Tuning the pores: towards engineeringplants for improved water useefficiencyL. Chaerle1,*, N. Saibo2,* and D. Van Der Straeten1

1Unit Plant Hormone Signaling and Bio-imaging, Ghent University, Ledeganckstraat 35, B-9000 Gent, Belgium2Present address: Plant Genetic Engineering Laboratory, Instituto de Tecnologia Quımica e Biologica, Av. da Republica,

2781-901 Oeiras, Portugal

The management of limited fresh water resources is a

major challenge facing society in the 21st century. The

agricultural sector accounts for more than two-thirds of

human water withdrawal and is therefore a prime area

to implement a more rational water use. Environmental

stresses are a major factor limiting stable food pro-

duction. Given the growing shortage of available water

for crops this will be an emerging factor in international

agricultural economy. The most environmentally

friendly and durable solution to the problem of water

shortage is to complement more efficient irrigation

approaches with crops with optimal water use effi-

ciency, achieved either through genetic engineering or

conventional breeding, combined with high yields.

Introduction

Plant leaves and stems have microscopic epidermal poresflanked by a pair of guard cells, called stomata, whichenable gas exchange – mainly of water vapour and carbondioxide – between inner leaf tissues and surrounding air.Environmental cues, such as light intensity, light quality,water status, temperature and the concentration ofatmospheric carbon dioxide (which influences the leafinternal CO2 concentration Ci), and also endogenous(mainlyhormonal) signals, control thedevelopment,densityand aperture of stomata [1,2]. These factors influence bothwater loss by transpiration and net photosynthesis. Wateruse efficiency (WUE) is therefore defined as CO2 assimila-tion per unit water transpired. Although reducing tran-spiration by stomatal closure is the most prominentresponse to drought, it could also be optimized through thecontrol of stomatal size and density [3].

Here we present an overview of recent advances inunderstanding stomatal development and response,which determine the actual gas exchange capacity of aplant and have an important impact on its final yield. Inaddition, we highlight major achievements in enhancingdehydration tolerance in plants. Finally, we illustrate howimaging technology can help in the process towards

Corresponding author: Van Der Straeten, D. (dominique.vanderstraeten@ugent.be).

* The first two authors contributed equally to this work.Available online 19 April 2005

www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved

engineering high yield crops tolerant to limited watersupplies.

Environmental control of stomatal development

Despite the fact that several genes involved in stomataldevelopment have been characterized (Box 1), little isknown about the environmental control thereof. In mostspecies, an increase in CO2 results in a lower stomataldensity [3,4]. The Arabidopsis gene HIC (high carbondioxide) was the first gene to be identified belonging to asignaling pathway that controls stomatal development inresponse to an environmental cue [5]. HIC codes for anenzyme involved in the synthesis of very-long-chain fattyacids and is a negative regulator of stomatal developmentin response to CO2 concentration. The hicK phenotype isnot different to wild type except when grown in elevatedCO2, in which case the mutant shows a higher stomatalindex (relative prevalence of stomatal cells). This suggeststhat the epidermal wax (very-long-chain fatty acid deriva-tives) composition of the cuticle of the guard cells controlsstomatal numbers at elevated CO2 [4]. Furthermore, plantsshow a more pronounced response (lower stomatal density)to high CO2 under drought conditions, compared with well-watered plants [3], suggesting that CO2 and droughtsignaling might interact. Whether HIC has a role in thetransduction of other external signals towards stomataldevelopment remains to be determined [6]. Plants grown atlow humidity have an increased cuticle wax load [6] and alower stomatal density than thosegrownathigherhumidity[7]. Moreover, it was shown that mutants with enhancedwax loadandalteredwaxcompositionshowa lower stomatalindex and higher drought tolerance [8], indicating thatcuticlewax compositionmightalsobe involved in the controlof stomatal development by water status. Other environ-mental cues, such as salt stress, also result in a reduction ofstomatal index [9].

