Tracy Lawson and James I. L. Morison, Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK

September, 2010

Introduction

As the leaf cuticle is almost impermeable to CO₂ and H₂O, stomata regulate gas exchange between the inside of the leaf and the external environment. Stomatal function is critical for controlling CO₂ uptake for photosynthesis and water loss through transpiration and therefore plant water use efficiency (WUE; amount of carbon gained per unit water lost, see Morison et al. 2008). Changing climatic conditions and the increasing global population is forcing plant breeders and researchers to find plants with improved yields and greater WUE. As stomata ultimately determine these two characteristics, they potentially provide a manipulation route to produce crop plants with improved yield, using less water. Before such a route could be considered viable the mechanism(s) that links mesophyll assimilation rate with stomatal conductance must first be established and the signalling and sensory mechanisms that allow stomatal guard cells to respond to the continually fluctuating environment must be understood. The fact that guard cells contain functional chloroplasts suggests this could be the location for sensory or regulatory mechanisms, but this is controversial. This essay briefly reviews the characteristics of guard cell chloroplasts and discusses their possible functions. In particular, we will examine the evidence for and against guard cell photosynthetic CO₂ fixation in relation to function, as well as illustrate some recent developments using transgenic plants. We will also illustrate how chlorophyll fluorescence has been used to assess photosynthetic function within the intact leaf (Lawson et al. 2002; 2003; von Caemmerer et al. 2004).

Guard Cell Chloroplasts

Chloroplasts are a common feature of guard cells in the majority of species examined, yet the function of these organelles has been the subject of debate and remains to be confirmed. The number of chloroplasts found in guard cells is species-dependent, but typically ranges between 8-15, compared with 30-70 in palisade mesophyll cells (Willmer & Fricker, 1996) resulting in between 25-100 fold lower chlorophyll content per cell in guard cells than in mesophyll cells. However, guard cell volume is about 10 fold lower than mesophyll, which means that the chloroplasts could provide a significant energy source. A noticeable difference in guard cell chloroplasts is that starch accumulates in the dark and disappears in the light, the opposite to mesophyll (Willmer & Fricker, 1996). Although this may also be species-dependent as Arabidopsis has been shown to be practically free of starch in the morning and accumulates it during the night.

A compelling argument supporting a role for guard cell chloroplasts in stomatal physiology has been the fact that stomata remain functional in epidermal strips or protoplasts where any mesophyll influence has been removed. Experimental evidence exists for four primary ways in which guard cell chloroplasts could contribute to stomatal function: (1) ATP and/or reductants produced by guard cell electron transport are used in osmoregulation; (2) chloroplasts are used in the blue-light signalling and responses; (3) starch stored in guard cell chloroplasts is used to synthesize malate to counter-balance K⁺ for stomatal opening; (4) guard cell photosynthesis produces osmotically active sugars (see Figure 1).

Figure 1   Schematic diagram of possible roles of guard cell chloroplasts in stomatal function and osmoregulation and the influence of mesophyll signals on stomatal behavior. Mesophyll driven signals include decreasing C_i or an unknown photosynthetic product, whilst mesophyll photosynthesis can also provide sucrose which may act as a solute. Guard cell chloroplasts could provide ATP from chloroplasts electron transport, or sucrose via Calvin cycle activity, or act as a blue light (BL) receptor (via zeaxanthin), or provide a store for starch, which can be broken down to provide malate to counter balance K⁺ uptake or provide sucrose. Diagram does not show all pathways, and is not drawn to scale.

