Stomatal Size, Speed, and Responsiveness Impact on Review on Stomata and Water Use Efﬁciency Stomatal Size, Speed, and Responsiveness Impact on Photosynthesis and Water Use Efﬁciency1[C]

Topical Review on Stomata and Water Use Efficiency

Stomatal Size, Speed, and Responsiveness Impact onPhotosynthesis and Water Use Efficiency1[C]

Tracy Lawson* and Michael R. Blatt

School of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom (T.L.); andLaboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ,United Kingdom (M.R.B.)

The control of gaseous exchange between the leaf and bulk atmosphere by stomata governs CO2 uptake for photosynthesis andtranspiration, determining plant productivity and water use efficiency. The balance between these two processes depends on stomatalresponses to environmental and internal cues and the synchrony of stomatal behavior relative to mesophyll demands for CO2. Here weexamine the rapidity of stomatal responses with attention to their relationship to photosynthetic CO2 uptake and the consequences forwater use. We discuss the influence of anatomical characteristics on the velocity of changes in stomatal conductance and explore thepotential for manipulating the physical as well as physiological characteristics of stomatal guard cells in order to accelerate stomatalmovements in synchrony with mesophyll CO2 demand and to improve water use efficiency without substantial cost to photosyntheticcarbon fixation. We conclude that manipulating guard cell transport and metabolism is just as, if not more likely to yield useful benefitsas manipulations of their physical and anatomical characteristics. Achieving these benefits should be greatly facilitated by quantitativesystems analysis that connects directly the molecular properties of the guard cells to their function in the field.

In order for plants to function efficiently, they mustbalance gaseous exchange between inside and outside theleaf to maximize CO2 uptake for photosynthetic carbonassimilation (A) and to minimize water loss throughtranspiration. Stomata are the “gatekeepers” responsiblefor all gaseous diffusion, and they adjust to both internaland external environmental stimuli governing CO2 up-take and water loss. The pathway for CO2 uptake fromthe bulk atmosphere to the site of fixation is determinedby a series of diffusional resistances, which start with thelayer of air immediately surrounding the leaf (theboundary layer). Stomatal pores provide a major resis-tance to flux from the atmosphere to the substomatalcavity within the leaf. Further resistance is encounteredby CO2 across the aqueous and lipid boundaries into themesophyll cell and chloroplasts (mesophyll resistance).Water leaving the leaf largely follows the same pathwayin reverse, but without the mesophyll resistance compo-nent. Guard cells surround the stomatal pore. They in-crease or decrease in volume in response to external andinternal stimuli, and the resulting changes in guard cellshape adjust stomatal aperture and thereby affect the flux

of gases between the leaf internal environment and thebulk atmosphere. Stomatal behavior, therefore, controlsthe volume of CO2 entering the intercellular air spaces ofthe leaf for photosynthesis. It also plays a key role inminimizing the amount of water lost. Transpiration, byvirtue of the concentration differences, is an order ofmagnitude greater than CO2 uptake, which is an inevi-table consequence of free diffusion across this pathway.Although the cumulative area of stomatal pores onlyrepresents a small fraction of the leaf surface, typicallyless than 3%, some 98% of all CO2 taken up and waterlost passes through these pores. When fully open, theycan mediate a rate of evaporation equivalent to one-halfthat of a wet surface of the same area (Willmer andFricker, 1996).

Early experiments illustrated that photosynthetic rateswere correlated with stomatal conductance (gs) whenother factors were not limiting (Wong et al., 1979). Lowgs limits assimilation rate by restricting CO2 diffusioninto the leaf, which, when integrated over the growingseason, will influence the carbohydrate status of the leafwith consequences for crop yield. Stomata of well-watered plants are thought to reduce photosyntheticrates by about 20% in most C3 species and by less in C4plants in the field (Farquhar and Sharkey, 1982; Jones,1987). However, even this restriction has been shown toimpact substantially on yield. For example, Fischer et al.(1998) demonstrated a close correlation between gs andyield in eight different wheat (Triticum aestivum) culti-vars. Those studies highlighted the effects gs can have oncrop yield, not only through reduced CO2 diffusion butalso through the impact on water loss and evaporativecooling of the leaf. Indeed, enhancing photosynthesisyields by only 2% to 3% is sufficient to substantially in-crease plant growth and biomass over the course of agrowing season (Lefebvre et al., 2005; Zhu et al., 2007).

1 This work was supported by the Natural Environment ResearchCouncil (studentship to T.L.) and the Biotechnology and BiologicalSciences Research Council (grant no. BB/I001187/1 to T.L. and grantnos. BB/H024867/1, BB/F001630/1, BB/H009817/1, and BB/L001276/1 to M.R.B.).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Tracy Lawson ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

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Stomata and their behavior profoundly affect the globalfluxes of CO2 and water, with an estimated 3003 1015 gof CO2 and 353 1018 g of water vapor passing throughstomata of leaves every year (Hetherington andWoodward, 2003). Changes in stomatal behavior inresponse to changing climatic conditions are thought toimpact on water levels and fluxes. For example, it is es-timated that partial stomatal closure driven by increasingCO2 concentration over the past two decades has led toincreased CO2 uptake and reduced evapotranspiration intemperate and boreal northern hemisphere forests(Keenan et al., 2013), with implications for continentalrunoff and freshwater availability associated with theglobal rise in CO2 (Gedney et al., 2006). Concurrently, theincrease in global water usage over the past 100 yearsand the expectation that this is set to double before 2030(UNESCO, 2009) has put pressure on breeders and sci-entists to find new crop varieties, breeding traits, orpotential targets for manipulation that would result incrop plants that are able to sustain yield with less waterinput. The fact that stomata are major players in plantwater use and the entire global water cycle makes thefunctional and physical attributes of stomata potentialtargets for manipulation to improve carbon gain andplant productivity as well as global water fluxes.There are several approaches for improving carbon

gain and plant water use efficiency (WUE) that focus onstomata. It is possible to increase or decrease the gaseousconductance of the ensemble of stomata per unit of leafarea (gs) through the manipulation of stomatal densities(Büssis et al., 2006). In addition, there is potential to alterthe stomatal response or sensitivity to environmentalsignals through the manipulation of guard cell charac-teristics that affect stomatal mechanics (e.g. OPENSTOMATA [ost] mutants; Merlot et al., 2002). Suchapproaches have produced an array of mutant plantswith altered characteristics and varying impacts on CO2uptake and transpiration, several of which we discussin greater detail below. An intuitive measure of the ef-ficacy of such manipulations is the WUE, commonlydefined as the amount of carbon fixed in photosynthesisper unit of water transpired. In general, higher WUEvalues have been observed in plants with lower gs, butthese gains are usually achieved together with a re-duction in A and slower plant growth. Plants withhigher gs have greater assimilation rates and grow fasterunder optimal conditions, but they generally exhibitlower WUE. An approach that has not been fully ex-plored or considered in any depth is to select plants fordifferences in the kinetics of stomatal response or tomanipulate stomatal kinetics in ways that improve thesynchrony with mesophyll CO2 demand (Lawson et al.,2010, 2012). To date, the majority of studies assessingthe impact of stomatal behavior on photosyntheticcarbon gain have focused on steady-state measure-ments of gs in relation to photosynthesis. These studiesdo not take account of the dynamic situation in thefield. As we discuss below, a cursory analysis of sto-matal synchrony with mesophyll CO2 demand suggeststhat gains of 20% to 30% are theoretically possible.

Here, we address the question of the kinetics of thestomatal response to the naturally fluctuating environ-ment, notably to fluctuations in light that are typical ofthe conditions experienced in the field. We focus onvascular seed plants, to which crop plants belong, anddo not address seedless vascular plants, such as ferns.The characteristics of the latter, and hence the issuesand challenges they present, are very different. In ourminds, of paramount importance is whether there ispotential for engineering guard cells of crop plants tomanipulate the dynamic behavior of stomata so as toimprove WUE without substantial cost in assimilation.Of course, in many circumstances, stomata are not theonly factor to limit water flux through the plant (seeother articles in this issue), but they are one of the mostimportant “gatekeepers” and therefore, serve as a goodstarting point for such considerations. Thus, we explorethe physical and functional attributes of stomata, theirsignaling, and the solute transport mechanisms that de-termine pore aperture as targets for potential manipulationof stomatal responses to changing environmental cues.

INFLUENCE OF ANATOMY ON gs

In addition to stomatal sensitivity and responses tophysiological drivers, gs is also dependent upon ana-tomical characteristics. Stomatal anatomical features de-fine the maximum theoretical conductance (Dow et al.,2014a) and also influence the speed of response. Maxi-mum gs is dictated by the size and density of stomata,which in turn can be influenced by the growth envi-ronment (Hetherington and Woodward, 2003; Franksand Farquhar, 2007). It is generally accepted that sto-matal density is altered by atmospheric CO2 concentra-tion (Woodward, 1987; Gray et al., 2000), light (Gay andHurd, 1975), and other environmental factors. More re-cent experimental evidence has demonstrated that sto-matal density is negatively correlated with stomatal size(Hetherington andWoodward, 2003; Franks and Beerling,2009). The interaction/correlation between stomatal sizeand density and the impact on stomatal function havereceived much attention, particularly with reference to theevolution of performance and plasticity in plants (Franksand Farquhar, 2007). The latest studies have also impliedthat physical attributes affect stomatal response timesfollowing environmental perturbations (Drake et al.,2013). Therefore, although it is possible to manipulatestomatal function, we must be fully aware of the in-teractions between stomatal size and numbers andthe impact they can have on rapidity of stomatalmovement.

Stomatal Density

Selecting crop plants with alterations in stomataldensity to improve/reduce plant water use was firstexplored in the 1970s and 1980s (Jones, 1977, 1987) invarious breeding programs with limited success. Theoriginal hypothesis was that increasing or decreas-ing stomatal numbers would, respectively, increase

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or decrease gs. However, several studies have demon-strated that this approach may be more complexthan the simple argument outlined above. Arabidopsis(Arabidopsis thaliana) stomatal density and distribution(sdd1-1) mutants have a point mutation in a single genethat encodes a subtilisin-like Ser protease that results inplants with a 2.5-fold higher stomatal density comparedwith their wild-type counterparts (Berger and Altmann,2000). The increase in stomatal density translates to anincrease in gs and 30% greater photosynthetic rate underhigh-light conditions (Schlüter et al., 2003). Transgenicplants overexpressing SDD1 have a 40% reduction instomatal densities compared with the wild type (VonGroll et al., 2002). In a comparative study, Büssis et al.(2006) showed no difference in gs or assimilation ratewhen measured under growth photosynthetically activephoton flux density (PPFD) conditions (180 mmol m22 s21)between the overexpressing SDD1 plants, the sdd1-1mutants, and wild-type controls. These findings indi-cated that increased stomatal aperture compensatedfor the lower stomatal density in the SDD1 plantswhile reduced aperture in the sdd1-1 plants offset thegreater number of stomata. However, at high lightintensities, low gs in the SDD1 overexpressors and re-stricted CO2 diffusion limited A to 80% of the wildtype (Büssis et al., 2006). These findings exemplify therole of both the physical and functional stomatal fea-tures in determining gs and that manipulation ofphysical attributes may be counterbalanced by modi-fications in function. However, more meaningfully,this work illustrates the importance of the surroundingenvironmental conditions on stomatal behavior and thesignificance of examining gs limitation on A at appro-priate light and CO2 concentrations. In the exampleabove, no significant impacts on carbon gain were ob-served under growth PPFD; however, when photosyn-thesis was driven by greater PPFD, Awas limited by CO2diffusion via reduced gs. Such studies also provide anexplanation for why breeding for increased WUE by al-tering stomatal density has so far been mostly unsuc-cessful (Jones, 1987). To date, there is only one reportclaiming a positive effect of stomatal density on A inplants maintained in their growth conditions: Tanakaet al. (2013) used overexpression of STOMAGEN, a pos-itive regulator of stomatal density, to produce plants witha 2- to 3-fold greater stomatal density than the wild type.A in these plants was increased by 30% due to greaterCO2 diffusion into the leaf rather than changes in photo-synthetic carboxylation capacity (Tanaka et al., 2013).However, the resultant 30% increase in A was at the ex-pense of transpiration, which was double that observed inthe wild-type plants, resulting in a 50% decrease in WUE.