Mitogen-activated protein kinases (MAPK) have beensuggested to integrate responses to stress, growth andcytokinesis [10]. The requirement for YODA (YDA) inmeristemoids (Box 1) might reflect the use of MAPKsignaling in response to a changing environment [11].Hence, YDA might have an important role in drought-induced stomatal development. Furthermore, overexpres-sion of protein kinases (such as SRK2C and NPK1 [12,13])

Review TRENDS in Biotechnology Vol.23 No.6 June 2005

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Box 1. Stomatal development (Figure I)

Stomata develop frommeristemoid mother cells (MMC), which divide

asymmetrically to form a nonstomatal subsidiary cell (SC) and a

smaller cell that becomes a meristemoid (M) [60]. The meristemoid

divides asymmetrically 1–3 times and then differentiates into a guard

mother cell (GMC), which produces two guard cells (GC). Stomata

formation follows strict spacing rules, leaving at least one pavement

cell in between two stomatal complexes. The first stomatal patterning

mutants isolated were too many mouths (tmm) and four lips (flp),

showing clustered stomata (i.e. stomata that are not separated by

intervening pavement cells) in the cotyledons [60]. TMM, a putative

cell surface receptor, participates in a signaling pathway that controls

the plane of patterning divisions as well as cell proliferation and

differentiation based on a positional context [61,62]. FLP limits the cell

division competence of the GMCs, acting only in later stages of the

meristemoid pathway [60]. A third mutant that exhibits increased

stomatal density and formation of clustered stomata was designated

sdd1 (stomatal density and distribution1) [63]. SDD1 acts through

control of cell fate and orientation of cell divisions. SDD1 is a predicted

subtilisin-like protease, probably modifying extracellular ligands that

bind to TMM and a co-receptor kinase. It was suggested that SDD1 is

less important for the one-cell spacing pattern than TMM but is more

crucial for determining stomatal density [7,60,62].

Recently, a cell-fate switch in stomatal development was

identified and designated YODA (YDA). YDA is a newly character-

ized mitogen-activated protein kinase kinase kinase (MAPKKK)

acting as a negative regulator of stomatal development [11,20].

Genetic analysis places YDA downstream of TMM and SDD. YDA

might alternatively act in an independent pathway controlling cell

fate in the same uncommitted cells as TMM and SDD – before

GMC specification [11]. YDA acts before FLP. A putative transcrip-

tion factor named FAMA is also involved in stomatal patterning

and development. A corresponding T-DNA insertion line shows no

stomata [11]. Whether FAMA is a target for MAPK signaling

remains to be determined.

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MMCM

scscGMC

sc

sc

scM

sc

sc

sc

sc

GC

GC

YDA

sc

sc

sc

GC

GC

FL P

FAMA

SDD1TMMYDASubsidiary cells

Meristemoidmother cell

Meristemoid Guardmother cell

Immatureguard cells

Maturestoma

Figure I. Stomatal development.

Review TRENDS in Biotechnology Vol.23 No.6 June 2005 309

resulted in enhanced dehydration tolerance confirmingtheir implication in the regulation of water usage. Inaddition, plants overexpressing other genes involved indrought tolerance, for example, the transcription factorCBF4, might display changes in stomatal control ordevelopment [14].

The role of the plant growth regulator abscisic acid(ABA) on stomatal development in plants exposed todrought is still not clear. ABA-treated Tradescantiavirginiana plants had significantly smaller stomata andhigher stomatal density in their lower epidermis, com-pared with non-treated plants [15]. These characteristicsmight enhanceWUE in drought conditions. Small stomatacan open and close more rapidly and thus provide thecapacity for a rapid decrease in stomatal conductance of aleaf, minimizing water loss upon drought [16]. InArabidopsis, the stomatal index of wild-type and ABA-insensitive or ABA-deficient mutants was similar [17].This suggests that ABA does not have an essential role instomatal development in normal environmental con-ditions; however, it does not imply that ABA is unnecess-ary for stomatal development under stress conditions.Besides ABA, ethylene is a signal implied in many stressresponses known to affect stomatal development [18,19].