1. Electron transport: Although guard cell chloroplasts are often smaller, often less well developed and contain less granal stacking than those found in the mesophyll, they have a similar pigment composition to mesophyll chloroplasts, functional photosystems I (PSI) and II (PSII) and evidence exists for both linear and cyclic electron flow. Photosynthetic electron transport, oxygen evolution and photophosphorylation have been measured in guard cell chloroplasts of several species and rates of photophosphorylation can be from 70-90% of those in mesophyll chloroplasts, on a chlorophyll basis. Studies have demonstrated that ATP produced by guard cell chloroplasts is used by the plasma membrane H⁺-ATPase for H⁺ pumping and stomatal opening under red light (Tominaga et al. 2001). Alternatively, it has been suggested that ATP produced during guard cell electron transport is used for the reduction of oxaloacetate (OAA) and 3-phosophoglycerate (3-PGA), which is exported to the cytosol via a 3-PGA-triose phosphate shuttle (Ritte & Raschke, 2003).
2. Blue-light signalling: Blue light induces a rapid highly sensitive opening response in the majority of stomata, correlated with the phosphorylation of a plasma membrane H⁺-ATPase pump (see Shimazaki et al. 2007). Evidence has been reported that both KCN and DCMU inhibit blue light induced stomatal opening, suggesting both mitochondrial and photosynthetic sources of ATP. Guard cell chloroplasts may also hold the receptor for blue light responses in the form of zeaxanthin, a carotenoid associated with dissipation of excess excitation within the photosynthetic pigment antenna. On the other hand, there is also significant evidence for phototropins as guard cell blue light receptors (see Shimazaki et al. 2007).
3. Starch storage: Guard cells can store starch, which generally accumulates in the dark and is hydrolyzed in the light. The starch can be provided from either mesophyll or guard cell photosynthesis and can be used to synthesize malate which acts as a counter-ion to K⁺. A quantitative relationship between malate accumulation and starch loss has been demonstrated along with light-stimulated activity of some of the key enzymes necessary for this process which correlated with stomatal opening. Malate production via PEPc activity has also been associated with stomatal responses to CO₂ concentration and the importance of this enzyme in osmoregulation has been demonstrated using transgenic plants (see references cited within Lawson 2009).
   
   
Guard cell photosynthesis: There are conflicting reports in the literature concerning the capacity for photosynthetic carbon reduction in guard cell chloroplasts, with (for further details see Lawson 2009). Despite earlier reports, it is now generally accepted that guard cell chloroplasts contain Rubisco and the majority of the Calvin cycle enzymes. However, the debate continues over the activity of these enzymes and the quantity (if any) of photosynthetic carbon fixation in guard cell chloroplasts, and its role in stomatal function. There are, however, several lines of evidence reporting significant Calvin cycle activity, namely: (1) similar chlorophyll fluorescence transients in guard and mesophyll cells; (2) guard cell sucrose synthesis sensitivity to DCMU, an inhibitor of PSII electron transport; (3) radio-labelled CO₂ incorporation into phosphorylated compounds under illumination. However, the contribution to osmoticum for stomatal opening has been reported in ranges from 2% to 40% (see references cited within Lawson 2009) with many reports suggesting that rates are too low for any functional significance, whilst conflicting evidence has shown Calvin cycle activity to be a major sink for the products of photosynthetic electron transport (Lawson et al. 2002; 2003). A possible explanation for the conflicting results was proposed by Zeiger et al. (2002), who emphasized the functional plasticity of guard cell chloroplasts, and demonstrated that several different osmoregulatory pathways exist in guard cells, changing depending upon species, environmental parameters, time of day and growth conditions.