As stomatal density has been correlated with gs(Franks and Beerling, 2009), the rationale behind ma-nipulating stomata anatomical characteristics to increaseor decrease stomatal density may be considered rela-tively straightforward, helped by studies that have il-lustrated that manipulation of a single gene can alterstomatal patterning (Doheny-Adams et al., 2012). How-ever, the manipulation of stomatal functional responses

is clearly more complicated. Before such approaches arepossible, we need a full understanding of the metabolicpathways (and underlying genetics) that form the basisof stomatal sensing, signaling, and response processes aswell as a comprehension of physiological responses atthe leaf level and at the cellular level. This understandingwill need to include an appreciation for the hierarchicalresponse of guard cells to internal and external signalsand is likely to benefit from exploring the natural var-iation that exists in the magnitude of changes betweenindividual stoma or groups of stomata within and be-tween leaves (Lawson et al., 1998). Additionally, if suchmanipulations are directed at improving WUE, it is im-portant to understand the coordination and synchroni-zation of gs responses with mesophyll carbon assimilation(Lawson et al., 2012).

Stomatal Patterning

Most research has focused on the physical behavioralaspects of stomata on gaseous diffusion, but investiga-tions using density and patterning mutants haveunderpinned the physiological importance of stomatalpatterning on CO2 uptake and water loss. An interest-ing observation in the Büssis et al. (2006) study was thehigh-resolution chlorophyll fluorescence imaging thatrevealed two distinct areas of mesophyll in the SDD1plants: one where mesophyll lay above stomata and theother where stomata were absent. Measurements ofmaximum and actual photosynthetic efficiency wereidentical to the wild type in areas with stomata; how-ever, leaf areas without stomata showed lower maxi-mum and actual photosynthetic efficiency, illustratingthat stomatal patterning determined CO2 concentrationand photosynthesis across the leaf lamina and that lat-eral fluxes of gas could not compensate for reducedvertical diffusion as a result of reduced stomatal num-bers (Büssis et al., 2006). This work agrees with reportsthat suggest that lateral fluxes can limit photosynthesis(Morison et al., 2005) but depend on species (Morisonet al., 2007; Morison and Lawson, 2007) and leaf anat-omy (Lawson and Morison, 2006).

There are many known density and patterning mu-tants (e.g. fama, spch, mute, and tmm) in which specificgene mutations have resulted in changes to cell divisionand differentiation and altered patterns of stomata andepidermal cells, resulting in stomatal pairing or clusteringin which the “one-cell-spacing” rule is broken (for review,see Lau and Bergmann, 2012). The one-cell-spacing rulerefers to the fact that stomata are separated from eachother by a minimum of one cell, enabling efficient sto-matal operation (Serna and Fenoll, 2000) and maintainingthe efficiency of gas fluxes (Nadeau and Sack, 2002). Thisis exemplified in a recent study by Dow et al. (2014b),who revealed that high stomatal density (due to stomatalclustering) restricted CO2 diffusion and lowered A. Thesefindings were explained by the reduced functional ca-pabilities of the guard cells from the reduced availabilityof ions required for driving membrane processes as wellas the influence of turgor pressure for adjacent guard cells

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and the disruption of signaling processes due to the closeproximity of the guard cells. This work reiterated theimportance of the one-cell-spacing rule and indicatedthat optimal stomatal function depended to a large ex-tent on the patterning of stomata. There is also a goodunderstanding of environmental regulation (Lake et al.,2001; Bergmann, 2004; Casson and Gray, 2008; Cassonand Hetherington, 2010) and what determines stomataldevelopment as well as the underlying genetic path-ways that lead to altered epidermal cell patterning(Bergmann and Sack, 2007; Casson and Hetherington,2010). However, less is known about how environ-mental regulation influences stomatal density (e.g. CO2concentration) and even less about the impact of pat-terning on leaf transpiration.

Stomatal Anatomy

Doheny-Adams et al. (2012) investigated the impact ofepidermal patterning factor (epf) mutants altered indensity and patterning of stomata on gs, A, and WUE.Alterations in the expression of the different members ofthe EPF family affected stomatal and, in some cases,epidermal cell densities as well as the spacing of cells,indicating an impact on division and differentiation. Ingeneral, plants with no expression of EPF1 or EPF2, anddouble mutants of epf1 or epf2, showed increases in sto-matal density; overexpressors showed reduced stomataldensities. It is noteworthy that the strong correlationbetween stomatal density and size was maintainedwithin these plants: plants with lower stomatal densitiesalso showed a greater mean stomatal size, whereassmaller stomata were found in leaves with greater sto-matal densities. Interestingly, those plants with reduceddensity and larger stomata also showed reduced tran-spiration, greater growth rates, and a larger biomass(Doheny-Adams et al., 2012). The authors ascribed theimproved growth rate to a combination of improvedwater status, higher metabolic temperatures, and lowermetabolic costs associated with the development ofguard cells. That growth rate was not enhanced in plantswith greater stomatal densities indicates that resistanceto CO2 diffusion was not limiting in these conditions andthat leaf water status dominated growth. It would beinstructive to determine productivity in these plantswhen PPFD is increased and mesophyll demand for CO2is elevated or the plants are subjected to fluctuating lightconditions. In contrast to the SDD1-expressing and ssd1mutant plants with altered stomatal densities describedabove, the epf1 and epf2 mutants showed no compensa-tory behavior in stomatal aperture. It is important toremember that reduced gs often decreases transpirationat the expense of carbon gain. It is necessary, therefore, toassess both CO2 uptake and water loss when consideringstrategies for improved WUE (McAusland et al., 2013).An excellent example of enhanced WUE driven by sto-matal numbers are the gtl1 mutants of Arabidopsis.GTL1 is a transcription factor that regulates trichomeand stomatal development through its interaction withSDD1 expression (Breuer et al., 2009). Physiological

analysis of these plants revealed no difference in pho-tosynthetic rates over a range of light levels but reducedgs and transpiration (Yoo et al., 2009, 2010), illustratingthat manipulating one gene related to stomatal devel-opment can “fine-tune” WUE.

Evidence from several studies has also suggestedthat smaller stomata respond faster than larger sto-mata, an observation that has been explained in thecontext of surface-to-volume ratios and the requirementfor solute transport to drive movement (Hetherington andWoodward, 2003; Franks and Beerling, 2009; Drake et al.,2013). There are some notable exceptions to this relation-ship. The stomata of ferns are relatively large comparedwith many angiosperm species but nonetheless respondrapidly to changes in vapor pressure difference (VPD),which is the difference in water vapor pressure betweeninside the leaf and outside; however, their response tolight is much slower than that of angiosperms (Brodribband Holbrook, 2004; McAdam and Brodribb, 2012b,2013). The differentiation in these responses betweenmodern angiosperms and earlier vascular seedless plantsmost likely arises from the passive hydraulic characteris-tics of ferns (McAdam and Brodribb, 2012a, 2013). It is ofinterest that the size-speed relationship holds for thecharacteristics predicted of stomatal behavior in Vicia fabaand Arabidopsis guard cells when modeled on the basisof quantitative information available for these two species(Chen et al., 2012c; Wang et al., 2012). In this case, thedifference can be ascribed directly to the surface-to-volume ratios, an observation that is broadly consistentwith the suggestions of Drake et al. (2013) and Hether-ington and Woodward (2003). However, it should beborne in mind that stomatal opening is a phys-icomechanical process and that, in order to open, guardcells must overcome the pressure exerted by the sur-rounding subsidiary cells. Franks and Farquhar (2007)illustrated that stomatal opening in some species wasonly possible with a substantial reduction in subsidiarycell osmotic pressure and that rapid opening in wheatand other grasses, which have dumbbell-shaped guardcells and subsidiary cells, was due to complementarychanges in turgor pressure between the guard cells andthese surrounding subsidiary cells. Furthermore, as wenote below, there can be substantial variations in thetransport activities of guard cells, even within one spe-cies. So, direct comparisons of surface-to-volume ratios islikely to be uninformative as often as not.

SPEED OF THE STOMATAL RESPONSE

The opening and closing of stomata is driven by anumber of external environmental and internal signalingcues (Blatt, 2000), and significant variation in sensitivityand responsiveness is known to exist among differentspecies (Lawson et al., 2003, 2012; Lawson, 2009). Ingeneral, stomata open in response to light (with the ex-ception of Crassulacean Acid Metabolism stomata), lowCO2 concentration, high temperatures, and low VPD,while closure is driven by low light or darkness, highCO2, and high VPD (Outlaw, 2003). In the natural





environment, these factors exert compound effects onstomatal movements (Sharkey and Raschke, 1981; Zeigerand Zhu 1998; Talbott et al., 2003; Wang et al., 2008);therefore, stomata must respond to multiple signals in anintegrated and sometimes hierarchical manner (Lawsonet al., 2010). Short-term stomatal responses to changes inVPD (and to some extent temperature) are often consid-ered to be related to the water status of the plant ratherthan the in situ photosynthetic carbon demand, whileresponses to CO2 concentration and irradiance are closelyassociated and correlated with mesophyll CO2 demand.

