard cells (36, 123). Despite the criti-cal role played by these cells in maintainingthe whole plant water status, the function ofaquaporins in stomatal movements is as yetunclear and deserves more attention for fu-ture research.

Kleaf: leaf hydraulicconductance

Tissue expansion. Although some aqua-porin isoforms seem to be specific to divid-ing cells (9), a strong link between PIP andTIP aquaporin expression and cell expansionhas been observed in numerous plant materi-als. For instance, expression of the AtTIP1;1promoter is associated with cell enlargementin Arabidopsis roots, hypocotyls, leaves, andflower stems (81), and transcript accumulationis enhanced by the growth-promoting hor-mone gibberellic acid (GA3) (109), suggest-ing that AtTIP1;1 may contribute to the dif-ferentiation of a large central vacuole in fullyelongated cells (81).

Because most plant cells have short half-times of water exchange (130), water influxinto a single plant cell can hardly be limit-ing during expansion growth. Membrane wa-ter transport in growing tissues should ratherbe envisioned in the context of a transcellu-lar delivery of water from vascular tissues to-ward peripheral expanding tissues (148). Thepresence, in certain physiological conditions,of significant water potential gradients withingrowing tissues supports the idea that this typeof water transport can be limiting. Accord-ingly, mercury blocks tissue growth in maizeroots, but exclusively in the older cells, distalto the apex (55). These cells are character-ized, with respect to younger cells, by a high,mercury-sensitive cell hydraulic conductivityand reduced symplastic connections with thephloem, suggesting that aquaporin-mediatedtranscellular water transport is necessary fordelivery of water from the phloem into thecells. In castor bean hypocotyls, the tran-script abundance of a specific PIP2 isoformand a high hydraulic conductivity of corticalcells both show the same light- and spatial-dependence as that of tissue expansion (30).Finally, diurnal epinastic movements (unfold-ing) of tobacco leaves can be accounted for bya differential growth of the upper and lowersurfaces of the petiole. A limiting role fortobacco aquaporin 1 (NtAQP1) in this pro-cess was proposed on the basis of observa-tions that expression of NtAQP1 in the peti-ole shows a diurnal rhythm that coincides


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gm: mesophyllconductance to CO2

with leaf unfolding and that antisense inhibi-tion of NtAQP1 impairs leaf movement (127).These studies draw a convincing, but qual-itative, picture of aquaporin function in ex-pansion growth. Future studies will have toquantitatively integrate growth rates, cell andtissue water relation parameters, and aqua-porin expression in time and space (148). Fruitdevelopment and ripening, which involve cellenlargement and cotransport of sugar and wa-ter, will also represent important processesin which to investigate aquaporin function(19, 110).

Tissue desiccation and imbibition. Plantreproduction requires the intense desiccationof certain organs, which then acquire spe-cific dissemination and resistance properties.TIP isoforms specific to pollen grains havebeen reported in Arabidopsis but their func-tion during pollen desiccation and/or pollentube growth is as yet unclear (111). By con-trast, antisense inhibition of PIP2 aquapor-ins in tobacco delays anther dehydration anddehiscence, suggesting that these aquaporinsare involved in water flow out of the anthervia the vascular bundle and/or evaporation(14). Seed germination represents another re-markable context, during early tissue imbibi-tion and subsequent embryo growth, in whichaquaporins may mediate a fine temporal andspatial control of water transport; evidence foraquaporin function in seeds is emerging. InBrassica napus, the germination rate of seedsthat have gone through various priming treat-ments is strongly correlated with the tran-script abundance of a PIP2 aquaporin (39).In pea and Arabidopsis, mercurials reduce thespeed of seed imbibition and seed germina-tion, respectively (145, 147). Finally, expres-sion in tobacco and rice of sense and antisensePIP1 transgenes shows that the speed and ex-tent of germination of seeds in normal and/orwater stress conditions is positively correlatedwith aquaporin expression (76, 156). Liu andcoworkers (76) suggested a role for PIP1s instimulating seed germination by nitric oxide(NO) in rice.

Nitrogen, Carbon, andMicronutrient Acquisition

Nitrogen fixation. A first link between aqua-porins and nitrogen (N) assimilation camefrom the observation that expression of someaquaporin genes is dependent on N com-pounds; some genes, such as ZmPIP1;5b, arestrongly induced by nitrate (40), whereasothers, such as AtTIP2;1, are induced un-der long-term N starvation or short termNH4

+ supply (79). This type of regulation wasfirst interpreted as a reflection of well-knownconnections between water relations and Nmetabolism. However, evidence was recentlypresented that aquaporins of the PIP, NIP, andTIP subfamilies can transport N compounds.Transport of urea by TIPs (42, 70, 140) maycontribute to urea equilibration within the celland storage into and remobilization from thevacuole (70). Wheat and Arabidopsis TIP2 ho-mologs also show a remarkable permeabilityto NH3 and may therefore contribute to sig-nificant loading of this compound and acid-trapping of the protonated form (NH4

+) inthe vacuole (53, 57, 79). However, studiesof transgenic Arabidopsis that overexpressesAtTIP2;1 failed to establish any significantrole for this aquaporin in NH4

+ accumulation(79).

CO2 transport and carbon metabolism.CO2 transport by aquaporins in planta wasfirst evaluated by treating Vicia faba and Phase-olus vulgaris leaves with mercury (136). Inthese two experiments, mercury altered thedependency of photosynthesis on intercellu-lar but not on chloroplastic CO2. This effectwas interpreted to mean that CO2 diffusioninto the chloroplast [i.e., mesophyll conduc-tance to CO2 ( gm)] is blocked and thereforeinvolves proteins, possibly aquaporins. Trans-genic plants with altered aquaporin expressionprovide systems in which to explore this is-sue further (47, 144). In tobacco plants withantisense inhibition or an antibiotic-inducibleoverexpression of NtAQP1, gm positively cor-relates with the NtAQP1 expression level

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(33, 144). In view of the CO2 transport ac-tivity of NtAQP1 in oocytes, this result wasinterpreted as evidence that this PIP1 ho-molog serves as a CO2 pore in tobacco leaves(33, 144). Interestingly, in various genetic andphysiological contexts gm is positively corre-lated with maximal stomatal conductance andCO2 assimilation capacity (see 33 and ref-erences therein). Up to now, it was unclearwhether changes in gm can be accounted forby changes in leaf anatomy or by changes incell permeability to CO2 (47). Following evi-dence that PIP contributes to both gm and leafhydraulic conductance, the hypothesis thataquaporins coregulate CO2 and H2O trans-port in the mesophyll emerged (Figure 4b).

A role for aquaporins in carbohydrate stor-age and compartmentation has also been sug-gested. In a first study, tomato fruits withantisense inhibition of a PIP homolog showedincreases in organic acid content and de-creases in sugar content; these defects areassociated with a marked alteration of theripening process (19). In another study,metabolomic analysis of Arabidopsis plantslacking AtTIP1;1 expression revealed com-plex alterations in the accumulation of var-ious sugars, organic acids, and starch (84).Although this provocative hypothesis re-mains to be confirmed, reduced expressionof AtTIP1;1 was proposed to alter vesi-cle trafficking and therefore carbohydratecompartmentation.

Nutrient uptake. Two recent studies unrav-eled novel functions of aquaporins in plant nu-trient uptake. Transcriptome analysis of Ara-bidopsis roots revealed striking upregulationof AtNIP5;1 in response to boron (B) defi-ciency (134). Interestingly, AtNIP5;1 trans-ports boric acid in Xenopus oocytes and signif-icantly contributes to root B uptake, as shownin two independent nip5;1 insertion lines. Thephysiological significance of AtNIP5;1 wasfurther underscored by mutant plants that,under B limitation, display a striking growthretardation of shoot and roots and an inhi-bition of flower and silique formation (134).

We note that a role for membrane channelsin B transport was first proposed in an earlystudy on squash roots (29). However, the roleof aquaporins had remained uncertain owingto the low B transport activity of the PIPs andNIPs investigated in that study.

