Control of stomatal aperture

Abstract

Stomata, functionally specialized small pores on the surfaces of leaves, regulate the flow of gases in and out of plants. The pore is opened by an increase in osmotic pressure in the guard cells, resulting in the uptake of water. The subsequent increase in cell volume inflates the guard cell and culminates with the opening of the pore. Although guard cells can be regarded as one of the most thoroughly investigated cell types, our knowledge of the signaling pathways which regulate guard cell function remains fragmented. Recent research in guard cells has led to several new hypotheses, however, it is still a matter of debate as to whether guard cells function autonomously or are subject to regulation by their neighboring mesophyll cells. This review synthesizes what is known about the mechanisms and genes critical for modulating stomatal movement. Recent progress on the regulation of guard cell function is reviewed here including the involvement of environmental signals such as light, the concentration of atmospheric CO₂ and endogenous plant hormones. In addition we re-evaluate the important role of organic acids such as malate and fumarate play in guard cell metabolism in this process.

Introduction

Stomata are formed by pairs of specialized epidermal guard cells which are generally surrounded by subsidiary cells. They serve as major gateways both for CO₂ influx from the atmosphere and transpirational water loss of plants.¹ Due to the impermeable wax layer on the epidermal surface, the stomatal complex, plays a major role in controlling gas exchange between plant and the surrounding atmosphere.² Stomata are, therefore, important regulators of the global atmospheric environment,³ and the developmental fate of epidermal cells to differentiate or not into a pair of mature guard cells is also intimately linked to environmental cues.^3,4 Moreover, environmental cues provided by the light intensity, concentration of atmospheric carbon dioxide or endogenous plant hormonal stimuli control stomatal aperture and development.³

Bearing the importance of guard cells in mind it is perhaps unsurprising that considerable research effort has been expended to better comprehend the structure, development and the physiology of stomata.^1,5–8 Given the relative ease of the isolation of guard cells, which gate stomata, our understanding of these cells exceeds that of many of the other 40 cell types described thus far in plants.^9,10 Although guard cells can be regarded as one of the most thoroughly investigated cell types, our knowledge of the signaling pathways that regulate guard cell function is still incomplete.^1,11,12 Despite the anatomical simplicity of the stomatal valve, there is little consensus on how stomata sense and respond to their extrinsic and intrinsic environment.¹³ One largely unresolved question is how environmental signals such as light intensity, the concentration of atmospheric CO₂ and endogenous plant hormones are able to modulate basal stomatal movements and gas exchange in accordance to prevailing environmental conditions. While it is well known that opening responses are achieved by coordination of light signaling, light-energy conversion, membrane ion transport, and metabolic activity in guard cells¹⁴ and current data suggest that light and carbon dioxide play major roles in the control of stomatal movement^1,14 the exact mechanisms which underlie this strict mechanistic control are yet to be unravelled.

Overview of Guard Cell Players and their Functions

Metabolism of guard cells continuously interacts with the environment, sensing biotic and abiotic stimuli and signals coming from both the atmosphere and from roots and responding with appropriate changes in pressure turgor. By this mechanism guard cells control stomata aperture in accordance with changes in the plants environment, in order to preserve water and CO₂ assimilation. Several environmental factors affect stomata opening, including hormone, light quality and intensity, air humidity, atmospheric CO₂ concentration, biotic and abiotic stresses.^3,11,14–17 Recent research progress has revealed that the guard cell response is co-ordinated by a complex of signal transduction networks including input from CO₂, abscisic acid (ABA) and Ca²⁺.^1,11,18 The signal transduction network appears to be intermediated by kinase/phosphatase regulation, secondary metabolites and the regulation of ion channels.^1,11,18 However, given their importance, some of the principal components of the signaling pathways are described in the following sections.

CO₂ Sensing and Signaling

The effects of CO₂ concentration upon stomata aperture has been known for decades.^19–21 At lower atmospheric partial pressure of CO₂ higher stomata aperture is observed in attempt to keep a minimum of CO₂ available in the sub-stomatal chambers and in order to maintain carbon assimilation in the mesophyll cells through operation of photosynthetic machinery. By contrast the opposite response is observed at high CO₂ concentrations in the intercellular spaces which lead to stomatal closure without reductions in the carbon assimilation. Recently CO₂-induced stomatal closure has been studied in detail and plant mutants with affected CO₂ stomatal response have been identified.^22–26