Stomatal responses to changes in light, CO2 concen-tration, and VPD have been studied extensively insteady-state conditions (Jones, 1994; Lawson et al., 2010).However, such steady-state situations are rarely ob-served in nature (Jones, 1994) or in isolation (Lawsonand Morison, 2004). Few studies have examined thedynamics of stomatal response and photosynthetic out-put in the face of environmental perturbations (Grantzand Zeiger, 1986; Knapp and Smith, 1987; Kirschbaumet al., 1988; Tinoco-Ojanguren and Pearcy, 1993; Barradaset al., 1994; Lawson et al., 2010; Wong et al., 2012;McAusland et al., 2013). The majority of these haveconcentrated on the impact of sun/shade flecks on car-bon gain in understory plants, often without reference tostomata, and even fewer have looked at the impact oncrop plants. Although grown in a monoculture, cropswill also experience sun and shade flecks across thecanopy from changes in cloud cover and sun angle toself-shading and shading from neighboring plants (Wayand Pearcy, 2012), overlapping leaves, and wind-drivenmovements (Lawson et al., 2010). In the naturally fluc-tuating environment, stomata and photosynthesisrespond continually to changing environmental cues,especially light and temperature. However, these re-sponses are not always synchronized, as stomatalmovements can be an order of magnitude slower thanthe more rapid photosynthetic responses to the sameenvironmental stimuli (Pearcy, 1990; Lawson et al.,2010). For example, over the diel period, plants experi-ence short-term fluctuations in light (sun/shade flecks)that drive the temporal and spatial dynamics of carbongain and water loss (Fig. 1A). The temporal disconnectbetween gs and A means that under natural fluctuatingenvironmental conditions, the coordination betweencarbon gain and water loss (and, therefore, WUE) is farfrom optimal (Fig. 1B) and can result in spatial hetero-geneity over individual leaves (Lawson and Weyers,1999) as well as throughout the crop canopy (Weyerset al., 1997). In C3 plants, photosynthetic rate can adjustin seconds to changes in irradiance (e.g. a sun fleck), butthe lag in stomatal responses will constrain photosyn-thesis by limiting CO2 uptake (Tinoco-Ojanguren andPearcy, 1993; Barradas et al., 1998; Lawson et al., 2010,2012). The example in Figure 1 is of natural changes inirradiance, A, and gs in a bean (Phaseolus vulgaris) leafover a 70-min period and clearly shows periods in whichgs and Awere uncoordinated, others in which the slowerstomatal reopening limited A, and periods in which gswas greater than required for the A achievable under the

available light. Intrinsic water use efficiency (WUEi;which used gs as an estimation of water loss rather thantranspiration [A/gs]) over this period was between 0.03 and0.11 mmol CO2 m

22 s21/mmol water m22 s21 (Fig. 1B)and yielded a time-averaged value of 0.071. Whenrecalculated assuming instantaneous responses andcoordination of gs with A for the theoretical maximumWUE (determined from steady-state measurements ofA and gs at different irradiances; Fig. 1B), this time-averaged value was improved by 22%, effectively ac-counting for the WUEi deficits illustrated by the shadedregions in Figure 1B. This simple experiment demon-strates the impact that the rapidity of stomatal responsesto changing light environment has on carbon gain andwater loss and illustrates the possible improvement inWUE if stomata respond rapidly (almost instantaneously)and in synchrony with mesophyll demands for CO2.

Pearcy and coworkers pioneered research on theimpact of sun flecks on carbon gain and stomatal dy-namics and dissected the photosynthetic response intoseveral different phases, attributing the initial inductionphase, periods of up to 10 min, to biochemical limita-tions (Barradas and Jones, 1996); induction was fol-lowed by a period dominated by stomatal limitation tothe photosynthetic maximum; a third phase was iden-tified in which gs remained high, effectively exceedingthat needed for maximum assimilation rates under thegiven light conditions (Kirschbaum et al., 1988; Tinoco-Ojanguren and Pearcy, 1993) and, hence, out of syn-chrony with A (Lawson et al., 2010). Figure 2 shows anexample of gs limiting photosynthesis, post induction,when irradiance was increased during a 2-min sunfleck. As the PPFD declined at the end of this period, gscontinued to rise over several minutes, leading to asurplus in transpiration without the benefit of enhancedassimilation. If we consider the impact of this short in-crease in illumination on instantaneous WUE (Fig. 2,gray squares), it is clear that gs has less impact on themeasurements of WUEi after transition to the higherlight intensity. However, it is also evident that assimi-lation was limited by gs during the sun fleck, with Arising by 0.6 mmol m22 s21, roughly 10% over the sec-ond half of the 2-min period, together with gs. Finally,Figure 2 illustrates the continued rise in gs following thesun fleck, leading to a significant decline in WUEi rel-ative to the mean starting value.

The effects of shade flecks are less well studied (Lawsonet al., 2010), but they yield dynamics similar to thoseobserved during sun flecks. A decrease in light results inan immediate drop in A, to a new value imposed by thelight level. gs rarely responds immediately, demonstrat-ing a lag phase, before the start of closing. Althoughclosing is often faster than the opening (Hetherington andWoodward, 2003), it can still take several tens of minutesto establish a new steady state in gs. Figure 3 illustratesdynamic responses in bean and V. faba subjected to 1-, 5-,and 15-min shade flecks. In agreement with previousobservations, a decrease in light intensity of 1 to 2 minresulted in very little or no change in gs and a near-instantrecovery in photosynthetic rate when light was restored.


Lawson and Blatt



For bean, longer shade flecks led to progressive declinesin gs and a slowing in the return of A to the initial valuewhen light was restored (Fig. 3B). With a shade fleck of15 min, the resulting drop in gs subsequently restrictedthe photosynthetic rate by some 35% in the first minutes

after light was restored (T. Lawson and L. McAusland,unpublished data). The same light regimes had little ef-fect on short-term gs inV. faba (Fig. 3, D–F). Consequently,these plants showed little evidence of CO2 limitationwhen irradiance was restored, but the much more

Figure 1. A, Variations in PPFD, A, andgs over a 1-h period in bean experi-encing natural light fluctuations in aglasshouse in Essex. PPFD is repre-sented by the solid gray line, A by thedashed black line, and gs by the solidblack line. Average temperature withinthe glasshouse was 25˚C and humiditywas 46%. Readings were recorded ev-ery 60 s. Shaded areas illustrate periodswhen (a) gs and A responses are uncoor-dinated and responding in opposite di-rections, (b) A is limited by the slowstomatal opening in response to increas-ing PPFD, and (c) gs is greater than nec-essary for the prevailing irradiance, due toa lag before reduced PPFD inducedclosing. B, WUEi calculated for the datacollected in A (solid gray line) and theo-retical maximum WUE (dashed blackline), assuming total synchrony and coor-dination in A and gs responses. MaximumWUEi was determined from light-response curves that were of a suffi-cient duration to ensure that both steadystomatal conductance and A were ach-ieved. Values for coordinated responseswere determined from steady-statemeasurement of A and gs in responseto changing irradiance (T. Lawson,unpublished data). [See online articlefor color version of this figure.]

Figure 2. Effects of sun flecks on A(white circles), gs (black circles), andWUEi (gray squares). PPFD was in-creased from 100 to 900mmol m22 s21 fora period of 120 s at time zero (yellowhighlighted section). Measurements ofA and gs were made every 10 s withcuvette CO2 concentration maintainedat 400 mmol mol21, temperature at 25˚C,and VPD at 1.34 kPa (T. Lawson andL. McAusland, unpublished data). [Seeonline article for color version of thisfigure.]





sluggish response of the stomata and gs meant a pro-portionally higher rate of transpiration during the periodsof reduced CO2 demand. In short, the longer stomata taketo close and reach a new gs value appropriate for the lightlevel and A, the greater the surplus in transpiration andreduction in WUE (Fig. 1B).

Of course, the speed and magnitude of changes in gsin response to sun and shade flecks are species specific

(Lawson et al., 2010) and may be dependent on dif-ferences in stomatal sensitivity or signaling mecha-nisms between species (Weyers et al., 1997; Mott andPeak, 2007; Mott, 2009). They will also depend on plantwater status (Fig. 4), the history of stress (Pearcy andWay, 2012; Porcar-Castell and Palmroth, 2012; Wonget al., 2012; Zhang et al., 2012), leaf age (Urban et al.,2008), and the magnitude and duration of the change

Figure 3. Effects of shadeflecksonAandgs inP. vulgaris (A–C) andV. faba (D–F). PPFDwasdecreased from900 to100mmolm22 s21 attime zero for 1 min (A and D), 5 min (B and E), or 15 min (C and F). Measurements of A (white circles) and gs (black circles) weremade every 10 s with cuvette CO2 concentration maintained at 400 mmol mol21, temperature at 25˚C, and VPD at 1.34 kPa(T. Lawson and L. McAusland, unpublished data). Insets show schematic diagrams that illustrate the difference in number andsize of stomata between the two species (not to scale). [See online article for color version of this figure.]


Lawson and Blatt



in irradiance (Lawson, 1997). As illustrated in Figure 3,V. faba showed little response to changes in irradiance.These plants were well watered and grown undermoderate light intensities and temperatures of 18°C to20°C to minimize stress. However, when the plants werechallenged with the same 15-min shade fleck following4 d without watering (Fig. 4), gs was significantly lowercompared with the well-watered plants (Fig. 3F) andshowed an immediate and more rapid decrease duringthe shade fleck. Furthermore, A recovered in parallelwith the relatively slow increase in gs when the light wasrestored to its initial level, both A and gs taking morethan 30 min to recover (Fig. 4). Experiments of this kindillustrate the range of variations in speed and amplitudein gs dynamics, and they suggest that stomatal size anddensity are often of secondary importance. Indeed, bean,which has smaller and more numerous stomata, hereresponded faster when well watered than well-wateredV. faba, which has larger and fewer stomata, but whenwater stressed, gs of V. faba responded in a manner similarto that of bean.One feature of note is an apparent correlation be-

tween opening and closing rates. Vico et al. (2011) used60 published gas-exchange data sets of the stomatalresponse to light variations to determine the impact ofstomatal delays on photosynthesis and transpirationand its relationship to the energetic costs of stomatalmovement. Unfortunately, the latter are compromisedby the use of incorrect stoichiometries for the couplingof ATP to transport and by the assumption that sto-matal closure is entirely passive, which it is not. How-ever, among other outcomes, their studies show ageneral parallel in rates of stomatal movement acrossdata sets: faster opening rates were generally associatedwith correspondingly faster closing rates. These find-ings agree with others (Hetherington and Woodward,

2003). Vialet-Chabrand et al. (2013) recently developeda new dynamic model to describe the diurnal timecourse of gs using empirical parameters to accuratelypredict daily variation in WUEi. This model uses non-linear methods to describe temporal changes in gsdriven by the fluctuating environment, and it under-lines a coupling between rates of opening and closing.

Overall, from these various studies, we have learned agreat deal about the manipulation of stomatal numbersand guard cell responses to environmental perturbations,but almost nothing is known about the mechanisms thatcontrol stomatal speed. However, research to date sug-gests that manipulating stomatal number and size is notnecessarily the simplest or best approach, and the ideathat manipulating the stomatal response to changingenvironmental conditions could provide a means to bothimprove the WUE of plants and, at the same time, in-crease the integrated photosynthetic carbon gain. Mostimportant, they indicate that the rapidity of the stomatalresponse influences both daily carbon gain andWUE andthat it is essential to consider the dynamics both of closingand opening together (Raschke, 1970; Kirschbaum et al.,1988; Knapp, 1993; Tinoco-Ojanguren and Pearcy, 1993;Cardon et al., 1994; Allen and Pearcy, 2000; Noe andGiersch, 2004; Pearcy and Way, 2012; Smith and Berry,2013). With these points in mind, guard cell ion transportis an obvious target for exploration with the aim of al-tering stomatal dynamics and the rates of opening andclosing.