Stomatal responses to drought

Stomatal pore opening and closure are regulated byenvironmental signals. This is a short-term adaptation

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that enables plants to retain water upon drought andmaximize CO2 uptake – essential to photosynthesis –during the day (Box 2) [20]. At night, stomatal aperturedecreases. The efficiency and speed of stomatal movementto fluctuating water availability [influenced by the watervapor pressure deficit (VPD) between the leaf andsurrounding air] are major factors in maximizing photo-synthesis and WUE [21,22]. To alleviate the ever-increas-ing water demand of agriculture, more water-efficientcrops are needed [23,24] (Stockholm International WaterInstitute; http://www.siwi.org). To engineer stomatalresponses and thus balance CO2 intake and plant waterloss, it is essential to understand the molecular mechan-isms underlying guard-cell responses to water deficit(Box 2) [25]. ABA is a primary signal in response todrought. Upon water deficit, ABA is synthesized in rootsand shoots [26,27] and is subsequently redistributed to theguard cells, where it triggers stomatal closure. Thisrequires the coordinated control of several cellularprocesses, such as guard-cell turgor, cytoskeleton organ-ization, membrane trafficking and gene expression[28,29]. ABA-deficiency (e.g. aba3/los5 [30]) or insensi-tivity (e.g. abi1 [31]) in vegetative tissues typically confersa wilting phenotype as a result of higher transpiration.Conversely, the gcr1 mutant (G-protein-coupled receptor,GPCR1) is hypersensitive to ABA and has a droughtresistance phenotype, as a result of lower rates of waterloss [32]. However, under normal growth conditions it is

Box 2. ABA signaling in stomatal closure (Figure II)

Stomatal movement is a finely tuned response modulated by a

complex network of control mechanisms, wherein abscisic acid (ABA)

has a prominent role (see stomatal level subfigure; http://isotope.bti.

cornell.edu/intro/intro_wue.html). Water shortage in the plant induces

ABA accumulation, which in turn reduces stomatal conductance (gs)

and thus transpiration (E). Leaf water use efficiency (WUE) is

expressed as the ratio of assimilation (A) and transpiration (E).

However, caution is required because this parameter is not always

correlated with crop WUE [24]. The stomatal perception of ABA leads

to a multitude of responses, such as cytosolic pH increase, accumu-

lation of reactive oxygen species (ROS), nitric oxide (NO) synthesis,

ion channel activity modulation (both at the vacuolar and plasma

membrane), increase in the concentration of cytosolic calcium ions,

synthesis of lipid-derived second messengers and activation of

protein kinases and phosphatases [64,65]. ABA is perceived by a yet

unidentified ABA receptor (ABA-R) activating Ca2C permeable chan-

nels (I). This activation is antagonized by both a protein phospha-

tase (PP) and the small G protein ROP10. ABA also activates Ca2C

channels via ROS and NO in a NAD(P)H-dependent manner (II).

Mobilization of Ca2C from internal stores is regulated by several

players, such as cADPR (by activation of ADP ribosyl cyclase),

Inositol-1,4,5- triphosphate (InsP3; derived from lipids through PLC

activity) and Inositol hexakisphosphate (InsP6) (III). The ABA signal

also triggers sphingosine kinase (SphK), which converts shingosine

(SPH) in sphingosine-1-P (S1P). S1P induces stomatal closure in a

process dependent on GPA1 (a Ga-subunit protein), whose function

is inhibited by GPCR1, a G-protein-coupled receptor-like protein

[32]. Most of these signals converge to affect Ca2C level [66].

Whether [Ca2C]cyt oscillations or calcium-independent pathways are

implicated in stomatal response to ABA is still under debate [66,67].

ABA signaling downstream of Ca2C is negatively regulated by the

protein phosphatases ABI1 and ABI2. ABI2 associates with a

calcineurin B-like protein (CBL1) and the CBL-interacting protein

kinase 15 (CIPK15) to form a trimeric protein complex, and is

inactivated by redox signals [64]. ABI1 is sequestered to the

plasmamembrane by phosphatidic acid (PA; derived from ABA-

activated PLD) [68]. This binding decreases ABI1 phosphatase

activity and, consequently, promotes ABA response. The serine-

threonine kinase OST1/SRK2E [43] functions downstream of ABI1 in

the stomatal closure response [69,70] but upstream of cytoplasmic

alkalinization [69]. ABA also induces the production of NO, which

modulates ion channel activities and Ca2C levels [67] (III).