Silicon is a major mineral componentof certain plants, such as Gramineae, andmore generally, helps plants withstand abi-otic stresses and pathogen attacks. Molecu-lar characterization of low silicon rice1 (lsi1),a mutant of rice defective in silicon uptakeinto roots, led to the unexpected finding thatLsi1 encodes a NIP homolog (82). Lsi1 trans-ports silicon after heterologous expression inoocytes. Lsi1 is expressed on the distal sideof exodermal and endodermal root cells andmay contribute, in combination with the ef-flux transporter Lsi2 (83), to a vectorial trans-port of silicic acid from the soil solution intothe xylem, a limiting step for translocationof silicon to the aerial parts (Figure 4a). Asimilar function can be expected in other im-portant crops such as maize, which also accu-mulates significant amounts of silicon and hasclose Lsi1 homologs (82).

AQUAPORINS IN A VARIABLEENVIRONMENT

Changes in Irradiance

Light is a key environmental parameter that,besides its long-term effects on plant growthand development, diurnally affects the plantmetabolic regime and therefore affects waterrelations. A primary effect of light is to con-trol stomatal aperture, and therefore transpi-ration. In guard cells of sunflower leaves, thetranscript abundance of a TIP homolog (Sun-TIP7) is under diurnal regulation and is max-imal at the end of the day, during stomatalclosure, suggesting that this aquaporin con-tributes to water efflux from the guard cell(123). Light also enhances Kleaf in many plantspecies (22, 103, 142). For instance in sun-flower, the ∼ 50% increase in Kleaf induced bylight is fully sensitive to mercury inhibition,


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suggesting that the variations in Kleaf are dueto changes in aquaporin activity (103). Thiswas confirmed in walnut twigs, where Kleaf

shows a very tight kinetic correlation with theabundance of two major PIP2 transcripts dur-ing a transition from dark to high light (22).Enhanced activity of leaf aquaporins duringthe day, and therefore increased Kleaf, may fa-vor water transport into the inner leaf tissueswhen transpiration is maximal. This processwould avoid excessive drops in leaf water po-tential, reduce xylem tensions, and thereforeprevent possible xylem embolization.

Light interception is optimized by diurnalmovements of leaves, a process in which aqua-porins also participate. In the Mimosaceae,the movement of leaves and leaflets is deter-mined by coordinate swelling and shrinking ofcells on opposing sides of a motor organ, thepulvinus. In Samanea saman, the osmotic wa-ter permeability of protoplasts isolated fromthe pulvinus shows diurnal regulation and ismaximal in the mornings and evenings, con-comitant with leaf movement (100). Accumu-lation of a PIP2 homolog in these cells is un-der circadian control and in phase with theserhythmic changes in water transport. In Mi-mosa pudica, motor cells harbor both a tanninand a central aqueous vacuole. Immunocyto-chemistry experiments indicate that the lattertype of vacuoles shows an approximate tenfoldhigher density of TIP1 aquaporins as com-pared with the former type, in agreement withthe major contribution of the central vacuoleto water exchanges during cell volume regu-lation (32).

Diurnal variations of root Lpr, with a two-to threefold increase during the day, have beenobserved in many plant species and may con-tribute, together with light-dependent regu-lation of Kleaf, to reducing xylem tension un-der conditions of high transpiration demand(16, 49, 78). In Lotus japonicus and maize, forinstance, root water transport is maximallyenhanced around midday and is matched orslightly preceded by an increase in the abun-dance of PIP1 and PIP2 transcripts (49, 78).In maize, ZmPIP1;5 transcripts are detected

in all root cell types during the day but re-stricted to the epidermis during the night (40).However, the abundance of PIP proteins inmaize roots shows a more complex diurnalvariation profile than that of transcripts, sug-gesting a role for posttranslational regulation(78). The mechanisms that allow light percep-tion and long-distance control of aquaporinsin the plant root deserve more precise inves-tigation.

Water, Salt, and Nutrient Stresses

Regulation of turgor and intracellular wa-ter movements. Because it is central forplant water relations, the regulation of wa-ter transport during water deficit has been theobject of extensive research. Understandingthe role of aquaporins in this context now re-quires integration of numerous observationsmade at the molecular, cell, and tissue lev-els. Fundamental regulation properties thatexplain the remarkable ability of plant cellsto withstand water deprivation have emergedfrom basic knowledge of plant cell water rela-tions and from more recent research on aqua-porins. Water deficit induces primarily an ef-flux of water, which can result in a markeddrop in cell turgor and ultimately, but morerarely, in cell plasmolysis or cytorrhysis. Inthis context, the cytosol, which contributesto a minor fraction of the plant cell volume,may be very sensitive to differential flow ofwater across the plasma membrane and thetonoplast. Abrupt changes in cytosolic volumecan theoretically be avoided if mobilization ofwater from or into the vacuole is nonlimit-ing (92, 140). Studies with membranes puri-fied from wheat and tobacco have confirmedthat, in these preparations at least, the tono-plast shows much higher water permeabilityand aquaporin activity than the plasma mem-brane (95, 104).

Osmotic stress also requires an adjustmentof plasma membrane surface area; recentresults link this process to the regulation ofmembrane water permeability. In Vicia fabaguard cells, a pretreatment with inhibitors

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of membrane trafficking (wortmannin,cytochalasin D) slows down cell shrinkagein response to hypertonicity, suggesting thata reduction of cell hydraulic conductivityand possibly aquaporin downregulation isinduced (126). When protoplasts isolatedfrom maize suspension cells are hypotonicallychallenged, a subset of these protoplastsexhibits a retarded swelling, which wasinterpreted to mean that their initial waterpermeability is extremely low and is dy-namically adjusted during the course of cellswelling and mobilization of membrane ma-terial at the cell surface (101). In agreementwith these functional data, dynamic changesin aquaporin subcellular localization wereobserved in osmotically challenged cells.These processes may also reflect transferof aquaporins to subcellular compartmentsdevoted to protein degradation. For instance,mannitol-induced osmotic stress in ice plantsuspension cells induces the relocalization ofMcTIP1;2 from the tonoplast to a putativeendosomal compartment (146). This processis dependent on aquaporin glycosylation anda cAMP-dependent pathway. In salt-treatedArabidopsis roots, AtTIP1;1, but not theAtTIP2;1 homolog, is relocalized in vacuolarbulbs (15). In addition, redistribution of PIPsfrom the plasma membrane to internal com-partments contributes to the downregulationof root water uptake (15).

The regulation of water transport in Characells in response to changing osmotic or hy-drostatic pressures has been interpreted asthe result of a direct gating of aquaporins bythese factors (152, 155). In higher plant cells,aquaporins are more likely under the controlof osmo- and pressure-sensing molecules anddownstream signaling cascades. For instance,the downregulation of water permeability inmelon protoplasts by salt can be counteractedby okadaic acid, a protein phosphatase in-hibitor (89). More specifically, phosphoryla-tion of SoPIP2;1 in spinach leaf fragmentsdecreases in response to a hyperosmotic treat-ment (64). A general model of cell osmoregu-lation involving stretch-activated Ca2+ chan-

nel and Ca2+-dependent phosphorylation ofSoPIP2;1 was proposed to explain that aqua-porin phosphorylation, and therefore cell hy-draulic conductivity, would be maximal athigh water potential to favor water entry infully turgid cells (63). Finally, several au-thors proposed that aquaporins themselvesmay function as osmosensors, but the molec-ular and cellular mechanisms involved remainelusive (51, 86).

Whole plant level. At the whole plant level,a major effect of drought is to reduce tran-spiration through stomatal closure. Yet, in ex-treme drought conditions, high tension in thexylem can lead to vessel occlusion by em-bolization. A specific role for aquaporins inembolism refilling and recovery of stem ax-ial conductance after drought was proposedin grapevine on the basis of mercury inhibi-tion experiments (80). In roots of most plantspecies investigated, drought or salt stressesalso result in a marked decrease in Lpr (61). InArabidopsis for instance, exposure to 100 mMNaCl reduces Lpr by 70% with a half-time ofapproximately 45 min. The fact that residualLpr of salt-stressed Arabidopsis or paprika rootsbecomes resistant to mercury was interpretedto mean that aquaporin activity is downreg-ulated in these conditions (17, 88). Duringthe day, this early response may provide a hy-draulic signal to the leaf to trigger stomatalclosure, whereas during the night, it may avoida backflow of water to the drying soil.