ION TRANSPORT, STOMATAL RESPONSE,AND WUE

There is no question that stomatal movements of seedplants, including crop plants, arise from the transport,accumulation, and release of osmotically active solutes. Avery large body of experimental evidence supports thecollective role of ion transport across the plasma mem-brane and tonoplast in both stomatal opening and closing(Willmer and Fricker, 1996; Blatt, 2000; Chen et al., 2012c;Hills et al., 2012). The primary inorganic species trans-ported are K+ and Cl2, which, with the organic anionmalate22 (Mal) and Suc, comprise the bulk of solute thatdrives water flux and guard cell turgor (Willmer andFricker, 1996; Roelfsema and Hedrich, 2005; McAinshand Pittman, 2009). Because mature guard cells lackfunctional plasmodesmata (Wille and Lucas, 1984), thesesolutes must be transported across the plasma mem-brane. Much of this solute uptake must also be trans-ported across the tonoplast. The guard cell vacuole, likethat of most mature plant cells, makes up the bulk of thecell volume and, hence, plays a very important role as a“repository” for osmotically active solutes (Gao et al.,2005; MacRobbie, 2006; Chen et al., 2012c). Mal metabo-lism (notably its synthesis within the guard cell cytosol)makes a substantial contribution to the osmotic content ofthe guard cell, while Mal loss during stomatal closureoccurs largely via efflux across the plasma membrane(Willmer and Fricker, 1996; Wang and Blatt, 2011). Forthis reason, questions of the extent and speed of stomatal

Figure 4. Effects of 15-min shade flecks on A and gs in V. faba plantsubjected to water stress by withholding water for 3 d. PPFD wasdecreased from 900 to 100 mmol m22 s21 at time zero for a period of15 min. Measurements of A (white circles) and gs (black circles) weremade every 10 s with cuvette CO2 concentration maintained at 400mmol mol21, temperature at 25˚C, and VPD at 1 kPa (T. Lawson andL. McAusland, unpublished data).





responses to environmental cues are intimately connectedwith characteristics of the guard cells, the most importantbeing (1) the capacity for solute transport and exchangewith the surroundings, and its relationship to the volumeof the guard cells, and (2) the speed with which transportresponds to environmental cues that determine stomatalmovements.

Transport capacity is determined primarily by thedensity and activity of the various transport proteins atthe membrane and is related secondarily to guard cellturgor and stomatal opening and closing by the surface-to-volume ratio of the cellular compartments. As notedbefore, guard cell size (and geometry) has been indicatedto affect the speed of stomatal movements, with largerstomata often exhibiting slower responses. The impor-tance of the size of the stomatal complex, as concludedfrom these studies, is predicated on the assumption thatsolute fluxes are largely uniform when normalized to theunit surface area; as a consequence, the time needed toadjust solute content within the cell volume is expectedto decrease roughly in proportion with guard cellvolume-to-surface ratio and, hence, the linear dimensionsof the guard cell. However, quantitative data from whichto compare solute fluxes are very limited (Willmer andFricker, 1996) and depend on factors such as the historyof water stress (Hiron and Wright, 1973; Figs. 3 and 4).The few studies that have examined specific transportactivities directly, for example, of outward-rectifying K+

currents in intact guard cells ofV. faba, tobacco (Nicotianatabacum), and Arabidopsis (Blatt, 1988b; Armstronget al., 1995; Blatt and Gradmann, 1997; Blatt et al., 1999;Ache et al., 2000; Bauly et al., 2000; Johansson et al.,2006; Chen et al., 2012b; Eisenach et al., 2012, 2014), in-dicate substantial variations between species indepen-dent of surface area. These comparisons call in questionthe validity of assuming a priori that transport activityremains constant on a unit-surface-area basis.

There are a large number of studies reporting on therates of net ion transport across the plasma membrane asthe principle barrier to solute flux. Attention has oftenturned to changes in pH, as, arguably, the rates of H+

fluxare a good measure of membrane energization by pri-mary H+-ATPases (Marre, 1979; Shimazaki and Kondo,1987; Goh et al., 1996; Kinoshita and Shimazaki, 1999;Merlot et al., 2007). By their nature, pH measurementsreport the net H+

flux rather than the true H+-ATPaseactivity and, therefore, are indirect, subject to the avail-ability of other ions to balance charge during transport(Blatt and Clint, 1989; Clint and Blatt, 1989; Gradmannet al., 1993). This caveat aside, the rates of H+-ATPaseactivity recorded directly under voltage clamp (Blatt,1987, 1988a; Lohse and Hedrich, 1992), and the corre-sponding fluxes of K+ and Cl2 (MacRobbie, 1981, 1983,1984; Clint and Blatt, 1989), have generally proven con-sistent with the changes in solute content (MacRobbieand Lettau, 1979, 1980; Blatt, 2000; for a quantitativesummary and comparison, see Hills et al., 2012).

Relating these data to the transport capacity of guardcells in mechanistic terms has proven much morecomplex, however. Difficulties arise in part because the

predominant transport pathways for H+, K+, Cl2, andMal are strongly dependent on membrane voltage overthe physiological voltage range, especially at the plasmamembrane. A second complication arises from the fun-damental physical requirement for charge balance acrossall biological membranes. As a result, the flux of any oneionic species is necessarily connected to that of all othersacross the samemembrane, unless this coupling is brokenby introducing a “shunt” through the circuit of a voltageclamp (Blatt, 1991, 2004). Experiments in which mem-brane voltage has been brought under experimentalcontrol address the question of transport capacity directlyand have proven quite revealing on several accounts.First and foremost, the dominant membrane transporters,including the K+ and Cl2 channels, remain functionalunder most physiological conditions. This observationcontrasts with a common misconception that transporterseither activate or shut down fully in response to variousstimuli. Their operation may be kinetically limited bysubstrate (ion) availability and especially by membranevoltage, but the activity of each transporter is generallyevident under voltage clamp. For example, currentsthrough the inward- and outward-rectifying K+ channelsare present when voltage is clamped, both in the absenceand presence of the water-stress hormone abscisic acid(ABA) that triggers stomatal closure (Blatt andArmstrong, 1993; Romano et al., 2000; Garcia-Mata et al.,2003). ABA affects the K+-carrying capacity of bothchannel populations; however, membrane depolarizationis the overriding factor in driving K+ efflux and stomatalclosure (Thiel et al., 1992; Romano et al., 2000). In otherwords, guard cells probably rarely, if ever, exist in a statein which the net flux of an osmotically active solute iszero. Instead, solute uptake and loss, and stomatalmovements, arise through a dynamic balance in soluteflux (Blatt and Armstrong, 1993; Gradmann et al., 1993;Chen et al., 2012c). A second point arises from the generalfinding that the ion transport underpinning stomatalmovements reflects a small fraction only of the maximalcapacity of the several transporters mediating these fluxes(Blatt et al., 1990; Thiel et al., 1992; Hamilton et al., 2000;Pottosin and Schönknecht, 2007; DeAngeli et al., 2009).The activities of these transporters are kinetically limitedby their inherent gating or other kinetic properties withinthe range of voltages typical for the plasma membraneand tonoplast. Two general conclusions may be drawnfrom these observations. First, the capacity for transport,especially through the several ion channels, is not inher-ently limiting for solute flux, although the balance oftransport often is. Second, any attempt to manipulatesolute accumulation and loss for accelerated stomatalmovements must therefore address the balance betweenionic fluxes at the plasma membrane and tonoplast, bothindividually and coordinately between membranes.

Information on the speed with which transport re-sponds to environmental cues, especially to light andCO2, is similarly complicated by the connections betweentransport at each membrane. Again, deconstructing thesequence of events in mechanistic terms must draw onthe voltage clamp to separate the individual transporter


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currents (Blatt, 2004) and is the focus of a number ofrecent reviews (Blatt, 2000; Hetherington and Brownlee,2004; Blatt et al., 2007; Pandey et al., 2007; Melotto et al.,2008; Wang and Song, 2008; McAinsh and Pittman, 2009;Hills et al., 2012). In general, however, stomatal move-ment arises as the cumulative sum of the net solutefluxes (i.e. of the rates of flux), especially for K+, Cl2, andMal integrated over time. Therefore, the stomata aper-ture responds over substantially longer time scales, typ-ically orders of magnitude greater than that needed forchanges in transport activity and membrane voltage ofthe guard cell. For example, membrane depolarization inABA stimulates K+ efflux within seconds throughoutward-rectifying K+ channels, in Arabidopsis theGORK K+ channel (Hosy et al., 2003; Suhita et al., 2004),and these K+ currents are enhanced during the subse-quent 3 to 5 min as a consequence of a rise in cytosolicpH (Blatt and Armstrong, 1993; Grabov and Blatt, 1997).Stomatal aperture responds more slowly, typically withhalf-times of 10 to 20 min, reaching a new stable, (near)closed state after 45 to 60 min (Raschke et al., 1975;Roelfsema and Prins, 1995; Zhang et al., 2001). Thus,making a connection to the speed and efficacy of sto-matal movements is necessarily indirect.One promising way forward draws on quantitative

systems modeling to relate transport and metabolicactivities to stomatal movements. Wang et al. (2014a),in this issue, have undertaken such an approach usingOnGuard models for V. faba and Arabidopsis (Chenet al., 2012a; Hills et al., 2012; Wang et al., 2012).OnGuard models incorporate all of our knowledge ofmolecular, biophysical, and kinetic characteristics ofguard cell transport, Mal metabolism, and H+ and Ca2+

buffering, and they link this knowledge to stomatalaperture. These models have demonstrated true pre-dictive power in uncovering previously unexpectedand emergent behaviors of guard cells, several ofwhich have been verified experimentally. OnGuardanalysis of the Arabidopsis slac1 mutant exposed anunexpected connection between the Cl2 channel andthe plasma membrane K+ channels that was subse-quently confirmed by experiments (Wang et al., 2012).This connection accounted fully for the effect of theslac1 mutant in slowing ion uptake and stomatalopening, even though the SLAC1 Cl2 channel con-tributes directly only to solute loss and stomatal clo-sure. Similarly, analysis of the Arabidopsis ost2mutant, which affects H+-ATPase activity at the plasmamembrane, has demonstrated an uncanny “commu-nication” of transport between the plasma membraneand tonoplast (Blatt et al., 2013), predictions that nowwarrant investigation. Most importantly, the model-ing underlines a commonality of kinetic limitationsthat arises from the sharing of substrates and mem-brane voltage across each membrane. These connec-tions underpin the emergent characteristics identifiedso far.So how might we approach the problem of im-

proving water use by the plant without a cost in car-bon gain? Different ion flux pathways are responsible