Guard Cell Fluorescence

The role of guard cell photosynthesis in stomatal function has been re-visited with the development of single cell chlorophyll fluorescence instrumentation. High-resolution chlorophyll fluorescence imaging of intact green leaves (Lawson et al. 2002; 2003) has the capability of resolving details of fluorescence including quenching parameters within individual chloroplasts. Early studies of guard cell chlorophyll fluorescence were restricted to work on white areas of variegated tissue or guard cell protoplasts, however this was extended to intact green leaves, and more recently to transgenic plants (Figure 2). Simultaneous examination of PSII operating efficiencies (Fq’/Fm’) of guard and mesophyll cells in intact green tissue revealed guard cell photosynthetic efficiency to be 70-80% that of mesophyll chloroplasts (Lawson et al. 2002). Similar findings have also been reported in transgenic plants with reduced Calvin cycle activity. For example, Figure 3(B), illustrates similar decreases in Fq’/Fm’ in both mesophyll and guard cells with increasing PPFD, in both wild type and transgenic plants with reduced amount of sedopheptulose-1,7-bisphosphatase (SBPase), with intact guard cells showing a 20% lower photosynthetic efficiency compared with the mesophyll and a high degree of correlation between the two cell types (see inset). The importance of Rubisco as a sink for ATP and NADPH, the end products of guard cell electron transport has been illustrated using different CO₂ concentrations at 2 and 21% O₂ concentrations (Fig. 3b). Both mesophyll and guard cells showed a decrease in photosynthetic efficiency at low CO₂ concentrations under low O₂, but when CO₂ concentration was high the effect of O₂ concentration was minimal. When CO₂ concentration is low, the sink activity from limited Rubisco carboxylation can be replaced by oxygenase activity at 21% O₂ concentrations thereby increasing photosynthetic efficiency. At low O₂ concentration both oxygenase and carboxylase activity are limited. These results demonstrated that photorespiration and Rubisco activity must act as a significant sink for the end-products of electron transport (ATP and NADPH) in guard cells, as it does in mesophyll. Such chlorophyll fluorescence studies have shown that guard cell chloroplasts have substantial photosynthetic activity and strongly indicate functional Calvin cycle, although they do not resolve the question of how much this activity contributes to stomatal opening. Using such imaging techniques under controlled environmental conditions it is possible to examine the impacts of gas concentrations and stomatal movement (through measurements of stomatal aperture) on guard cell photosynthetic efficiency. Although rates of electron transport could not be calculated from the photosynthetic due to uncertainties in the exact absorption and the contribution of PSI fluorescence in guard and mesophyll cells, these values indicate the influence of environmental parameters on stomatal function and electron transport in the two cell types (Figure 4).

Figure 2   (a) One reflected light image of Nicotiana tabacum stoma catrued from transgenic plants with reduced sedoheptulose-1, 7-bisphosphatase (SBPase) activity. (b) Image of steady state fluorescence (F′) of stoma taken using a high resolution chlorophyll fluorescence imaging system. (c) Corresponding image of maximum fluorescence (F′). (d) Isolation of guard cell chloroplasts from the F′ image created using the editing softwar developed for the imager. (e) Image of Fq′/ Fm′ from guard cell chloroplasts built using only F′ and Fm′ images; color scale is from red (highest), through blue and green (lowest).

Figure 3   (A) Response of Fq′/Fm′ of guard and mesophyll cells from wild type and transgenic tobacco with reduced levels of sedopheptulose-1, 7-bisphosphatase (SBPase) to PFD. Data were obtained from wild type (WT) guard cells (open circles), WT mesophyll (open squares), guard cells of antisense SBPase (solid squares) and mesophyll cells of antisense SBPase plants(solid circles). Measurements were made at a CO₂ of 360 μmol mol^-1. The inset in A shows the relationship between Fq′/Fm′ for mesophyll and guard cells in WT (open symbols) and transgenic plants (closed symbols). (B) Response of Fq′/Fm′ of mesophyll (solid squares and open squares) and guard cells (solid circles and open circles) to increasing CO₂ in the green areas of a Tradescantia leaf in an atompshere contatin 2% (open symbols) or 21% (closed symbols) O₂. Measurements were made at a PFD of 215 μmol m^-2s^-1.

Figure 4   The effect of changing VPD on stomatal aperture (solid circles) and Fq′/Fm′ from mesophyll (solid triangles) and guard cells (solid squares) in a Commelina communis leaf at a CO₂ concentration of 180 µmol mol^-1. ↑ indicates the time when VPD was increased from 1.0 to 1.5 kPa and ↓ when VPD was decreased back to 1.0 kPa.