++

- -

T

∆w

E

ROS

NRNO

cADPR

GPA1

PA

PP

ABA responses

pH

NOS

OST1

k

A

ψsoil

(I)

(III)

(I)

(II)

ABA-R: ABA-receptor

PP: Phosphatase

NO: nitric oxide

PLC/PLD: phospholipase C/D

NR: nitrate reductase

PA: phosphatidic acid

InsP3: Inositol 1,4,5-Triphosphate

InsP6: inositol hexakisphosphate

S1P: sphingosine-1-P

ROS: reactive oxygen species

Anion channels

S Slow

R Rapid

WUE = A / E: Leaf water use efficiency [28]

A: CO2 assimilation

Ci: leaf internal CO2 concentration

E: transpiration

gs: stomatal conductance

ψleaf: leaf water potential

ψsoil: soil water potential

∆w: leaf to air water vapour pressure difference

k: xylem hydraulic conductance

T: temperature

ψleaf

ABAci

Lightgs

Plant level Stomatal level

ABAABA

ABA

ABA

ABAR

PLD

Ca2+ permeable channel

ABAR

ABAR

NADPHoxidase

ABI1

ABI1

PLC

SP

HK

GP

CR

1

Anion channels

S-type R-type GORK1K+

out K+in

Ca2+Ca2+

Reduction in stomatalaperture

[Ca2+]Ca2+

Ca2+

Ca2+

ROP10

Farnesylation

CIPK?/PKS?

Redox signals

ABI2CBL1/

SCaBP5

CIPK15/PKS3

S1P

SPH

InsP3

InsP6 ROP10

Figure II. ABA signaling in stomatal closure.

Review TRENDS in Biotechnology Vol.23 No.6 June 2005310

indiscernible from the wild type. This indicates thatGPCR1 is a key factor in the regulation of stomatalclosure and opening under drought stress.

The reduction in guard-cell turgor pressure thatmediates stomatal closure involves the efflux of potassiumand anions, and sucrose and malate removal [22,33]. A

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knockout mutant of the major voltage-gated potassiumchannel in the guard-cell membrane (guard cell outwardrectifying KC channel, gork1) displays an increased levelof transpiration (as expected from its inability to close itsstomata) [22]. Importantly, this increased water loss isamplified under drought conditions, hinting at a

Review TRENDS in Biotechnology Vol.23 No.6 June 2005 311

regulatory role of GORK1 in water retention. Further-more, there is evidence that ABC-transporter proteins(multidrug resistance-associated proteins with an ATPbinding cassette, MRP) are implicated in modulation ofguard-cell ion-channel activities. Knockout mutants ofMRP4 and MRP5 were assessed for effects on stomatalregulation [34,35]. Disruption of MRP5 resulted inreduced opening of stomata in the light and, hence,reduced transpiration. In addition, stomatal closure inmrp5 mutants was insensitive to ABA and Ca2C.Importantly, MRP5 deficiency also led to a higher leafWUE compared to the wild type. By contrast, MRP4knockout mutants leave their stomata (that are sensitiveto ABA) more opened both in light and darkness, and areless tolerant to dehydration. Likewise, the ABA-deficientnced3 mutant, affected in a key-regulatory step in ABAsynthesis [36], and gork1 [22] displayed an increasedwater loss both in dark and light.

AlthoughABA is known to initiate the reactions of guardcells to drought (Box 2), it is likely that other signals cancontrol stomatal movement independently of ABA. This isexemplifiedby the limited stomatal opening inducedby lightin the ABA-insensitive mrp5 mutant. In addition, it wasshown that stomatal closure can be induced by Cd2C in theABA insensitive mutant abi1–1 [37].

Imaging techniques to quantify plant water usage

The basic question in monitoring stomatal responses todrought is: how can aberrant stomatal regulation inmutant plants be revealed efficiently? Modified waterstatus can be highlighted by several methods: (i) visual orcamera-based [38] assessment of wilting; (ii) recordingweight loss (an integrative measurement of water lossover several hours) [8,32,36]; (iii) water uptake (poto-metric) measurements [34]; and (iv) monitoring of airhumidity at the leaf level (porometry) [39]. However, allthese procedures have the drawback of either having lowprecision, or being labor intensive and not fit for screeningof an entire plant population [40]. Thermal imagingovercomes these hurdles and permits automated, non-invasive monitoring of evaporation at the leaf surface.

Similar to the function of sweat glands in animals, theevaporation of water from leaf stomata has a cooling effect.