for solute uptake during the opening and solute lossduring the closing of stomata (Blatt, 2000; Roelfsemaand Hedrich, 2005; Shimazaki et al., 2007), and at firstglance, this separation should be a simplifying factor.However, a primary difficulty is that of transport in-teractions, such as those described above. Anticipatingfully the consequences of such manipulations is gen-erally beyond intuitive understanding and can only beaddressed through quantitative modeling. Wang et al.(2014a) have explored systematically the most prom-ising targets for genetic manipulation that will enhancethe speed of stomatal opening and closing using theOnGuard platform. Their results indicate that, withfew exceptions, modest changes to the populations ofindividual ion channels are largely ineffective. Onlyprimary H+ transport, and those transporters affectingCa2+ homeostasis directly, are predicted to have anysubstantial effects on stomatal movements. Surpris-ingly, the effects of manipulating almost all transporterpopulations were compound and, in part, at odds withthe desired effects of accelerating opening and closingwhile maintaining the dynamic range of apertures. Tothe extent of our current knowledge, the results con-firm past observations from a number of mutants, in-cluding the K+ channel gork (Hosy et al., 2003), theanion channel slac1 (Negi et al., 2008; Wang et al.,2012), the ost2 H+-ATPase mutant (Merlot et al., 2007),the clca H+-Cl2 antiporter mutant (De Angeli et al.,2006), and the tpk1 K+ channel mutant (Gobert et al.,2007). They are supported, additionally, by the work ofWang et al. (2014b), who concurrent with this publi-cation reported the effects of overexpressing theseveral K+ channels and the AHA2 H+-ATPase inArabidopsis guard cells. Their results confirm the lackof effect on channel overexpression; they also demon-strate enhanced apertures, gs, and A with H+-ATPaseoverexpression. However, these gains come with acorresponding reduction in WUE, suggesting that theacceleration in stomatal opening was not reflected inan increase in the rates of stomatal closure. Again,these findings confirm modeling predictions (Wanget al., 2014a) that interactions between transporterspreempt most, if not all, of the intuitive solutions toaccelerating stomatal movements. The most promisingtargets for manipulation to arise from these studies arethe voltage-dependent characteristics of the two classesof K+ channels at the plasma membrane, rather thansimple changes to the populations of these transporters.Wang et al. (2014a) note that a 218-mV shift in thegating of the outward-rectifying K+ channel (GORK inArabidopsis) could be sufficient to accelerate closingby more than 30%, whereas doubling the number ofthese K+ channels counterintuitively slows the rate ofclosing in simulations. Other effective solutions mayinclude manipulations of more than one transportpathway. In short, even at this simplified level, theproblem of manipulating stomatal behavior throws upa surprising degree of complexity that is likely tochallenge practical solutions to future efforts in “re-verse engineering” of stomata.





ENGINEERING STOMATAL SIGNALINGAND METABOLISM

Many of the challenges associated with manipulatingion transport in guard cells have analogs in efforts fo-cused on their signaling, cell biology, and metabolism.Indeed, there are obvious functional overlaps, for exam-ple in the contributions of organic acid synthesis to theosmotic content of the cells. Thus, it is not surprising thataltered Mal metabolism and transport will affect stomatalmovements (Gruber et al., 2011). More still, a survey ofthe literature shows that the lessons learned from thesystems biological approach of OnGuard about the (oftencounterintuitive) connections between transport andmetabolism (Wang and Blatt, 2011; Wang et al., 2012)have direct bearing here, too. A thorough discussion ofthis literature is not possible here. Thus, we have selecteda few of the more recent articles addressing leaf-level gsand A that illustrate both the difficulties and the potentialfor manipulation of stomatal responses, especially in re-lation to stomatal kinetics. We stress that, to date, nosingle mutations of any structural gene products havesurfaced that enhance stomatal function to improvedWUE without a cost to carbon gain.

The overlaps with ion transport and the importance ofstomatal dynamics are well illustrated by the vesicle-trafficking protein SYP121 in Arabidopsis. Eisenachet al. (2012) reported that the syp121 mutant impairsstomatal reopening following closing signals associatedwith elevated cytosolic free Ca2+ concentration. Themutant mimicked the phenomenon of so-called “pro-grammed closure,” previously ascribed to a “memory”of stress that leads stomata to open only slowly there-after. The authors demonstrated the underlying mech-anism that arises from cycling of the KAT1 K+ channelbetween the plasma membrane and endosomal mem-branes, thereby suppressing channel-mediated K+ up-take by the guard cells and slowing stomatal reopening.Stomatal reopening was slowed from a half-time ofapproximately 20 min to half-times of more than 60 min,sufficient to reduce long-term carbon assimilation andslow growth at low relative humidity. Slowed stomatalopening in the light has also been observed in the highleaf temperature1 mutant (Hashimoto et al., 2006), whichexhibits alterations in responsiveness to CO2 and thusmay have important implications for synchronizingstomata with mesophyll-based photosynthesis. Finally,as noted above, Wang et al. (2012) analyzed the slac1mutant that eliminates the predominant anion channelin Arabidopsis guard cells and greatly slows stomatalclosure. They found that the mutation also slowed sto-matal opening by altering the two K+ channel currents.These observations were traced directly to changes inthe baseline of cytosolic free Ca2+ concentration and el-evated cytosolic pH, the latter arising from the accu-mulation of Mal and suppression of its synthesis. Inother words, mutation of a Cl2 channel has unexpectedand profound effects that extend well beyond Cl2

transport, both to other ion channels and to organic acidmetabolism.

Manipulation of photosynthetic carbon metabolismhas likewise revealed some informative interactions be-tween mesophyll and stomatal guard cells, although theconclusions are often seemingly contradictory. Transgenicplants with reduced photosynthesis showed that gs waslargely unaffected (Quick et al., 1991; Stitt et al., 1991;Hudson et al., 1992; Evans et al., 1994; Price et al., 1998),thereby breaking the long-standing correlation betweenA and gs that is usually conserved. More recent studieshave similarly demonstrated that gs andA can be uncoupledwhen carbon fixation via Rubisco (von Caemmerer et al.,2004) and electron transport (Baroli et al., 2008) aredown-regulated. However, accelerated stomatal openingwas observed when the regeneration capacity of ribulose1,5-bisphosphate was suppressed in transgenic tobaccowith reduced levels of Sedoheptulose-1,7-bisphos-phatase (Lawson et al., 2008). Overexpression of maize(Zea mays) NAD-malic enzyme in tobacco resulted inplants with a decrease in gs but gains in biomass, sug-gesting that manipulation of both stomata and mesophyllprocesses could improve plant WUE (La Porte et al.,2002). Finally, Kelly et al. (2012) increased the expressionof hexokinase in guard cells with the counterintuitive ef-fect of decreasing gs. Hexokinase drives starch breakdownthrough the phosphorylation of Glc, which then feeds intoglycolysis, the tricarboxylic acid cycle, and malic acidsynthesis. Analysis of these transgenics suggested a Sucfeedback mechanism that coordinated stomatal behaviorwith mesophyll photosynthesis. Other, seemingly moreindirect, manipulations can have unexpected conse-quences for stomata and WUE. Some examples includemanipulations to SUC2 that increased guard cell invertaseactivity (Antunes et al., 2012). Araújo et al. (2011) observedan elevated gs and a 25% increase in A in tomato (Solanumlycopersicum) plants with altered succinate dehydrogenaselevels. This response was only observed on ectopic over-expression with a 35S promoter, not when expression wasrestricted to the guard cells only. In short, these studieshighlight the potential for manipulating metabolic pro-cesses, both of mesophyll and guard cells. However, theyalso underscore the need, in addressing complex behaviorsuch as WUE, for quantitative systems approaches on atissue-wide scale if we are to effectively target and exploitstomatal guard cell physiology.

CONCLUSION

Improving plant WUE and a plant’s ability to copewith reduced water availability is high on the scientificagenda. Stomata ultimately control 95% of all gaseousfluxes between the leaf and the environment. It followsthat stomata represent an attractive target for manipu-lations aimed at reducing water loss. Given that stomataalso regulate CO2 access to the photosynthetic tissues ofthe leaf, the challenge will be to achieve this goal withoutcompromising carbon gain (Lawson et al., 2012). Equallyimportant, any manipulations to stomatal function, sen-sitivity, or responsiveness should not render the plantsmore vulnerable to environmental extremes or biotic


Lawson and Blatt



challenge. Progress to these ends is most likely to comenow from combinations of physiological and moleculargenetic methods together with those of quantitativesystems analysis and, therefore, will benefit from addi-tional information about the quantitative kinetics, espe-cially of signal transduction.Significant advances have been made in our under-

standing of the biochemical response pathways of guardcells and the underlying molecular biology and geneticbasis that underpin these functional responses and ana-tomical characteristics. Recent advances in molecularbiology tools and approaches to manipulate key bio-chemical processes or select for desirable traits mean thatwe can reconsider these earlier ideas and the manipula-tions of guard cell metabolism or specific stomatal traitsas tangible targets, with real potential to deliver plantswith improved WUE and yield. For example, transcrip-tional analysis of guard cells from epidermal fragments(Wang et al., 2012) or microdisection of individual guardcells (Gandotra et al., 2013), along with single-cell met-abolic profiling (Burrell et al., 2007), have greatly assistedin our understanding of the metabolic signaling and re-sponse pathways within guard cells. The identification ofhighly specific guard cell promoters (Müller-Röber et al.,1994; Cominelli et al., 2005) and transcription factors(Cominelli et al., 2010; Yoo et al., 2010), the production ofguard cell enhancer trap lines (Gardner et al., 2009), andthe ability to induce transient expression in guard cells(Rusconi et al., 2013) open up new exciting potentialapproaches to not only fully understand signaling andtransduction pathways in guard cells but also to provideus with the tools to manipulate guard cell metabolism inorder to improve plant WUE. Such tools have greatlyimproved our understanding of the molecular networksthat control guard cell perception of and response tointernal and external environmental cues and have pro-vided possible candidates for manipulation (Galbiatiet al., 2008; Rusconi et al., 2013).It is equally important that plant phenotyping

approaches keep pace with the molecular and genetictechnical developments described above to deliverrapid and practical screening approaches and tools toidentify plants with faster stomata. McAusland et al.(2013) recently used combined chlorophyll fluorescenceand thermal imaging to develop the first imaging ap-proach to screen plants for alterations in intrinsic WUE.This approach provides both spatial and temporal im-ages of gs and A as well as dynamic protocol capabil-ities. Using this technique, stomatal responsiveness andspeed can be assessed simultaneously with rates ofphotosynthesis, which would be ideal for identifyingplants with faster stomata with no impairment in car-bon assimilation.Received February 1, 2014; accepted February 25, 2014; published February27, 2014.