Numerous early studies reported on wa-ter stress–dependent expression of aquaporingenes and a large variety of individual regula-tion profiles were described (92). A more com-prehensive understanding of the processes in-volved has emerged from recent studies inmaize, rice, radish, and Arabidopsis, in whichexpression of the whole aquaporin family wasconsidered (3, 15, 44, 58, 73, 85, 131, 159).In salt-stressed roots of Arabidopsis and maize,a coordinated downregulation of most aqua-porin transcripts occurs, which over the first24 h of stress can contribute to Lpr downreg-ulation (3, 15, 85). A recovery toward initial


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transcript abundance occurs over longer termstresses (85, 159). Transcriptional control ofaquaporins in drought-stressed leaves appearsto be more complex and, although a tendencyto overall aquaporin gene downregulation isalso observed, specific upregulation of certainPIP transcripts occurs in rice and Arabidop-sis leaves (3, 44). Interestingly, the two tran-scripts that are upregulated in Arabidopsis arespecifically expressed in aerial parts (3). Al-though their tissue expression pattern is as yetunknown, these isoforms may facilitate wa-ter flow toward critical cell types. Studies inHordeum vulgare (barley) leaves suggest thatincreased abundance of HvPIP1;6 transcriptsin response to salt may reflect a role for thisaquaporin in promoting residual growth ofthe leaf under stress (37).

In agreement with its central role in plantresponses to water stress, abscisic acid (ABA)seems to mediate, at least in part, drought-and salt stress-induced aquaporin regulation.For instance, treatment of maize roots withABA results over 1–2 h in a transient increasein hydraulic conductivity of the whole organand of cortical cells, by factors of 3–4 and 7–27, respectively (54). Consistent with these ef-fects, ABA also rapidly enhances the expres-sion of some PIP isoforms (159). In rice roots,a strong induction of several PIPs is observedin response to water deficit, specifically in anupland cultivar that shows an enhanced pro-duction of ABA (73). This response may opti-mize uptake of residual soil water at the onsetof soil drying.

Genetic approaches have also been usefulto investigate the function of aquaporins dur-ing drought. Transgenic tobacco plants withantisense inhibition of PIP1 and transgenicArabidopsis plants with antisense inhibition ofPIP1 expression and PIP2 expression showedlower leaf water potentials than wild-typeplants under drought stress conditions (90,128). Most strikingly, the recovery followingrewatering of leaf wilting in tobacco and leafwater potential and plant hydraulic conduc-tance in Arabidopsis is significantly delayedin the antisense plants (90, 128). Therefore,

PIP aquaporins contribute to adaptation ofthe plants to drought by mechanisms thatremain to be determined, and even moresignificantly, contribute to rehydration ofthe whole plant body after drought. Anothergenetic strategy is to enhance aquaporinexpression in transgenic plants. Althoughspectacular phenotypes are observed in moststudies, aquaporin overexpression has eitherbeneficial (44, 72, 156) or adverse (2, 68)effects on drought tolerance, dependingon the aquaporin gene or the plant speciesinvestigated. Therefore, the relevance of thisapproach for biotechnological improvementof plant tolerance to water stress remains un-certain. One reason may be that many studiesrelied on overexpression of an aquaporin in aheterologous plant species (2, 44, 68, 156). In-adequate regulation of the foreign aquaporinmay disturb the endogenous stress response.In these respects, more relevant insights wereprovided by a study showing that Oryza sativaOsPIP1;3 is specifically induced by waterstress in an upland, drought-avoidant cultivarof rice (72). Furthermore, the performanceof a lowland cultivar under drought can besignificantly enhanced by expression of thisaquaporin under a stress-responsive promoter(72). In future studies it will be important toevaluate the capacity to recover from waterstress after rewatering in transgenic plantsthat ectopically express an aquaporin.

Responses to nutrient stress. Strong inter-actions exist between the nutrient and waterstatus of plants; integration of these two as-pects seems to be critical for a deeper un-derstanding of plant stress responses. For in-stance, deprivation of N, phosphorus (P), orsulfur (S) in plants results after a few days ina significant inhibition of water transport inwhole roots or individual root cells; initial rootwater transport properties can be restored inthe 24 h following nutrient resupply (16, 21,125). A downregulation of water channels un-der N and P deprivation is invoked on the basisof the insensitivity of residual Lpr to mercury(16). The adverse effects of nutrient starvation

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on plant water relations have also been stud-ied in sorghum under drought stress. As com-pared with replete conditions, P starvation en-hances the inhibition of Lpr by a polyethyleneglycol treatment and slows down its recoveryafter water resupply (125).

The molecular and cellular mechanismsinvolved in these regulations remain unclear.Stimulation of maize Lpr by NO3

− is blockedin the presence of tungstate, an inhibitor ofnitrate reductase, suggesting that products ofthe N assimilation pathway are required foractivation of aquaporin functions (10). A gen-eral transcriptional control of aquaporins bynutrient stress is also observed in the Arabidop-sis root and, for instance, calcium deprivationresults in an overall transcriptional downregu-lation of aquaporins (85). The effects of potas-sium (K) starvation are more moderate buta downregulation of Arabidopsis PIPs is alsoinduced in the long term. By contrast, K de-privation in rice induces a twofold stimula-tion of Lpr after 4–6 h and enhances ex-pression of some PIP isoforms, in parallel toexpression of K channels (75). Coregulationof aquaporins and K transport systems hasalso been observed in roots treated with CsCl,which in addition to blocking K transport,reduces Lpr and aquaporin expression (75).These data suggest tight interactions betweenwater and K transport during cell turgorregulation.

Cold Stress

Chilling of plant roots (i.e., exposure to 4◦C–8◦C) reduces root pressure, sap flow, andLpr in a few hours (5, 71, 157). These ef-fects in turn induce water deficit symptomsin shoots, such as decreased leaf water poten-tial and stomatal closure (157). In maize, botha chilling-tolerant and a chilling-sensitive va-riety show an initial, >80% decline in Lpr

but the tolerant variety shows a unique ca-pacity to spontaneously overcompensate Lpr

upon prolonged (>3 d) chilling (5). The speedand reversibility of inhibition of Lpr by chill-ing in cucumber and rice and a concomitant

six- to ninefold reduction in cortex cell hy-draulic conductivity in cucumber roots sug-gest that inhibition of aquaporin activity isinvolved (71, 157).

Comprehensive gene expression analysesin roots and shoots of rice, maize, and Ara-bidopsis show that chilling induces a marked(two- to fourfold) decrease in abundance formost PIP transcripts (5, 58, 120, 157). Normalgene expression is restored in the 24 h fol-lowing the return to permissive temperature.However, in maize and rice roots under coldstress or recovery, the abundance of aquaporintranscripts and proteins is not always corre-lated, suggesting the occurrence of posttran-scriptional regulation (5, 157). In addition, theabundance of PIP1s and of phosphorylatedPIP2 forms (as monitored by immunodetec-tion) increases during prolonged chilling inroots of both a chilling-tolerant and a chilling-sensitive variety of maize; intriguingly, this re-sponse is not correlated to their differentialLpr regulation (5). Because the sensitivities tochilling and H2O2 are correlated in the tol-erant and sensitive varieties, it was proposedthat ROS-induced damage probably domi-nates the aquaporin response and determinesthe poor performance of the sensitive varietyduring stress (5). A more direct relationshipbetween aquaporins and ROS was determinedin cucumber (71). In this species, H2O2 accu-mulates in response to chilling and treatmentof roots by exogenous H2O2 inhibits Lpr tothe same extent as chilling.

Flowers can also perceive temperature.In tulip, diurnal movements of petals arecontrolled in part by changes in temperature(7). Petal opening and an associated waterretention can be induced at 20◦C and arelinked to phosphorylation in a microsomalmembrane fraction of a 31-kDa protein,tentatively identified as a PIP isoform. Petalclosure is induced at 5◦C and is associatedwith decreased phosphorylation of the pu-tative aquaporin. The effects of a calciumchelator [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, or BAPTA]and a calcium channel blocker on petal


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AM: arbuscularmycorrhiza

movement and the associated phosphory-lation of the aquaporin suggest a role forcalcium signaling in this process (7).