+1d +2d

+4d +5d

Figure 1. Thermal monitoring of drought stress. Thermal images of attached sugar beet l

after irrigation was stopped (upper panels), the leaf temperature in the indicated circula

temperature increase was apparent. Wilting occurred after six days (lower right panel)

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As a consequence of stomatal closure, transpiration (E)decreases, as does the associated heat loss, causing anincrease in surface temperature. Thermography revealsthe temperature distribution of objects by visualizing theiremitted long-wave infrared radiation (i.e. thermal radi-ation typically near 10 mm) [41]. As water transport inplants is driven by leaf transpiration, thermal imagingcan quantitatively estimate plant water usage. Undercontrolled environment conditions, VPD, air speed andlight level are kept constant. Transpiration is then mainlydetermined by stomatal conductance because transpira-tion through the remaining part of the leaf surface(98.0–99.8%) represents only a fraction of the totaltranspiration (10–100 times lower) owing to the presenceof a waxy cuticula [41]. However, leaf surface morphology(e.g. the absence of hairs) can alter the boundary layerconditions, which in turn influences leaf temperature.Stomatal conductance is the last step in the ‘chain ofevents’ controlling water use, but is influenced by thewater availability within the plant (Box 2). Water uptakeand transport also influence WUE; for instance, modifiedroot architecture (e.g. more lateral roots [8]) could, inaddition to an altered stomatal index and cuticularpermeability, determine the final characteristics of theplant.

Thermography has been applied since the 1970s todetect disturbed plant water relations [42]. As an example,the progressive onset of drought stress can be visualizedbefore visible wilting occurs (Figure 1). The use of thermalimaging to screen for mutants with altered transpirationis gainingmomentum; specific responses to changes in keyenvironmental factors, such as CO2, humidity and light,can be monitored in real-time [31,43,44] (Figure 2).Thermography can also help to characterize transpirationkinetics in mutants isolated by alternative screeningapproaches. Thus, this technique provides new leads tothe characterization of the signaling cascade for stomatalcontrol. However, the implications of stomatal closureextend beyond the limitation of transpiration (E). Depen-dent on the light level and CO2 availability, decreased CO2

uptake can limit photosynthetic assimilation [45]. Thus,screening for water-conservation traits only (i.e. efficientstomatal closure), will probably be detrimental to crop

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+3d

+6d

21.120.820.520.219.919.619.319.0

eaves have a temperature span of 2 8C as indicated by the scale. The first three days

r region was 19.2 8C. Four days after irrigation was stopped (lower left panel), a leaf

when the surface temperature had approached 20 8C.

25

21

Figure 2. Mutant screening by thermal imaging. Highlighting abi1–1 (abscisic acid

insensitive) Arabidopsis mutants with a low leaf temperature phenotype within a

population of wild-type plants. Top panel: thermal image. Lower panel: corre-

sponding image in the visible spectrum.Temperature scale from 21 to 25 8C;

mutants are more than 1 8C colder than the surrounding wild-type plants.

Figure reproduced with permission from Ref. [31].

Review TRENDS in Biotechnology Vol.23 No.6 June 2005312

yield. Hence, additional monitoring tools are needed toensure optimal photosynthetic yield.

Assessing photosynthetic yield

Assimilation can be quantified by measuring changes inCO2 concentration at the level of leaves or plants enclosedin a cuvette [34]; however, this approach is not amenableto screening. Destructive determination of the carbonisotope signature of a plant at the end of the growth period(DELTA technique; http://www.csiro.au) yields an inte-grative account of assimilation efficiency [24], and is usedto assess WUE. Using this approach, high yielding wheatcultivars were obtained. However, combined imaging oftranspiration and assimilation would enable non-contactmeasurements throughout plant development.

Light absorbed by higher plants is only partially usedfor photo-assimilation. Dependent on temperature, light,