LITERATURE CITED

Ache P, Becker D, Ivashikina N, Dietrich P, Roelfsema MRG, Hedrich R(2000) GORK, a delayed outward rectifier expressed in guard cells of

Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel. FEBS Lett486: 93–98

Allen MT, Pearcy RW (2000) Stomatal behavior and photosynthetic per-formance under dynamic light regimes in a seasonally dry tropical rainforest. Oecologia 122: 470–478

Antunes WC, Provart NJ, Williams TCR, Loureiro ME (2012) Changes instomatal function and water use efficiency in potato plants with alteredsucrolytic activity. Plant Cell Environ 35: 747–759

Araújo WL, Nunes-Nesi A, Osorio S, Usadel B, Fuentes D, Nagy R, Balbo I,Lehmann M, Studart-Witkowski C, Tohge T, et al (2011) Antisense in-hibition of the iron-sulphur subunit of succinate dehydrogenase enhancesphotosynthesis and growth in tomato via an organic acid-mediated effecton stomatal aperture. Plant Cell 23: 600–627

Armstrong F, Leung J, Grabov A, Brearley J, Giraudat J, Blatt MR (1995)Sensitivity to abscisic acid of guard-cell K+ channels is suppressed byabi1-1, a mutant Arabidopsis gene encoding a putative protein phos-phatase. Proc Natl Acad Sci USA 92: 9520–9524

Baroli I, Price GD, Badger MR, von Caemmerer S (2008) The contributionof photosynthesis to the red light response of stomatal conductance.Plant Physiol 146: 737–747

Barradas VL, Jones HG (1996) Responses of CO2 assimilation to changes inirradiance: laboratory and field data and a model for beans (Phaseolusvulgaris L.). J Exp Bot 47: 639–645

Barradas VL, Jones HG, Clark JA (1994) Stomatal responses to changingirradiance in Phaseolus vulgaris L. J Exp Bot 45: 931–936

Barradas VL, Jones HG, Clark JA (1998) Sunfleck dynamics and canopystructure in a Phaseolus vulgaris L. canopy. Int J Biometeorol 42: 34–43

Bauly JM, Sealy IM, Macdonald H, Brearley J, Dröge S, Hillmer S,Robinson DG, Venis MA, Blatt MR, Lazarus CM, et al (2000) Over-expression of auxin-binding protein enhances the sensitivity of guardcells to auxin. Plant Physiol 124: 1229–1238

Berger D, Altmann T (2000) A subtilisin-like serine protease involved inthe regulation of stomatal density and distribution in Arabidopsisthaliana. Genes Dev 14: 1119–1131

Bergmann DC (2004) Integrating signals in stomatal development. CurrOpin Plant Biol 7: 26–32

Bergmann DC, Sack FD (2007) Stomatal development. Annu Rev Plant Biol58: 163–181

Blatt MR (1987) Electrical characteristics of stomatal guard cells: the con-tribution of ATP-dependent, “electrogenic” transport revealed bycurrent-voltage and difference-current-voltage analysis. J Membr Biol98: 257–274

Blatt MR (1988a) Mechanisms of fusicoccin action: a dominant role forsecondary transport in a higher-plant cell. Planta 174: 187–200

Blatt MR (1988b) Potassium-dependent bipolar gating of potassium chan-nels in guard cells. J Membr Biol 102: 235–246

Blatt MR (1991) A primer in plant electrophysiological methods. InK Hostettmann, ed, Methods in Plant Biochemistry. Academic Press,London, pp 281–321

Blatt MR (2000) Cellular signaling and volume control in stomatal move-ments in plants. Annu Rev Cell Dev Biol 16: 221–241

Blatt MR (2004) Concepts and techniques in plant membrane physiology.In MR Blatt, ed, Membrane Transport in Plants, Vol 15. Blackwell,Oxford, UK, pp 1–39

Blatt MR, Armstrong F (1993) K+ channels of stomatal guard cells: abscisicacid-evoked control of the outward rectifier mediated by cytoplasmicpH. Planta 191: 330–341

Blatt MR, Clint GM (1989) Mechanisms of fusicoccin action: kinetic mod-ification and inactivation of K+ channels in guard cells. Planta 178: 509–523

Blatt MR, Garcia-Mata C, Sokolovski S (2007) Membrane transport andCa2+ oscillations in guard cells. In S Mancuso, S Shabala, eds, Rhythmsin Plants: Phenomenology, Mechanisms, and Adaptive Significance.Springer, Berlin, pp 115–134

Blatt MR, Grabov A, Brearley J, Hammond-Kosack K, Jones JD (1999) K+

channels of Cf-9 transgenic tobacco guard cells as targets for Clado-sporium fulvum Avr9 elicitor-dependent signal transduction. Plant J 19:453–462

Blatt MR, Gradmann D (1997) K+-sensitive gating of the K+ outward rec-tifier in Vicia guard cells. J Membr Biol 158: 241–256

Blatt MR, Thiel G, Trentham DR (1990) Reversible inactivation of K+

channels of Vicia stomatal guard cells following the photolysis of cagedinositol 1,4,5-trisphosphate. Nature 346: 766–769





Blatt MR, Wang Y, Leonhardt N, Hills A (2013) Exploring emergentproperties in cellular homeostasis using OnGuard to model K+ and otherion transport in guard cells. J Plant Physiol (in press)

Breuer C, Kawamura A, Ichikawa T, Tominaga-Wada R, Wada T, Kondou Y,Muto S, Matsui M, Sugimoto K (2009) The trihelix transcription factorGTL1 regulates ploidy-dependent cell growth in the Arabidopsis trichome.Plant Cell 21: 2307–2322

Brodribb TJ, Holbrook NM (2004) Stomatal protection against hydraulicfailure: a comparison of coexisting ferns and angiosperms. New Phytol162: 663–670

Burrell MM, Earnshaw C, Clench MR (2007) Imaging matrix assisted laserdesorption ionization mass spectrometry: a technique to map plantmetabolites within tissues at high spatial resolution. J Exp Bot 58: 757–763

Büssis D, von Groll U, Fisahn J, Altmann TA (2006) Stomatal aperture cancompensate altered stomatal density in Arabidopsis thaliana at growthlight conditions. Funct Plant Biol 33: 1037–1043

Cardon ZG, Berry JA, Woodrow IE (1994) Dependence of the extent anddirection of average stomatal response in Zea mays L. and Phaseolusvulgaris L. on the frequency of fluctuations in environmental stimuli.Plant Physiol 105: 1007–1013

Casson S, Gray JE (2008) Influence of environmental factors on stomataldevelopment. New Phytol 178: 9–23

Casson SA, Hetherington AM (2010) Environmental regulation of stomataldevelopment. Curr Opin Plant Biol 13: 90–95

Chen C, Xiao YG, Li X, Ni M (2012a) Light-regulated stomatal aperture inArabidopsis. Mol Plant 5: 566–572

Chen ZH, Eisenach C, Xu XQ, Hills A, Blatt MR (2012b) Protocol: opti-mised electrophysiological analysis of intact guard cells from Arabi-dopsis. Plant Methods 8: 15–25

Chen ZH, Hills A, Bätz U, Amtmann A, Lew VL, Blatt MR (2012c) Systemsdynamic modeling of the stomatal guard cell predicts emergent be-haviors in transport, signaling, and volume control. Plant Physiol 159:1235–1251

Clint GM, Blatt MR (1989) Mechanisms of fusicoccin action: evidence forconcerted modulations of secondary K+ transport in a higher plant cell.Planta 178: 495–508

Cominelli E, Galbiati M, Tonelli C (2010) Transcription factors controllingstomatal movements and drought tolerance. Transcription 1: 41–45

Cominelli E, Galbiati M, Vavasseur A, Conti L, Sala T, Vuylsteke M,Leonhardt N, Dellaporta SL, Tonelli C (2005) A guard-cell-specificMYB transcription factor regulates stomatal movements and plantdrought tolerance. Curr Biol 15: 1196–1200

De Angeli A, Monachello D, Ephritikhine G, Frachisse JM, Thomine S,Gambale F, Barbier-Brygoo H (2006) The nitrate/proton antiporterAtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442:939–942

De Angeli A, Monachello D, Ephritikhine G, Frachisse JM, Thomine S,Gambale F, Barbier-Brygoo H (2009) Review: CLC-mediated aniontransport in plant cells. Philos Trans R Soc Lond B Biol Sci 364: 195–201

Doheny-Adams T, Hunt L, Franks PJ, Beerling DJ, Gray JE (2012) Geneticmanipulation of stomatal density influences stomatal size, plant growthand tolerance to restricted water supply across a growth carbon dioxidegradient. Philos Trans R Soc Lond B Biol Sci 367: 547–555

Dow GJ, Bergmann DC, Berry JA (2014a) An integrated model of stomataldevelopment and leaf physiology. New Phytol 201: 1218–1226

Dow GJ, Berry JA, Bergmann DC (2014b) The physiological importance ofdevelopmental mechanisms that enforce proper stomatal spacing inArabidopsis thaliana. New Phytol 201: 1205–1217

Drake PL, Froend RH, Franks PJ (2013) Smaller, faster stomata: scaling ofstomatal size, rate of response, and stomatal conductance. J Exp Bot 64:495–505

Eisenach C, Chen ZH, Grefen C, Blatt MR (2012) The trafficking proteinSYP121 of Arabidopsis connects programmed stomatal closure and K+

channel activity with vegetative growth. Plant J 69: 241–251Eisenach C, Papantsiou M, Hillert EK, Blatt MR (2014) Clustering of the K+

channel GORJ parallels its gating by extracellular K+. Plant J (in press)Evans JR, von Caemmerer S, Setchell BA, Hudson GS (1994) The rela-

tionship between CO2 transfer conductance and leaf anatomy in trans-genic tobacco with a reduced content of Rubisco. Aust J Plant Physiol 21:475–495

Farquhar DG, Sharkey TD (1982) Stomatal conductance and photosyn-thesis. Annu Rev Plant Physiol 33: 317–345

Fischer KA, Ress D, Sayre KD, Lu ZM, Condon AG, Saavedra AL (1998)Wheat yield progress associated with higher stomatal conductance andphotosynthetic rate and cooler canopies. Crop Sci 38: 1466–1475

Franks PJ, Beerling DJ (2009) CO2-forced evolution of plant gas exchangecapacity and water-use efficiency over the Phanerozoic. Geobiology 7:227–236

Franks PJ, Farquhar GD (2007) The mechanical diversity of stomata and itssignificance in gas-exchange control. Plant Physiol 143: 78–87

Galbiati M, Simoni L, Pavesi G, Cominelli E, Francia P, Vavasseur A,Nelson T, Bevan M, Tonelli C (2008) Gene trap lines identify Arabi-dopsis genes expressed in stomatal guard cells. Plant J 53: 750–762

Gandotra N, Coughlan SJ, Nelson T (2013) The Arabidopsis leaf provas-cular cell transcriptome is enriched in genes with roles in vein pattern-ing. Plant J 74: 48–58

Gao XQ, Li CG, Wei PC, Zhang XY, Chen J, Wang XC (2005) The dynamicchanges of tonoplasts in guard cells are important for stomatal move-ment in Vicia faba. Plant Physiol 139: 1207–1216

Garcia-Mata C, Gay R, Sokolovski S, Hills A, Lamattina L, Blatt MR(2003) Nitric oxide regulates K+ and Cl2 channels in guard cells througha subset of abscisic acid-evoked signaling pathways. Proc Natl Acad SciUSA 100: 11116–11121

Gardner MJ, Baker AJ, Assie JM, Poethig RS, Haseloff JP, Webb AA(2009) GAL4 GFP enhancer trap lines for analysis of stomatal guard celldevelopment and gene expression. J Exp Bot 60: 213–226

Gay AP, Hurd RG (1975) The influence of light on stomatal density in thetomato. New Phytol 75: 37–46