Finally, freeze-thaw cycles occurring dur-ing winter can, similar to severe droughtstress, embolize xylem vessels of woody plants(119). Embolism repair may be achieved byhydrolysis of starch in adjacent parenchymacells, exudation of the resulting sugars inthe vessel, and concomitant water influx tochase out the air bubble. In walnut twigs,the transcripts and proteins corresponding totwo PIP2 homologs show seasonal variationsand preferentially accumulate in the xylemparenchyma during winter, suggesting a rolefor these aquaporins in embolism repair (119).

Anoxia

Flooding or compaction of soils results inacute oxygen deprivation (anoxia) of plantroots, which is a major stress for cultivatedplants. Most plant species investigated show arapid inhibition of Lpr in response to anoxia.Tournaire-Roux and coworkers (138) delin-eated the organ and cell bases of this processin Arabidopsis roots. They showed that anoxicstress results in acidosis of root cells and thatthe Lpr of excised roots diminishes in paral-lel to cytosolic pH. These observations canbe linked to the molecular mechanism of PIPaquaporin gating by cytosolic protons, whichis conserved in PIPs of all plants and there-fore can explain how an anoxic stress resultsin a massive inhibition of root water uptake.This regulation may avoid excessive dilutionof xylem sap after flooding or, on a longerterm, favor ethylene accumulation, which inturn induces aerenchyma differentiation (52).Ethylene, which enhances Lpr in hypoxic as-pen roots, may also compensate for the ini-tial inhibition of water transport in responseto oxygen deprivation (65). Consistent withthe physiological inhibition of Lpr, a gen-eral downregulation of PIP and TIP genesin response to hypoxia occurs in Arabidopsis(74). By contrast, expression of AtNIP2;1 ismarkedly induced upon flooding stress and

hypoxia (20, 74). AtNIP2;1 transports lacticacid and may therefore provide a path for re-lease of this fermentation product from rootcells, to contribute to cytosolic pH regula-tion and metabolic adaptation to long-termhypoxia (20).

Biotic Interactions

Rhizobia-legume symbiosis. Interactionsof plants with soil microorganisms, whichhave long been known as central for plantmineral nutrition and metabolism, more re-cently appear to play an important role inplant water relations and tolerance to en-vironmental stresses (6, 43, 143). Notably,GmNOD26, the first plant aquaporin to beidentified, is specifically expressed in symbi-otic nitrogen-fixing nodules formed after in-fection of soybean by Rhizobiaceae bacteria(149). Similar nodulins have been identified inother legumes. GmNOD26 is a major compo-nent of the peribacteroid membrane, a mem-brane of plant origin that surrounds the bac-teroid and mediates exchanges with the rootcell. Antibodies raised against either the na-tive or phosphorylated form of GmNOD26reveal that maximal expression of the proteinand its subsequent phosphorylation coincidewith bacteroid maturation (43). Because of itssolute transporting activity, GmNOD26 hasbeen tentatively linked to a channel-mediatedimport of NH3 from the bacteroid, but un-equivocal evidence for such function is stilllacking (105, 149). The water transport activ-ity of GmNOD26 may also help the plant cellto couple osmoregulation of the plant cytosoland peribacteroid space. Accordingly, droughtand salt stress result in a threefold increase inGmNOD26 phosphorylation, suggesting thatenhanced water permeability is required fornodule osmoregulation and adaptation to wa-ter stress. (43).

Mycorrhiza. Arbuscular mycorrhizas (AM)represent the most common form of symbio-sis between land plants and soil fungi. Sim-ilar to the Rhizobia-legume symbiosis, this

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interaction results in deep anatomical changesof root cells, involving in this case thedifferentiation of convoluted, periarbuscu-lar membrane structures that are the site ofextensive exchanges of mineral nutrient(phosphate), carbohydrates (photosynthates),and water with the fungus. This membranespecialization results in marked changes inPIP and TIP gene expression, with a specificprofile depending on the plant host or thesymbiotic fungus (reviewed in 143). In poplar,mycorrhized plants show, with respect to non-mycorrhized plants, a 55% increase in Lpr;besides changes in root anatomy (internal sur-face), this increase can be accounted for byenhanced expression of most of the PIPs ex-pressed in roots (87). By contrast, the Lpr

of mycorrhized Phaseolus vulgaris plants is re-duced approximately threefold and this reduc-tion is associated with a decreased abundanceof PIP2s and their phosphorylated forms (6).Plant aquaporins expressed during AM sym-biosis may also contribute to NH3 importfrom the fungus (143).

AM symbiosis also exerts beneficial effectson tolerance of plants to water stress, whetherinduced by drought, salinity, or chilling (6,143). These effects may be mediated throughalteration of both root water uptake and tran-spiration to promote water economy. A spe-cific role for aquaporins was deduced in astudy on transgenic tobacco, showing that an-tisense inhibition of NtAQP1 reduces the pos-itive effects of mycorrhiza on root and leafgrowth under drought (112). Two recent stud-ies in lettuce, soybean, and tomato drew aninteresting parallel between the effects of AMand drought stress, which synergistically reg-ulate PIP genes in roots and leaves (reviewedin 143).

Nematode and other infections. Specificaquaporins also seem to be involved in plant-pathogen interactions as an adaptive responseto infection-induced changes in plant cellmorphology. For instance, infection of rootsby nematodes leads to the differentiation ofgiant cells that serve as feeding sites for the

parasite. Regulatory sequences that are specif-ically responsive to nematode infection wereidentified in the promoter sequence of a to-bacco TIP gene (107). Enhanced expression ofthis aquaporin might be necessary to achieveextensive delivery of water and solutes to theparasite, together with proper osmotic regu-lation of the giant cells. In tomato, incompati-ble interaction with the parasite Cuscuta reflexainduces the expression of a PIP1 homolog,probably in relation to the auxin-dependentelongation of hypodermal cells induced afterpathogen attachment (153).

CONCLUSIONS

Aquaporins have provided a unique molecu-lar entry into plant water relations and theirstudy has significantly improved our under-standing of integrated mechanisms of watertransport, in roots in particular. Yet, in viewof the complex expression and regulation pro-files of aquaporins, their role in regulating wa-ter transport in many other physiological anddevelopmental contexts, including seed ger-mination, stomatal regulation, and leaf wa-ter transport, deserves further investigation(Figure 5). In most studies, aquaporin func-tion is experimentally monitored through wa-ter flow intensities and kinetics. However, inthe whole plant the overall flow of water acrossplant tissues is determined by stomatal regula-tion and/or solute transport. Therefore, it willbe important to consider how, in this context,aquaporins may critically determine local wa-ter potential gradients rather than water flowintensities. These new considerations maylead to a better understanding of the role ofaquaporins during cell elongation and waterstress.

Studies on aquaporins have also led usfar beyond membrane water transport. Thetransport of solutes of great physiologicalsignificance, such as CO2, H2O2, B, or silicicacid, is now well established and has linkedaquaporins to many functions, including car-bon metabolism, oxidative stress responses,and plant mineral nutrition (Figure 5). Yet,


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Leaf movement

Embolism repair

Cell expansion

Water uptake

Seed germination

Biotic interaction

Micro-nutrientuptake

Circadian & diurnal controlsDroughtSaltNutrient availabilityColdAnoxiaPathogen-interactions...

DroughtFreezing

LightGA3

Circadian & diurnal controlsStomatal movement

Extra-vascular transport

Nutrient availability

Nitric oxide

DroughtSalt

CO2 transport

Antherdehiscence

Water uptakeWater uptake

Figure 5Integrated functions of plant aquaporins. The endogenous and environmental factors acting on each ofthe indicated aquaporin functions are shown. GA3, gibberellin.

novel putative substrates of plant aquaporinssuch as arsenate, NO, and NO3

− await fur-ther investigation. We also note that severalplant aquaporins, in the NIP and SIP sub-groups in particular, have unknown functionsand that new aquaporin subclasses are beingdiscovered (139).

Finally, aquaporin functions need to befurther integrated in the whole plant phys-iology. This will first require a betterunderstanding of how the various transportactivities of aquaporins are coupled with those

of other transport proteins (75, 83, 134). Thechains of events that lead to control of aqua-porin functions by local or long-distance sig-nals, during development or in response tobiotic or abiotic signals, will also have to beelucidated. Finally, and although the field ofaquaporin research has already enlarged con-siderably, we may not be at the end of oursurprises because novel primary functions asdiverse as cell proliferation (117) or virusreplication (69) might be anticipated for plantaquaporins.