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CO2 level and the photosynthetic capacity of plants, avariable share of the captured light energy is dissipated asheat (xanthophyll and/or lutein cycles) [46]. This thermalemission from leaves can be measured with a photo-acoustic setup [47], which is non-imaging and requires theenclosure of the studied leaf-region in a gas-tight cuvette.In addition, a small fraction of the light quanta absorbed isre-emitted as chlorophyll fluorescence. This emission canbe revealed after blue light excitation by imaging theemitted red fluorescence light [48], and has been usedextensively to monitor a plethora of stress conditions [42].The information derived from chlorophyll fluorescenceemission at different light intensity levels (low non-photosynthetically active light, optimal light for photo-synthesis and saturating light levels) gives an indicationof the share of photochemical and non-photochemical‘quenching’ (NPQ) processes [42,48]. From these measure-ments, parameters are derived that display a linearrelationship with photosynthetic electron transport, thusreflecting carbon assimilation [49,50], which contributesto crop yield. As illustrated by Figure 3, local ABA-inducedstomatal closure imposes a limitation on photosynthesisunder high light conditions, which induces a concomitantincrease in thermal dissipation (NPQ). Conversely, asexpected from its higher stomatal density, the sdd1mutant (Box 1) displayed less NPQ in response toincreasing light, compared with the wild type [51], whichcould be beneficial in field conditions. As a drawback, sdd1has a higher transpiration rate. By contrast, the lsd1(lesion simulating disease) mutant shows a 50% reductionin transpiration and is unable to dissipate the excessexcitation energy by NPQ, eventually leading to itsrunaway cell death (rcd) phenotype [39].

Finally, the response of plants targeted for field cultureto combinations of stresses needs to be considered. Morespecifically, a thorough insight is needed into how plantstress-response networks interact [52]. Engineeringdrought resistance needs to be reconciled with optimalplant performance, which implicates fine-tuning of sto-matal reactions under diverse environmental conditions,avoiding deleterious side-effects.

Towards screening for optimal WUE: perspectives for

the future

Safeguards are required against relying solely on thermo-graphy to quantify stomatal conductance for screeningpurposes. Preferably, quantitative yield parametersshould be followed up simultaneously. In this respect, ascreening for alterations in chlorophyll fluorescenceemission could help to highlight lowered photosyntheticefficiency. Based on the results of a first screening,candidate plants can be further submitted to short-termplant growth assays [42]. The combination of thermal andchlorophyll fluorescence imaging has been exploitedpreviously to determine the quantitative link betweenstomatal conductance and chlorophyll fluorescence par-ameters as NPQ and photochemical yield of PSII [53]. Toapply such screening strategy, cameras must be able tomonitor populations of plants. Moving either plants ([54],http://www.lemnatec.com/scanalyzer_gh.htm, Traitmill)or camera systems [55] greatly expands the screening

10 mm 0 600mmol H2O m–2s–1

Total stomatal conductance, gs

NPQ0 2.0

0 1.0ΦPSII

0 15 50 90

Figure 3. Combined thermal and chlorophyll fluorescence imaging. A kinetic analysis of stomatal conductance (gs) – as a measure of transpiration – and the chlorophyll

fluorescence parameters, non-photochemical quenching (NPQ) and photochemical yield of photosystem II (F PSII) is shown. From left to right: just before abscisic acid (ABA)

application, 15, 50 and 90 minutes after treatment. A decrease in gs and F PSII, and an increase in NPQ were visualized at the site of ABA treatment on an attached bean leaf.

At lower illumination levels, no decrease in photochemical parameters is evident, while the response at higher light levels is exacerbated. Figure reproducedwith permission

from [53].

Review TRENDS in Biotechnology Vol.23 No.6 June 2005 313

capacity of a setup. Quantification of performance isobjectively assessed through image processing [38,56].Such combined screening setups could be a ‘test-bed’ toassess the agricultural performance of engineered crops.Screening under stress-free conditions produces cultivarswith superior performance also under mild drought stress,as exemplified by breeding in wheat [57]. Moreover,engineering drought resistance by inducible promoterconstructs [25,46,58] will avoid the growth penaltyassociated with constitutive expression of the targetedgenes. Plant engineering based on mutant analysis or,ultimately, on in silico testing of virtual plants [25,59]combined with crop simulation modeling [24], will besupported by the quantitative feedback from imaging-based assessment.

Acknowledgements

L.C. is a post-doctoral fellow of the Fund for Scientific Research(Flanders). N.S. is indebted to Fundacao para a Ciencia e Tecnologia(SFRH/BPD/14541/2003) and Ghent University (BOF/2001/144).

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68 Zhang, W. et al. (2004) Phospholipase D alpha 1-derived phosphatidicacid interacts with ABI1 phosphatase 2C and regulates abscisic acidsignaling. Proc. Natl. Acad. Sci. U. S. A. 101, 9508–9513

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