Gedney NPM, Cox PM, Betts RA, Boucher O, Huntingford C, Stott PA(2006) Detection of a direct carbon dioxide effect in continental riverrunoff records. Nature 439: 835–838

Gobert A, Isayenkov S, Voelker C, Czempinski K, Maathuis FJM (2007)The two-pore channel TPK1 gene encodes the vacuolar K+ conductanceand plays a role in K+ homeostasis. Proc Natl Acad Sci USA 104: 10726–10731

Goh CH, Kinoshita T, Oku T, Shimazaki KI (1996) Inhibition of blue light-dependent H+ pumping by abscisic acid in Vicia guard-cell protoplasts.Plant Physiol 111: 433–440

Grabov A, Blatt MR (1997) Parallel control of the inward-rectifier K+

channel by cytosolic-free Ca2+ and pH in Vicia guard cells. Planta 201:84–95

Gradmann D, Blatt MR, Thiel G (1993) Electrocoupling of ion transportersin plants. J Membr Biol 136: 327–332

Grantz DA, Zeiger E (1986) Stomatal responses to light and leaf-air watervapor pressure difference show similar kinetics in sugarcane and soy-bean. Plant Physiol 81: 865–868

Gray JE, Holroyd GH, van der Lee FM, Bahrami AR, Sijmons PC,Woodward FI, Schuch W, Hetherington AM (2000) The HIC signallingpathway links CO2 perception to stomatal development. Nature 408:713–716

Gruber BD, Delhaize E, Richardson AE, Roessner U, James RA, Howitt SM,Ryan RA (2011) Characterisation of HvALMT1 function in transgenicbarley plants. Funct Plant Biol 38: 163–175

Hamilton DWA, Hills A, Kohler B, Blatt MR (2000) Ca2+ channels at theplasma membrane of stomatal guard cells are activated by hyperpo-larization and abscisic acid. Proc Natl Acad Sci USA 97: 4967–4972

Hashimoto M, Negi J, Young J, Israelsson M, Schroeder JI, Iba K (2006)Arabidopsis HT1 kinase controls stomatal movements in response toCO2. Nat Cell Biol 8: 391–397

Hetherington AM, Brownlee C (2004) The generation of Ca2+ signals inplants. Annu Rev Plant Biol 55: 401–427

Hetherington AM, Woodward FI (2003) The role of stomata in sensing anddriving environmental change. Nature 424: 901–908

Hills A, Chen ZH, Amtmann A, Blatt MR, Lew VL (2012) OnGuard, acomputational platform for quantitative kinetic modeling of guard cellphysiology. Plant Physiol 159: 1026–1042

Hiron RWP, Wright STC (1973) Role of endogenous abscisic acid in re-sponse of plants to stress. J Exp Bot 24: 769–781

Hosy E, Vavasseur A, Mouline K, Dreyer I, Gaymard F, Porée F,Boucherez J, Lebaudy A, Bouchez D, Very AA, et al (2003) The Ara-bidopsis outward K+ channel GORK is involved in regulation of sto-matal movements and plant transpiration. Proc Natl Acad Sci USA 100:5549–5554

Hudson GS, Evans JR, von Caemmerer S, Arvidsson YBC, Andrews TJ(1992) Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase


Lawson and Blatt



content by antisense RNA reduces photosynthesis in transgenic tobaccoplants. Plant Physiol 98: 294–302

Johansson I, Wulfetange K, Porée F, Michard E, Gajdanowicz P, Lacombe B,Sentenac H, Thibaud JB, Mueller-Roeber B, Blatt MR, et al (2006) Ex-ternal K+ modulates the activity of the Arabidopsis potassium channelSKOR via an unusual mechanism. Plant J 46: 269–281

Jones HG (1977) Transpiration in barley lines with differing stomatal fre-quencies. J Exp Bot 23: 162–168

Jones HG (1987) Breeding for stomatal characters. In E Zeiger, GD Farqu-har, IR Cowan, eds, Stomatal Function. Stanford University Press,Stanford, CA, pp 431–443

Jones HG (1994) Plants and Microclimate: A Quantitative Approach toEnvironmental Plant Physiology. Cambridge University Press, Cam-bridge, UK

Keenan TF, Hollinger DY, Bohrer G, Dragoni D, Munger JW, Schmid HP,Richardson AD (2013) Increase in forest water-use efficiency as atmo-spheric carbon dioxide concentrations rise. Nature 499: 324–327

Kelly G, David-Schwartz R, Sade N, Moshelion M, Levi A, Alchanatis V,Granot D (2012) The pitfalls of transgenic selection and new roles of AtHXK1:a high level of AtHXK1 expression uncouples hexokinase1-dependent sugarsignaling from exogenous sugar. Plant Physiol 159: 47–51

Kinoshita T, Shimazaki Ki (1999) Blue light activates the plasma mem-brane H+-ATPase by phosphorylation of the C-terminus in stomatalguard cells. EMBO J 18: 5548–5558

Kirschbaum MUF, Gross LJ, Pearcy RW (1988) Observed and modelledstomatal responses to dynamic light environments in the shade plantAlocasia macrorrhiza. Plant Cell Environ 11: 111–121

Knapp AK (1993) Gas-exchange dynamics in C3 and C4 grasses: conse-quences of differences in stomatal conductance. Ecology 74: 113–123

Knapp AK, Smith WK (1987) Stomatal and photosynthetic responsesduring sun/shade transitions in subalpine species: influence on wateruse efficiency. Oecologia 74: 62–67

Lake JA, Quick WP, Beerling DJ, Woodward FI (2001) Plant development:signals from mature to new leaves. Nature 411: 154–155

Laporte MM, Shen B, Tarczynski MC (2002) Engineering for droughtavoidance: expression of maize NADP-malic enzyme in tobacco resultsin altered stomatal function. J Exp Bot 53: 699–705

Lau OS, Bergmann DC (2012) Stomatal development: a plant’s perspectiveon cell polarity, cell fate transitions and intercellular communication.Development 139: 3683–3692

Lawson T (1997) Heterogeneity in stomatal characteristics. PhD thesis.University of Dundee, UK

Lawson T (2009) Guard cell photosynthesis and stomatal function. NewPhytol 181: 13–34

Lawson T, Kramer DM, Raines CA (2012) Improving yield by exploitingmechanisms underlying natural variation of photosynthesis. Curr OpinBiotechnol 23: 215–220

Lawson T, Lefebvre S, Baker NR, Morison JIL, Raines CA (2008) Re-ductions in mesophyll and guard cell photosynthesis impact on thecontrol of stomatal responses to light and CO2. J Exp Bot 59: 3609–3619

Lawson T,Morison JIL (2004) Stomatal function and physiology. InARHemsley,I Poole, eds, The Evolution of Plant Physiology: From Whole Plants to Eco-system. Elsevier Academic Press, Cambridge, UK, pp 217–242

Lawson T, Morison JIL (2006) Visualising patterns of CO2 diffusion inleaves. New Phytol 169: 641–643

Lawson T, Oxborough K, Morison JIL, Baker NR (2003) The response ofguard cell photosynthesis to CO2, O2, light and water stress in a range ofspecies are similar. J Exp Bot 54: 1734–1752

Lawson T, von Caemmerer S, Baroli I (2010) Photosynthesis and stomatalbehaviour. Prog Bot 72: 265–304

Lawson T, Weyers JDB (1999) Spatial and temporal variation in gas ex-change over the lower surface of Phaseolus vulgaris L. primary leaves. JExp Bot 50: 1381–1391

Lawson T, Weyers JDB, A’Brook R (1998) The nature of heterogeneity inthe stomatal behaviour of Phaseolus vulgaris L. primary leaves. J Exp Bot49: 1387–1395

Lefebvre S, Lawson T, Zakhleniuk OV, Lloyd JC, Raines CA, Fryer M(2005) Increased sedoheptulose-1,7-bisphosphatase activity in trans-genic tobacco plants stimulates photosynthesis and growth from anearly stage in development. Plant Physiol 138: 451–460

Lohse G, Hedrich R (1992) Characterization of the plasma-membrane H+-ATPasefrom Vicia faba guard cells: modulation by extracellular factors and sea-sonal changes. Planta 188: 206–214

MacRobbie EAC (1981) Ion fluxes in ‘isolated’ guard cells of Commelinacommunis L. J Exp Bot 32: 545–562

MacRobbie EAC (1983) Effects of light/dark on cation fluxes in guard cellsof Commelina communis L. J Exp Bot 34: 1695–1710

MacRobbie EAC (1984) Effects of light/dark on anion fluxes in isolatedguard cells of Commelina communis. J Exp Bot 35: 707–726

MacRobbie EAC (2006) Osmotic effects on vacuolar ion release in guardcells. Proc Natl Acad Sci USA 103: 1135–1140

MacRobbie EAC, Lettau J (1979) Potassium accumulation and stomatalopening. Plant Physiol 63: 60–66

MacRobbie EAC, Lettau J (1980) Ion content and aperture in isolated guardcells of Commelina communis L. J Membr Biol 53: 199–205

Marre E (1979) Fusicoccin: a tool in plant physiology. Annu Rev PlantPhysiol Plant Mol Biol 30: 273–288

McAdam SA, Brodribb TJ (2012a) Fern and lycophyte guard cells do notrespond to endogenous abscisic acid. Plant Cell 24: 1510–1521

McAdam SA, Brodribb TJ (2012b) Stomatal innovation and the rise of seedplants. Ecol Lett 15: 1–8

McAdam SA, Brodribb TJ (2013) Ancestral stomatal control results in acanalization of fern and lycophyte adaptation to drought. New Phytol198: 429–441

McAinsh MR, Pittman JK (2009) Shaping the calcium signature. NewPhytol 181: 275–294

McAusland L, Davey PA, Kanwal N, Baker NR, Lawson T (2013) A novelsystem for spatial and temporal imaging of intrinsic plant water use. JExp Bot 64: 4993–5007

Melotto M, Underwood W, He SY (2008) Role of stomata in plant innateimmunity and foliar bacterial diseases. Annu Rev Phytopathol 46: 101–122

Merlot S, Leonhardt N, Fenzi F, Valon C, Costa M, Piette L, Vavasseur A,Genty B, Boivin K, Müller A, et al (2007) Constitutive activation of aplasma membrane H+-ATPase prevents abscisic acid-mediated stomatalclosure. EMBO J 26: 3216–3226

Merlot S, Mustilli AC, Genty B, North H, Lefebvre V, Sotta B, Vavasseur A,Giraudat J (2002) Use of infrared thermal imaging to isolate Arabidopsismutants defective in stomatal regulation. Plant J 30: 601–609

Morison JI, Gallouët E, Lawson T, Cornic G, Herbin R, Baker NR (2005)Lateral diffusion of CO2 in leaves is not sufficient to support photo-synthesis. Plant Physiol 139: 254–266

Morison JIL, Lawson T (2007) Does lateral gas diffusion in leaves matter?Plant Cell Environ 30: 1072–1085