SUMMARY POINTS

1. Aquaporins are membrane channels that have a conserved structure and facilitate thetransport of water and/or small neutral solutes (urea, boric acid, silicic acid) or gases(ammonia, carbon dioxide).

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2. Aquaporins exhibit a high isoform multiplicity that reflects distinct transport speci-ficities and subcellular localizations.

3. Aquaporin transport activity can be regulated by multiple mechanisms, includingregulation of transcript or protein abundance, subcellular trafficking, or gating byphosphorylation or cytosolic protons.

4. Aquaporins play a central role in plant water relations. They mediate the regulationof root water transport in response to a variety of environmental stimuli. They facili-tate water transport through inner leaf tissues during transpiration and in expandingtissues.

5. Multiple integrated roles of aquaporins in carbon and nitrogen assimilation and mi-cronutrient uptake are being uncovered.

FUTURE ISSUES

1. The transport specificity of aquaporins lacking function and, in particular, of novelclasses of aquaporins recently discovered in certain plant species should be investi-gated.

2. The mechanisms governing aquaporin subcellular trafficking should be investigated,and in particular it will be important to evaluate in planta how the functional expressionof aquaporins of the plasma membrane intrinsic proteins 1 and 2 (PIP1 and PIP2)subclasses is determined by mutual physical interactions.

3. Investigation of the function and regulation of aquaporins in poorly explored physi-ological contexts, such as stomatal regulation or seed germination, will be required.

4. The mechanisms that determine regulation of aquaporins by light in inner leaf tissuesshould be dissected, and the role of aquaporins in coregulating CO2 and H2O transportin these tissues should be deciphered.

5. The relevance of altered aquaporin expression for biotechnological improvement ofplant tolerance to water stress must be explored. The role of aquaporins during waterstress recovery and in conditions similar to those experienced by crops in the field willhave to be specified.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTS

Work in our laboratory is supported by grants from INRA (AgroBI AIP300) and Genoplante(ANR-05-GPLA-034-06). We thank members of our group for fruitful discussions and apol-ogize to all colleagues whose valuable work could not be cited owing to space limitations.


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Annual Review ofPlant Biology

Volume 59, 2008Contents

Our Work with Cyanogenic PlantsEric E. Conn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

New Insights into Nitric Oxide Signaling in PlantsAngelique Besson-Bard, Alain Pugin, and David Wendehenne � � � � � � � � � � � � � � � � � � � � � � � � � 21

Plant Immunity to Insect HerbivoresGregg A. Howe and Georg Jander � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 41

Patterning and Polarity in Seed Plant ShootsJohn L. Bowman and Sandra K. Floyd � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 67

Chlorophyll Fluorescence: A Probe of Photosynthesis In VivoNeil R. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 89

Seed Storage Oil MobilizationIan A. Graham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �115

The Role of Glutathione in Photosynthetic Organisms:Emerging Functions for Glutaredoxins and GlutathionylationNicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot � � � � � � � � � � � � � � � � � � � � �143

Algal Sensory PhotoreceptorsPeter Hegemann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �167

Plant Proteases: From Phenotypes to Molecular MechanismsRenier A.L. van der Hoorn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �191

Gibberellin Metabolism and its RegulationShinjiro Yamaguchi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �225

Molecular Basis of Plant ArchitectureYonghong Wang and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �253

Decoding of Light Signals by Plant Phytochromesand Their Interacting ProteinsGabyong Bae and Giltsu Choi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �281

Flooding Stress: Acclimations and Genetic DiversityJ. Bailey-Serres and L.A.C.J. Voesenek � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

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AR342-FM ARI 26 March 2008 19:43

Roots, Nitrogen Transformations, and Ecosystem ServicesLouise E. Jackson, Martin Burger, and Timothy R. Cavagnaro � � � � � � � � � � � � � � � � � � � � � � �341

A Genetic Regulatory Network in the Development of Trichomesand Root HairsTetsuya Ishida, Tetsuya Kurata, Kiyotaka Okada, and Takuji Wada � � � � � � � � � � � � � � � � � �365

Molecular Aspects of Seed DormancyRuth Finkelstein, Wendy Reeves, Tohru Ariizumi, and Camille Steber � � � � � � � � � � � � � � �387

Trehalose Metabolism and SignalingMatthew J. Paul, Lucia F. Primavesi, Deveraj Jhurreea, and Yuhua Zhang � � � � � � � �417

Auxin: The Looping Star in Plant DevelopmentRene Benjamins and Ben Scheres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �443

Regulation of Cullin RING LigasesSara K. Hotton and Judy Callis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �467

Plastid EvolutionSven B. Gould, Ross F. Waller, and Geoffrey I. McFadden � � � � � � � � � � � � � � � � � � � � � � � � � � � � �491

Coordinating Nodule Morphogenesis with Rhizobial Infectionin LegumesGiles E.D. Oldroyd and J. Allan Downie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �519

Structural and Signaling Networks for the Polar Cell GrowthMachinery in Pollen TubesAlice Y. Cheung and Hen-ming Wu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �547

Regulation and Identity of Florigen: FLOWERING LOCUS T MovesCenter StageFranziska Turck, Fabio Fornara, and George Coupland � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �573

Plant Aquaporins: Membrane Channels with Multiple IntegratedFunctionsChristophe Maurel, Lionel Verdoucq, Doan-Trung Luu, and Veronique Santoni � � � �595

Metabolic Flux Analysis in Plants: From Intelligent Designto Rational EngineeringIgor G.L. Libourel and Yair Shachar-Hill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �625

Mechanisms of Salinity ToleranceRana Munns and Mark Tester � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �651

Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal CellsLacey Samuels, Ljerka Kunst, and Reinhard Jetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �683

Ionomics and the Study of the Plant IonomeDavid E. Salt, Ivan Baxter, and Brett Lahner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �709

vi Contents

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Alkaloid Biosynthesis: Metabolism and TraffickingJorg Ziegler and Peter J. Facchini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �735

Genetically Engineered Plants and Foods: A Scientist’s Analysisof the Issues (Part I)Peggy G. Lemaux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �771

Indexes

Cumulative Index of Contributing Authors, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � �813

Cumulative Index of Chapter Titles, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �818

Errata

An online log of corrections to Annual Review of Plant Biology articles may be foundat http://plant.annualreviews.org/

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Plant Response to Water-deficit StressElizabeth A Bray, University of Chicago, Chicago, Illinois, USA

When plants do not receive sufficient water they are subjected to a stress called water

deficit.Water deficit in the plant disruptsmany cellular andwhole plant functions, having a

negative impact on plant growth and reproduction. Plants have evolved many different

mechanisms to deal with the occurrence of this stress as it occurs in their environments.

Availability of water is the most important factor in the environment that reduces the

production of our crops.

Effects ofWater Deficit on Plant Growthand Development

Drought, a period of abnormally dry weather, results insoil-water deficit and subsequently plant-water deficit. Thelack ofwater in the environment constitutes a stresswhen itinduces an injury in the plant. Water deficit in the plantdisrupts many cellular and whole plant functions, having anegative impact on plant growth and reproduction. Cropyields are reduced by 69% on average when plants areexposed to unfavourable conditions in the field (Boyer,1982). Availability of water is the most important factor inthe environment that reduces the production of our crops.Aswater is increasingly needed for humanpopulations andprime agricultural lands are used for housing, the availa-bility of water will have a greater impact on our ability toproduce crops. See also: Agricultural Production

In nature, certain species are adapted to dry environ-ments. The genotype determines the ability of the plant tosurvive and thrive in environments with low water avail-ability. In addition, the duration of the water-deficit stress,the rate of stress imposition and the developmental stage ofthe plant at the time of stress imposition also affect plantgrowth and the ability of the plant to produce a crop.Whether the amount of water present in the environment isa stress is different for each species. Sensing mechanisms,yet to be identified, initiate the responses to water deficit,which occur at the molecular, metabolic, cellular, physi-ological and developmental levels. Many of theseresponses are driven by changes in gene expression.See also: Hot Deserts