Studies on Transgenic Plants

Transgenic plants with impairments in photosynthetic function have recently been used to address the role of guard cell chloroplasts and guard cell photosynthesis in stomatal function. Despite severe reductions in either electron transport or Calvin cycle processes, stomata in transgenic plants were still able to achieve similar stomatal conductances as wild type controls. Tobacco plants with reduced amounts of Rubisco (von Caemmerer et al. 2004) revealed similar reduction in guard cell photosynthetic efficiency to those found in mesophyll cells, although no differences in stomatal behaviour were observed. The fact that stomata opened in response to a step-change in light despite high internal CO₂ concentration (C_i) implied that the stomata in these plants were insensitive to C_i. Major reductions in carboxylation capacity of photosynthesis and impaired rates of electron transport (via reductions in the b₆f complex) also resulted in no phenotypic stomatal response despite decreases in sucrose content, suggesting that something other than sucrose concentration acts as the osmoregulator during opening (Baroli et al. 2008). A minor regulatory role for photosynthetic electron transport was suggested in studies conducted on antisense SBPase plants in which stomatal opening in response to red light was greater in transgenic plants compared with wild type controls, possibly due to increased ATP availability (Lawson et al. 2008). However, reduced ATP availability in tobacco plants with reductions in the cytochrome b6f complex showed no effect on red light induced stomatal opening (Baroli et al. 2008). Although reduction in guard cell photosynthesis and Calvin cycle activity have either shown no or only minor effects on stomatal function and behaviour, studies on transgenic antisense PEPc potato plants have supported a role for malate and PEPc activity in guard cells, with reduced PEPc activity showing delays in stomatal opening which was accelerated in over-expressing plants (Gehlen et al. 1996). This work is supported by recent findings that show reduced rates of stomatal opening and final conductance in Amaranthus edulis mutants deficient in PEPc (Cousins et al. 2007). Additionally, stomata in plants with 12% wild-type fructose-1,6-bishpatase (FBPase) activity showed significantly faster opening responses and higher final conductance with increasing irradiance, despite lower photosynthetic rates and elevated C_i concentrations (Muschak et al. 1999). Zea mays plants with increased amount of NADP-malic enzyme (ME) which converts malate to pyruvate have also been shown to have a reduced stomatal conductance (Laporte et al. 2002). Guard cell chloroplasts do not necessarily need to play a role in providing energy or osmotica for stomatal function, they could act as part of the sensory or signalling pathways. Such pathways may involve reactive oxygen species such as H₂O₂, which has been shown to be involved in ABA signalling. Alterations of stomatal conductance in plants with changes in the redox state of ascorbic acid, which is an important component of the antioxidant system, suggest a regulatory role of hydrogen peroxide production.

Mesophyll Photosynthesis Drives Stomatal Behaviour

The link between photosynthetic CO₂demand and stomatal behaviour has often been considered to be guard cell sensing of C_i or the C_i/C_a ratio. However as described above, transgenic plants with elevated C_i concentrations show no differences in stomatal conductance compared with wild type controls, questioning not only the role of C_i linking mesophyll photosynthesis with stomatal behaviour, but whether stomata respond to internal or external CO₂ concentration (von Caemmerer et al. 2004). Alternatively, it has been proposed that guard cells sense the metabolic status of mesophyll via a diffusible factor that is a product of mesophyll photosynthetic activity, such as ATP, NADPH or an unknown substance which was named stomatin by Lee & Bowling (1992). The balance between electron transport and carboxylation reactions has also been postulated as a possible mechanism (see Messinger et al. 2006). However, studies on transgenic plants with impaired photosynthetic rates have demonstrated that stomatal opening is not influenced by photosynthesis alone, and therefore support the conclusion that neither guard nor mesophyll cell photosynthesis are essential for stomatal function and therefore do not support a mesophyll or C_i driven signal. More recently, Mott et al. (2008) demonstrated mesophyll influences on stomatal behaviour, by grafting isolated peels onto mesophyll and showed stomatal responses to light and CO₂. These results led Mott and co-workers to renew the suggestion that stomata respond to a signal generated in mesophyll in response to changes in light and CO₂. Mesophyll photosynthetic sucrose accumulated in the guard cell apoplast has also been hypothesized as the signal that links stomatal aperture with mesophyll photosynthesis (Outlaw and De Vlieghere, 2001).

Although the role of the guard cell chloroplast in stomatal function is still unclear, it is apparent that substantial photosynthetic electron transport and functional Calvin cycle activity takes place within guard cells, although their contributions to stomatal responses has not been resolved. Transgenic plants with impairments in components of electron transport and down stream processes have and continue to provide an ideal opportunity to explore some of the unanswered questions regarding the link between both mesophyll and guard cell photosynthesis and stomatal function as well as providing key information on stomatal responses and mechanisms to changing environmental parameters. The advancement of in situ techniques and the production of mutant and transgenic plants along with the identification of gene trap lines, guard cell specific promoters and single cell transcriptomics is providing new opportunities to address many of the questions that remain regarding guard cell chloroplast function, guard cell metabolism and signalling pathways that enable stomata and photosynthesis to be in tune with each other and the environment.

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