Morison JIL, Lawson T, Cornic G (2007) Lateral CO2 diffusion inside di-cotyledonous leaves can be substantial: quantification in different lightintensities. Plant Physiol 145: 680–690

Mott KA (2009) Opinion: stomatal responses to light and CO2 depend onthe mesophyll. Plant Cell Environ 32: 1479–1486

Mott KA, Peak D (2007) Stomatal patchiness and task-performing net-works. Ann Bot (Lond) 99: 219–226

Müller-Röber B, La Cognata U, Sonnewald U, Willmitzer L (1994) Atruncated version of an ADP-glucose pyrophosphorylase promoter frompotato specifies guard cell-selective expression in transgenic plants.Plant Cell 6: 601–612

Nadeau JA, Sack FD (2002) Control of stomatal distribution on the Ara-bidopsis leaf surface. Science 296: 1697–1700

Negi J, Matsuda O, Nagasawa T, Oba Y, Takahashi H, Kawai-Yamada M,Uchimiya H, Hashimoto M, Iba K (2008) CO2 regulator SLAC1 and itshomologues are essential for anion homeostasis in plant cells. Nature452: 483–486

Noe SM, Giersch C (2004) A simple dynamic model of photosynthesis inoak leaves: coupling leaf conductance and photosynthetic carbon fixa-tion by a variable intracellular CO2 pool. Funct Plant Biol 31: 1195–1204

Outlaw WH Jr (2003) Integration of cellular and physiological functions ofguard cells. Crit Rev Plant Sci 22: 503–529

Pandey S, Zhang W, Assmann SM (2007) Roles of ion channels andtransporters in guard cell signal transduction. FEBS Lett 581: 2325–2336

Pearcy RW (1990) Sunflecks and photosynthesis in plant canopies. AnnuRev Plant Physiol Plant Mol Biol 41: 421–453

Pearcy RW, Way DA (2012) Two decades of sunfleck research: looking backto move forward. Tree Physiol 32: 1059–1061

Porcar-Castell A, Palmroth S (2012) Modelling photosynthesis in highlydynamic environments: the case of sunflecks. Tree Physiol 32: 1062–1065

Pottosin II, Schönknecht G (2007) Vacuolar calcium channels. J Exp Bot 58:1559–1569





Price GD, von Caemmerer S, Evans JR, Siebke K, Anderson JM, Badger MR(1998) Photosynthesis is strongly reduced by antisense suppression ofchloroplastic cytochrome bf complex in transgenic tobacco. Aust J PlantPhysiol 25: 445–452

Quick WP, Schurr U, Scheibe R, Schulze ED, Rodermel SR, Bogorad L,Stitt M (1991) Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS. I.Impact on photosynthesis in ambient growth conditions. Planta 183:542–554

Raschke K (1970) Temperature dependencies and apparent activation en-ergies of stomatal opening and closing. Planta 95: 1–17

Raschke K, Firn RD, Pierce M (1975) Stomatal closure in response toxanthoxin and abscisic acid. Planta 125: 149–160

Roelfsema MG, Prins HA (1995) Effect of abscisic acid on stomatal openingin isolated epidermal strips of abi mutants of Arabidopsis thaliana.Physiol Plant 95: 373–378

Roelfsema MRG, Hedrich R (2005) In the light of stomatal opening: newinsights into ‘the Watergate.’ New Phytol 167: 665–691

Romano LA, Jacob T, Gilroy S, Assmann SM (2000) Increases in cytosolicCa2+ are not required for abscisic acid-inhibition of inward K+ currentsin guard cells of Vicia faba L. Planta 211: 209–217

Rusconi F, Simeoni F, Francia P, Cominelli E, Conti L, Riboni M, Simoni L,Martin CR, Tonelli C, Galbiati M (2013) The Arabidopsis thaliana MYB60promoter provides a tool for the spatio-temporal control of gene expressionin stomatal guard cells. J Exp Bot 64: 3361–3371

Schlüter U, Muschak M, Berger D, Altmann T (2003) Photosyntheticperformance of an Arabidopsis mutant with elevated stomatal density(sdd1-1) under different light regimes. J Exp Bot 54: 867–874

Serna L, Fenoll C (2000) Plant biology: coping with human CO2 emissions.Nature 408: 656–657

Sharkey TD, Raschke K (1981) Separation and measurement of direct andindirect effects of light on stomata. Plant Physiol 68: 33–40

Shimazaki K, Doi M, Assmann SM, Kinoshita T (2007) Light regulation ofstomatal movement. Annu Rev Plant Biol 58: 219–247

Shimazaki K, Kondo N (1987) Plasma membrane H+-ATPase in guard cellprotoplasts from Vicia faba L. Plant Cell Physiol 28: 893–900

Smith WK, Berry ZC (2013) Sunflecks? Tree Physiol 33: 233–237Stitt M, Quick WP, Schurr U, Schulze ED, Rodermel SR, Bogorad L (1991)

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in trans-genic tobacco transformed with ‘antisense’ rbcS. II. Flux-control coeffi-cients for photosynthesis in varying light, CO2, and air humidity. Planta183: 555–566

Suhita D, Raghavendra AS, Kwak JM, Vavasseur A (2004) Cytoplasmicalkalization precedes reactive oxygen species production during methyljasmonate- and abscisic acid-induced stomatal closure. Plant Physiol134: 1536–1545

Talbott LD, Rahveh E, Zeiger E (2003) Relative humidity is a key factor inthe acclimation of the stomatal response to CO2. J Exp Bot 54: 2141–2147

Tanaka Y, Sugano SS, Shimada T, Hara-Nishimura I (2013) Enhancementof leaf photosynthetic capacity through increased stomatal density inArabidopsis. New Phytol 198: 757–764

Thiel G, MacRobbie EAC, Blatt MR (1992) Membrane transport in sto-matal guard cells: the importance of voltage control. J Membr Biol 126:1–18

Tinoco-Ojanguren C, Pearcy RW (1993) Stomatal dynamics and its im-portance to carbon gain in 2 rain forest Piper species. 1. VPD effects onthe transient stomatal response to light flecks. Oecologia 94: 388–394

UNESCO (2009) http://www.unesco.org/new/en/natural-sciences/environment/water/wwap/wwdr/wwdr3-2009/downloads-wwdr3

Urban O, Sprtová M, Kosvancová M, Tomásková I, Lichtenthaler HK,Marek MV (2008) Comparison of photosynthetic induction and tran-sient limitations during the induction phase in young and mature leavesfrom three poplar clones. Tree Physiol 28: 1189–1197

Vialet-Chabrand S, Dreyer E, Brendel O (2013) Performance of a newdynamic model for predicting diurnal time courses of stomatal con-ductance at the leaf level. Plant Cell Environ 36: 1529–1546

Vico G, Manzoni S, Palmroth S, Katul G (2011) Effects of stomatal delayson the economics of leaf gas exchange under intermittent light regimes.New Phytol 192: 640–652

von Caemmerer S, Lawson T, Oxborough K, Baker NR, Andrews TJ,Raines CA (2004) Stomatal conductance does not correlate with pho-tosynthetic capacity in transgenic tobacco with reduced amounts ofRubisco. J Exp Bot 55: 1157–1166

Von Groll U, Berger D, Altmann T (2002) The subtilisin-like serine pro-tease SDD1 mediates cell-to-cell signaling during Arabidopsis stomataldevelopment. Plant Cell 14: 1527–1539

Wang PT, Song CP (2008) Guard-cell signalling for hydrogen peroxide andabscisic acid. New Phytol 178: 703–718

Wang Y, Blatt MR (2011) Anion channel sensitivity to cytosolic organicacids implicates a central role for oxaloacetate in integrating ion fluxwith metabolism in stomatal guard cells. Biochem J 439: 161–170

Wang Y, Hills A, Blatt MR (2014a) Systems analysis of guard cell mem-brane transport for enhanced stomatal dynamics and water use effi-ciency. Plant Physiol 164: 0000-0000

Wang Y, Noguchi K, Ono N, Inoue S, Terashima I, Kinoshita T (2014b)Overexpression of plasma membrane H+-ATPase in guard cells pro-motes light-induced stomatal opening and enhances plant growth. ProcNatl Acad Sci USA 111: 533–538

Wang Y, Noguchi KO, Terashima I (2008) Distinct light responses of theadaxial and abaxial stomata in intact leaves of Helianthus annuus L.Plant Cell Environ 31: 1307–1316

Wang Y, Papanatsiou M, Eisenach C, Karnik R, Williams M, Hills A, Lew VL,Blatt MR (2012) Systems dynamic modeling of a guard cell Cl2 channelmutant uncovers an emergent homeostatic network regulating stomataltranspiration. Plant Physiol 160: 1956–1967

Way DA, Pearcy RW (2012) Sunflecks in trees and forests: from photosyntheticphysiology to global change biology. Tree Physiol 32: 1066–1081

Weyers JDB, Lawson T, Peng ZY (1997) Variation in stomatal character-istics at the whole-leaf level. In PR van Gardingen, GM Foody, PJ Cur-ran, eds, SEB Seminar Series: Scaling Up. Cambridge University Press,Cambridge, UK, pp 129–149

Wille AC, Lucas WJ (1984) Ultrastructural and histochemical studies onguard cells. Planta 160: 129–142

Willmer C, Fricker MD (1996) Stomata. Chapman and Hall, LondonWong SC, Cowan IR, Farquhar GD (1979) Stomatal conductance correlates

with photosynthetic capacity. Nature 282: 424–426Wong SL, Chen CW, Huang HW, Weng JH (2012) Using combined mea-

surements for comparison of light induction of stomatal conductance,electron transport rate and CO2 fixation in woody and fern speciesadapted to different light regimes. Tree Physiol 32: 535–544

Woodward FI (1987) Stomatal numbers are sensitive to increases in CO2from pre-industrial levels. Nature 327: 617–618

Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV (2009) Regulation oftranspiration to improve crop water use. Crit Rev Plant Sci 28: 410–431

Yoo CY, Pence HE, Jin JB, Miura K, Gosney MJ, Hasegawa PM,Mickelbart MV (2010) The Arabidopsis GTL1 transcription factor regu-lates water use efficiency and drought tolerance by modulating stomataldensity via transrepression of SDD1. Plant Cell 22: 4128–4141

Zeiger E, Zhu JX (1998) Role of zeaxanthin in blue light photoreception andthe modulation of light-CO2 interactions in guard cells. J Exp Bot 49:433–442

Zhang Q, Chen YJ, Song LY, Liu N, Sun LL, Peng CL (2012) Utilization oflightflecks by seedlings of five dominant tree species of different sub-tropical forest successional stages under low-light growth conditions.Tree Physiol 32: 545–553

Zhang X, Miao YC, An GY, Zhou Y, Shangguan ZP, Gao JF, Song CP(2001) K+ channels inhibited by hydrogen peroxide mediate abscisic acidsignaling in Vicia guard cells. Cell Res 11: 195–202

Zhu XG, de Sturler E, Long SP (2007) Optimizing the distribution of re-sources between enzymes of carbon metabolism can dramatically increasephotosynthetic rate: a numerical simulation using an evolutionary algorithm.Plant Physiol 145: 513–526


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