Resistance can Occur throughAvoidance or Tolerance ofWater-deficitStress

Resistance to water deficit may arise from the ability totolerate water deficit or from mechanisms that allow

avoidance of the water deficit (Figure 1). Some species,such as desert ephemerals, are able to escape drought bycompleting their life cycle when water is plentiful. Othersavoid water deficit with the development of a large rootsystem that permits improved extraction of water fromthe soil. Avoidance of water deficit may also be achievedby using mechanisms that save water as in succulents.Some plants may also have improved water-use effi-ciency, such as found in crassulacean acid metabolismplants, in which stomata are open at night and an alter-native form of carbon assimilation promotes the use ofless water. However, these plants do not tolerate waterdeficit. Other plants have biochemical and morphologicalmechanisms such as found in mosses and resurrection

Article Contents

Advanced article

. Effects of Water Deficit on Plant Growth and

Development

. Resistance can Occur through Avoidance or Tolerance

of Water-deficit Stress

. Water Dynamics of the Plant Cell

. Whole Plant and Physiological Responses

. OsmoticAdjustmentPermitsWaterUptakeandTurgor

Recovery

. The Role of the Signalling Molecule Abscisic Acid

. Genes Regulated byWater Deficit Affect Water-deficit

Resistance

. Mechanisms of Gene Regulation

. Deduction of Gene Function through Overexpression

in Transgenic Plants

. Bringing our Knowledge to Crop Plants in the Field

doi: 10.1002/9780470015902.a0001298.pub2

Escape by completinglife cycle in a wet season

Avoidance ofwater deficit

Resistance towater-deficit stress

Tolerance ofwater deficit

Avoid water deficitusing strategies of

maximum water acquisition

Avoid water deficitby maintaining water

in the cells

Tolerate low waterpotential while maintaining

turgor pressure ( �p)

Tolerate cellulardehydration using biochemicaland morphological strategiesto protect cells from injury

Figure 1 Resistance to water-deficit stress can arise from mechanismsinvolving avoidance or tolerance of the water deficit.

1ENCYCLOPEDIA OF LIFE SCIENCES & 2007, John Wiley & Sons, Ltd. www.els.net

plants that permit the plants to withstand dehydration.See also: Crassulacean Acid Metabolism; Plant StressPhysiology

Morphological characteristics of some species permitcontinued survival in arid environments, while other plantsmust acclimate to the stress to permit survival. Someresponses may promote physiological adaptation to waterdeficit; other responses may indicate that an injury hasoccurred. It is difficult to distinguish functions of theresponses to water-deficit stress – Which responses are adirect result of water deficit and are effective in preparingthe cell to function in a water-deficit condition? Somemechanisms promote survival and limit injury, butmay dothis using amechanism that slows cropproduction, and aretherefore not useful strategies to utilize in crop plants. It isimportant to identify mechanisms that permit continuedgrowth during periods of water deficit to promote cropproduction.

Water Dynamics of the Plant Cell

The water within the cell is defined in terms of its freeenergy content or ability to do work. The free energy perunit volume of water is the water potential (cw). Water istaken into the plant if thewater potential is less than that ofthe environment surrounding the cell (Figure 2), since watermoves down a chemical gradient. If the water potential inthe soil solution is higher than that of the cells, water can betransported into the cells of the root. Thewater potential ofthe cell is dependent upon two important parameters: theosmotic potential (cs) and the turgor pressure (cp). Thecontent of solutes in the water of the cell (cs) and the pres-sure of the cellular contents against the cell wall (cp)decrease the water potential. An additional component,

the matric potential (cm), or the binding of water to sur-faces, also reduces the cell water potential. Equation (1) isused to describe cell water potential.

cw ¼ cs þ cp þ cm ½1�

Changes in cellular water relations trigger further eventsthat are manifested in plant responses at the molecular,metabolic, cellular, physiological and developmental lev-els. See also: Plant–Water Relations

Whole Plant and PhysiologicalResponses

The water of the plant can also be viewed in the contextof a soil–plant–air continuum: the plant is a column ofwater between the soil and the air. Transpiration, releaseof water from the plant, will continue when water isavailable. Under drought conditions in the field, soil-water content drops, which does not favour water move-ment into the root cells. Water will be lost throughtranspiration and will not be fully replaced, causing aloss in turgor in the plant cells. As a defence againstwater loss, transpiration decreases. In the leaf, the poreof the stomatal complex closes in response to soil-waterdeficit. As turgor decreases in the guard cells surroundingthe stomatal pore, the cells fill the pore, thus reducingthe stomatal aperture; this is the main cause ofreduced transpiration. An Arabidopsis leucine-rich re-peat receptor-like kinase (ERECTA) regulates leaf tran-spiration efficiency through a combined effect onstomatal density and photosynthetic capacity (Masleet al., 2005). When the stomata are closed, uptake ofcarbon dioxide is also reduced, reducing the carbon as-similation rate of the plant. Depending upon the dura-tion of the water deficit, this may reduce crop productionand cause injury to the chloroplasts through the processof photoinhibition. There may also be an interactionwith other stresses, such as heat stress, when transpira-tion is reduced that will also contribute to the strain onthe plant. See also: Forest Ecosystems; Photosynthesis:Ecology; Plant Stress Physiology; Plant TemperatureStressAs cellular turgor approaches zero, growth will be

inhibited. Cellular expansion or growth depends upon cel-lular turgor. The pressure of the cellular contents againstthe cell wall is the driving force for cell expansion andturgor is dependent upon uptake of water. The stage ofdevelopment of the plant at the time of the occurrenceof water deficit will alter the outcome.Growthwill return ifturgor is restored as the plant acclimatizes to water-deficitstress.See also: Plant Cell Growth andElongation; TurgorPressure

H2O

�w outside > �w inside

H2O

�w outside < �w inside

H2O

H2O

Osmoticadjustment

Figure 2 Cellular and soil-water potential control water uptake into thecell. Osmotic adjustment, a lowering of cellular osmotic potential, can

permit water uptake and restore cellular turgor.

Plant Response to Water-deficit Stress

2

Osmotic Adjustment Permits WaterUptake and Turgor Recovery

In a process called osmotic adjustment, metabolism maybe altered to maintain cellular water content through anincrease in the concentration of solutes. In these cells, theosmotic potential is lowered and thus the water potentialof the cell is lowered, permitting water uptake to bemaintained (Figure 2). Turgor will fully or partiallyrecover depending upon the external water potential.The cells will avoid a loss of water, yet they must be ableto withstand low cellular water potential. The solutesthat accumulate, called osmolytes, include sugars, prolineand quaternary ammonium compounds such as glycinebetaine. They are generally thought to be neutral tometabolic processes, and therefore do not disrupt plantfunction. The ability to adjust osmotically is dependentupon the genotype and is a more successful defencein resistant genotypes. Interestingly, osmolytes may haveadditional functions in stress resistance includingthe ability to stabilize proteins and ameliorate oxidativestress that may arise when plants are subjected to waterdeficit. Plants engineered to produce more glycine beta-ine have improved photosynthetic capacity when grownunder water-deficit conditions. Engineering to increaseaccumulation of the osmolyte mannitol has not resultedin a level of accumulation that is sufficient to alterosmotic adjustment, but plant performance in responseto stress is improved nonetheless. These overexpressionstudies may indicate that the alternative roles, ratherthan the first studied role in osmotic adjustment, may bethe more important role for osmolytes. See also: Ozoneand Reactive Oxygen Species

Membrane permeability to water and ions is alsoinvolved in the control of cellular water potential andturgor. Thewater channels, called aquaporins, are proteinsthat form a channel in the membrane that specifically faci-litates transport of water across the membrane. Thesechannels facilitate water transport using osmotic or hy-draulic driving force. There are four different classes ofthese proteins. Plasma membrane intrinsic (PIP) andtonoplast intrinsic (TIP) are water channels in the plasmamembrane and tonoplast, respectively. NOD26-like in-trinsic proteins have glycerol transport activity and thefunction of the final class is uncharacterized. All but 4 ofthe 35 aquaporin-like genes in the Arabidopsis genome aredownregulated at the RNA level by water-deficit condi-tions. This decrease in transcript abundance may limit lossof water from the cells. Plants engineered to reduce theexpression of aquaporins have implicated them in roothydraulic conductivity, water-deficit stress resistance andin the ability of plants to recover from water-deficit stress.Other transport proteins including K+ channels are alsolikely to be involved in the response to water deficit. Seealso: Plant Ion Transport; Water Channels: Aquaporins

The Role of the Signalling MoleculeAbscisic Acid

Loss of water is a physical stress in the environment thatinitiates biochemical events. The mechanism to sense thestress and the signal transduction events that follow are notunderstood. However, it is certain that the cell must have amechanism to recognize a decrease in water content, whichis probably related to turgor pressure. One intermediary inthe signalling pathway is the plant hormone abscisic acid(ABA). TheABAcontent of the plant increases in responseto water deficit by alterations in ABA biosynthesis andcatabolism. ABA accumulates in all of the plant organsand this response is important for physiological andmolecular responses to water deficit, with the moststudied response being stomatal closure.The pathway of ABA biosynthesis is complex and

required a number of dedicated scientists using genetic,biochemical and molecular approaches to unravel it. Thebreakdown of carotenoids, rather than synthesis from asmaller carbon backbone, is the pathway taken. Duringperiods of water deficit, the enzyme, 9-cis-epoxycarotenoiddioxygenase (encoded by a family of genes namedNCED),completes this oxidative cleavage of cis-xanthophylls toxanthoxin (Figure 3). The Arabidopsis gene family containsnine members, and five are likely involved in ABA biosyn-thesis, with the one member NCED3, playing a major roleduring stress. There are two steps remaining in the synthesisof ABAwith ABA-aldehyde as the immediate precursor toabscisic acid. The gene ABA2 from Arabidopsis encodesa short-chain alcohol dehydrogenase that convertsxanthoxin into abscisic aldehyde. Abscisic aldehyde oxi-dase 3, AAO3, one of four such oxidases in Arabidopsis,oxidizes abscisic aldehyde to ABA. This enzyme requires asulfurylated form of molybdenum cofactor (MoCo), andthus the Arabidopsis gene ABA3, a MoCo sulfurase is alsorequired forABAbiosynthesis. This requirementwas origi-nally identified in the tomatomutantflaccawhich also has amutatedMoCo sulfurase. In response to water-deficit con-ditions, NCED3, AAO3 and ABA3 are all induced.Since the plant hormone is rapidly degraded, the break-

down of the molecule also has an important role in con-trolling the concentration of ABA in the plant. ABA iscatabolized by hydroxylation and conjugation (Figure 3).The hydroxylation pathway results in oxidation of the 7’, 8’or 9’-carbon of the ring structure. The major pathway ofcatabolism begins with 8’ hydroxylation by the cytochromeP450 monooxygenase CYP707A, followed by spontaneousconversion to phaseic acid.Dihydrophaseic acid (DPA)andDPA glucoside are further catabolites. ABA and its hydro-xylated catabolites may also be conjugated with glucose.The accumulation of ABA in turn initiates a series of

events (Figure 3), many of which promote plant adaptationto the conditions of water deficit. First, ABA must be rec-ognized in the cell. One ABA receptor, encoded by a gene


3

controlling flowering time inArabidopsis,FCA, has recentlybeen identified (Razem et al., 2006), although, this ABAreceptor isunlikely tobe theonly receptor involved inwater-deficit responses since this receptor does not function in thecontrol of stomatal conductance. FCA is an RNA-bindingprotein that promotes transition to flowering. ABA recog-nition initiates a signal transduction pathway that is com-posed of such signalling components as kinase/phosphatasecascades, RNA-processing proteins and calcium. Manymutants in ABA signalling pathways are being used to sortout the complex interactions that control ABA responses.Finally, signal transduction fulfils the action of ABA byactivating gene expression. See also: Floral Meristems

Genes RegulatedbyWaterDeficit AffectWater-deficit Resistance

The information contained within the genome of eachspecies dictates the plant response. The genome controls

the regulation of the response to water deficit as well asthe effectiveness of the response. Microarrays, largelydone using the model plant Arabidopsis thaliana, havebeen used to catalogue the many genes that are inducedand repressed in response to conditions that may lead tocellular water-deficit stress (e.g. Seki et al., 2002). In-duced genes are candidates for those that function in theregulation of the plant response or in the adaptation ofthe plant to the stress. Different research groups, usingdifferent methods of exposing Arabidopsis plants tocellular water-deficit stress, have identified more than800 induced genes (Bray, 2004). These genes can beplaced in at least four different functional groups: signaltransduction, transcriptional regulation, cellular meta-bolism and transport and protection of cellular structures(Figure 4). There also remain a substantial group of geneswithout a known or predicted function. The many differ-ent gene products are predicted to function in all of theorganelles throughout the cell. See also: FunctionalGenomics in Plants; Gene Expression in Plants

O

OH

HO

I

HO

OH

or

9-cis Xanthophylls

Neoxanthin Violaxanthin

OHO

OO

OO −O

OO

OH−O

HO

OO

OH−O

O

Phaseic acid

8′ Hydroxy ABA

Abscisic acid

ABA aldehyde

cis-Xanthoxin

Recognition of ABA

Signal transduction

ABA action

O O

2

OH

OH

O2Cleavagesite 1

4

8′ 9′

7′

6′1′

2′3′4′

5′

5

6

3

42

1

5

3MoCo

Figure 3 ABA biosynthesis beginning with a carotenoid and proceeding through the major pathway for catabolism. Both synthesis and breakdowncontribute to the level of ABA in a particular organ of theplant in response towater deficit. The numbers in boxes represent enzymes that havebeen cloned in

Arabidopsis. (1) 9-cis-epoxycarotenoids dioxygenase 3 (NCED3) catalyses the cleavage of cis-xanthophylls during water-deficit stress. (2) The productxanthoxin is converted into ABA aldehyde by a short-chain alcohol dehydrogenase, ABA2. (3) Abscisic aldehyde oxidase AAO3, an enzyme that requires a

sulfurylated formofMoCo (synthesizedby (4)MoCo sulfurase (ABA3)), completes the final stepof ABAbiosynthesis. (5) The key step of ABA catabolism is thehydroxylation of the 8’metyl group to yeild 8’ hydroxy ABA by a cytochrome P450monooxygenase CYP707A3. Phaseic acid is then formed spontaneously.

The increasedconcentrationofABA initiates a signal transductionpathway throughanunknownsensingmechanism.This leads to inductionof specificgenes.


4

Many proteins that function in signal transduction path-ways are induced by stress. Multiple kinase/phosphatasecascades function to regulate the stress response, includinga subunit of G proteins and type 2C protein phosphatases(e.g. ABI1) that are induced under multiple laboratoryconditions. There are at least six different classes oftranscription factors that participate in gene induction orrepression in response to water deficit. Homeobox domainand NAC domain containing transcription factors areinduced by multiple experimental treatments that mimicwater-deficit stress. Accumulation of proteins encoded bygenes that have metabolic or structural functions arethought to promote adaptation to the stress. One class ofgenes that may play a role in protection is called thelate embryogenesis abundant (lea) genes. The lea genes arealso developmentally programmed for expression in desic-cating seeds. These genes encode small hydrophilic proteinsthat are predicted to protect proteins and membranesthrough chaperone-like functions. Plant genomes encodemany of these genes that fall into several different classesbased on their amino acid structure and several of them arecommonly induced by water-deficit treatments. One ofthese classes is known as dehydrin.Metabolismmayalso bealtered by cellular water deficit with genes encodingenzymes involved in osmotic adjustment and repair ordegradation of damaged cellular contents being induced.

Many other enzymes are also induced, but their potentialfunctions are not as readily deduced. Genes that arerepressed by water deficit include those involved in photo-synthesis and growth, although these genes are not com-monly repressed under all water stress conditions. See also:Regulatory Genes in Plant Development: Homeobox;SeedsThe microarray experiments also highlight that the

expression of sets of genes are timed differently. The ex-pression profiles indicate that some genes are induced earlyand transiently while others are gradually increased.Genesinduced early largely encode transcription factors andcomponents of signalling cascades. Those in the later cat-egory may be those involved in the adaptation of the plantto prolonged water deficit.

Mechanisms of Gene Regulation

The expression of genes in response to water deficit can beregulated at the transcriptional, post-transcriptional andtranslational levels. Themajority of researchhasbeendoneto explore themechanisms of transcriptional regulation. Inresponse to water-deficit stress, there are two major tran-scriptional pathways of gene expression defined by the

Soil-water-deficit stress

H2O SignallingMetabolism and transport

Protection Unknown function ?

Transcriptionalregulation

[ABA] ?DREB2A/B

DREB2A/B

DREB1DMYC

MYB

ANAC019/055/072

AREB1/2,ABF3

AZF1/2/3,STZ

Stress-induced genesRepressed gene

AREB1/2,ABF3

Osmolyte synthesiscell wall metabolismplasma membrane andorganellar transporters

Multiple kinase/phosphatasecascadesPhosphoinositidesCalcium

Mitochondrion

LEA proteins

ChloroplastNucleus

Figure 4 Classes of genes that are induced by water-deficit stress. The inset shows the different types of transcription factors that induce/repress sets ofgenes, called regulons.


5

involvement of ABA. The ABA-independent pathway iscontrolled largely by a family of transcription factorscalled drought response element binding protein (DREB),which contains a DNA binding motif originally identifiedin a flower patterning protein called APETALA2, AP2(Figure 4). The consensus promoter element TACCGA-CAT was originally identified in cold response genes andcalled the C repeat. There are two main families of DREBtranscription factors, DREB1 (3 genes) and DREB2 (8genes). In Arabidopsis, DREB2A and B and DREB1D areinduced by water deficit and salinity stress, but not by lowtemperature stress. DREB2A and B proteins require post-translational modification for activation by a mechanismthat is not understood.

ABA-dependent gene induction during water deficit iscontrolled by at least five different classes of transcriptionfactors. The ABA response element (ABRE) with the con-sensusACGTGG/TC is bound by bZIP-type transcriptionfactors (Figure 4). Three Arabidopsis bZIP transcriptionfactors (AREB1/ABF2, AREB2/ABF4, ABF3) are ex-pressed in response to water-deficit stress and ABA treat-ment. Activation of the transcription factors requiresABAaccumulation and the induction of an ABA-responsiveprotein kinase which activates the transcription factorthrough phosphorylation.

Other transcription factors are also involved in ABAregulation of gene expression during cellular water deficit.Three genes encoding a class of transcription factors that isunique to plants, the NAC domain proteins ANAC019,ANAC055, and ANAC072, are induced by water deficitand ABA treatment. The NAC domain is a 60 bp DNAbinding domain that is predicted to form a helix-turn-helixmotif. MYB, MYC and homeodomain transcription fac-tors, and a family of transcriptional repressors (Cys2/His2-type zinc-finger proteins) are also involved in the ABAresponse to water deficit.

Deduction of Gene Function throughOverexpression in Transgenic Plants

The function of an individual gene can be tested by over-expressing or knocking out the expression of that gene andexposing the plant to stress conditions. The performance ofthe transgenic plants can then be used to deduce the func-tion of specific genes. However, the conclusions are only asgood as the stress test used to evaluate the performance ofthe plants.

A number of different types of proteins are likely tofunction to improve stress resistance. Genes encoding theenzymes of osmolyte biosynthesis permit the synthesis ofthese osmotic compounds in response to stress. Over-expression of genes promoting the synthesis of many po-tential osmolytes has resulted in transgenic plants withimproved survivability or growth compared to the wild

type. The same result has been obtained for a number ofLEA proteins as well as scavengers for reactive oxygenspecies. Thus these individual genes have been shown tofunction as determinants of the water-deficit response to adefined laboratory stress. Regulatory genes, such as sig-nallingmolecules and transcription factors, control a set ofgenes, and overexpression studies can also be used todetermine if individuals in this class of genes are functionaldeterminants of stress resistance. Overexpression of genesencoding components of signal transduction pathways in-volving protein kinases, phosphoinositides and calciumhave also improved survivability. In addition, five differentclasses of transcription factors have been shown to havethis attribute. Since some of the transcription factorsrequire posttranslational modification that occurs only incells that are subjected to water deficit, these genes must bemodified prior to construction of the transgenic plants toproduce protein that do not require activation.

Bringing our Knowledge to Crop Plantsin the Field

Biotechnological approaches utilizing our basic knowledgeof plant stress response may provide a means to developcrops that respond to stress in a manner that improves tol-erance or avoidance of water deficit. In the process of char-acterizing the function of individual genes, many geneshave been identified that improve the response to water-deficit stress in the laboratory situation. However, theultimate test, the response of a transgenic crop plant todrought in the field, has rarely been reported, with the ma-jority of this research being completed in the private sector.Strategies involve altering the expression of single genes,which have the potential to alter a particular aspect of cel-lular metabolism or to alter the expression of a large set ofgenes (Figure 5). Given our current state of knowledge, only

Individual transgene Predicted size of the set of genes regulatedby the transgene

Osmosensor

Signalling molecule

ABA content or sensitivity

Transcription factor

LEA protein

Osmolyte biosynthesis

Figure 5 Approaches to improve crops using our current knowledge ofplant gene expression.When individual genes are expressed the size of the

regulatedgene set dependson the functionof the transgene.ManipulatingABA levels or sensitivity may also result in the induction of some genes that

are not normally induced under stress conditions.


6

trial-and-error can determine if a particular gene set willpromote plant adaptation to drought conditions in thefield. See also: Plant Breeding and Crop Improvement;Transgenic Plants

The largest set of genes would be induced if the initialsensor of cellular water deficit were activated inappropri-ately. Currently, this is not feasible since the initial sen-sor(s) of the cellular changes in water content has not beenidentified. Yet, overexpression of signalling molecules thatare likely to bedownstreamof the initial sensor has resultedin improved response of plants to laboratory-imposedstress. Since ABA is one of the most important signallingmolecules acting after the stress has been initiated, meth-ods that alter ABA accumulation or ABA sensitivity arepromising means to increase crop adaptation to stress.Plant sensitivity to ABA is decreased by the activity offarnesyl transferase. Transgenic Brassica napus, in whichdownregulation of farnesyltransferase is driven by adrought-inducible promoter, have greater seed yield thanthe parental line when grown in the field (Wang et al.,2005). Therewas no yield penalty reported under nonstressconditions. Increased expression of one of the many tran-scription factors that act during water-deficit stress causesa subset of the genes that are responsive to water deficit tobe induced.TheDREBgenes are thought to be apromisingsource of stress resistance and are being transferred to cropplants for field testing.

Future studies optimizing promoters and protein accu-mulation characteristics can be used to further the utility ofthis approach for the farmer.

References

Boyer JS (1982) Plant productivity and environment. Science 218: 443–

448.

Bray EA (2004) Genes commonly regulated by water-deficit stress in

Arabidopsis thaliana. Journal of Experimental Botany 55: 2331–2341.

Masle J, Gilmore SR and Farquhar GD (2005) The ERECTA gene

regulates plant transpiration efficiency in Arabidopsis. Nature 436:

866–870.

Razem FA, El-Kareamy A, Abrams SR et al. (2006) The RNA-binding

protein FCA is an abscisic acid receptor. Nature 439: 290–294.

Seki M, Narusaka M, Ishida J et al. (2002) Monitoring the expression

profiles of 7000 Arabidopsis genes under drought, cold and high-

salinity stresses using a full-length cDNAmicroarray. Plant Journal 3:

279–292.

Wang Y, Ying J, Kuzma M et al. (2005) Molecular tailoring of far-

nesylation for plant drought tolerance and yield protection.The Plant

Journal 43: 413–424.

Further Reading

Maggio A, Zhu J-K, Hasegawa M et al. (2006) Osmogenetics: Aristotle

to Arabidopsis. Plant Cell 18: 1542–1557.

Nambara E and Marion-Poll A (2005) Abscisic acid biosynthesis and

catabolism.Annual Review of Plant Biology 56: 165–185. doi: 10.1146/

annurev.arplant.56.032604.144046.

Umezawa T, Fujita M, Fujita Y et al. (2006) Engineering drought tol-

erance in plants: discovering and tailoring genes to unlock the future.

Current Opinion in Biotechnology 17: 113–122.

Yamaguchi-Shinozaki K and Shinozaki K (2006) Transcriptional reg-

ulatory networks in cellular responses and tolerance to dehydration

and cold stresses. Annual Review of Plant Biology 57: 781–803.


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