The physiological mechanism by which elevated CO₂ regulates stomatal aperture has been extensively documented.^11,27,28 In brief, high atmospheric CO₂ concentrations activate anion channels and K⁺ out efflux channels in guard cells,^29,30 and promote chloride release from guard cells and membrane depolarization.³¹ Indeed considerable experimental evidence has been accrued that a ion Ca²⁺ is required for CO₂-induced stomatal closure.^32,33 It has been reported that elevated CO₂ activates plasma membrane anion channels of guard cells.²⁷ Interestingly it was found that after a rise in CO₂ from 150 to 800 µmol mol-1, it took a few minutes before the apoplastic Cl⁻ increased.³¹ The authors suggested that the accumulation of an intermediate effector is necessary to activate anion channels. Malate has been proposed as such an effector that mediates between CO₂ and anion channel activation.³⁴ These authors reported that changes in the CO₂ concentration modify the apoplastic malate concentration and that elevated malate enhanced the probability of opening of anion channels by shifting their activation potentials. Although the nature of the intermediate effector that drives anion efflux is unresolved, its accumulation may increase with increasing CO₂ concentration, which induces concentration-dependent anion efflux and stomatal closure.

Despite the fact that the mechanism by which CO₂ regulates stomatal closure is relatively well accepted, the cell types wherein CO₂ is sensed is still a matter of discussion. Potentially, CO₂ could be directly sensed by the guard cells^22,35 or remotely via communication from the mesophyll cells.^15,36,37 It is additionally possible that both cells types could be directly involved in the stomatal CO₂ response.¹ Nevertheless the recent study of Araújo and colleagues³⁷ clearly indicate that mesophyll derived malate plays an important role in controlling stomatal aperture.

Several attempts have recently been made to identify the proteins responsible for binding CO₂ and thus triggering a signal transduction pathway which could control stomatal responses. One of these studies allowed the isolation of Arabidopsis mutants (high leaf temperature 1; ht1-1 and ht1-2) with altered stomatal movements in response to CO₂.²⁵ Further analyses demonstrated that HT1 is a protein kinase expressed mainly in guard cells and suggested that it is important in the control of stomatal movement with its function being more evident in response to CO₂ than to ABA or light. A recent study demonstrated that carbonic anhydrases (CA) can be involved in CO₂ sensing without alteration in ABA and light responses.²⁴ This study additionally characterised a double-mutant β-carbonic anhydrases βCA1 and βCA4 Arabidopsis plant which was affected in both stomatal movement and density but did not display alterations in either ABA or blue light responses.²⁴ Moreover it was demonstrated that βCA-mediated stomatal movements were not linked to leaf photosynthesis. In summary this study suggests that CO₂-binding carbonic anhydrase proteins might function early in CO₂ signaling pathway, controlling gas exchange between plants and their surrounding atmosphere.

Elevated atmospheric CO₂ concentration is known to promote activity of anion channels which mediate efflux of the osmoregulatory anions, Cl⁻ and malate^2-, during stomatal closure.²⁹ For this reason several efforts have recently been made to identify genes encoding guard cell anion channels. However, only recently the first guard cell anion channel was identified in Arabidopsis thaliana. The gene SLAC1 (Slow Anion Channel Associated 1), which encodes a protein homologue to bacterial and fungal dicarboxylate/malate^2- transporter protein, localized specifically in the plasma membrane of guard cells was isolated.^23,38 Recent evidence however demonstrated by functional expression in Xenopus laevis oocytes that guard cell-expressed Arabidopsis SLAC1 encodes a weak voltage-dependent, anion-selective plasma membrane channel rather than a malate transporter.³⁹ Analyses of the slac1 mutants suggest that SLAC1 protein function as a mediator of the CO₂ sensitivity and regulation of stomatal closure. Further analysis revealed that SLAC1 belongs to a protein family of four related members with the same plasma membrane localization but different tissue specificity expression patterns.²³ Additional experiments suggest that the SLAC1-protein family function is essential to maintain organic/inorganic anion homeostasis on the cellular level²³ and is required for stomatal closure in response ABA, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide.³⁸ The lack in SLAC1 protein was shown to impair slow (S-type) anion channel currents that are activated by cytosolic Ca²⁺ and ABA, but do not affect rapid (R-type) anion channel currents or Ca²⁺ channel function. Taken together these results associated with the permeability of S-type anion channels to malate⁴⁰ suggest that SLAC1 plays an important role in the function of S-type anion channels. Additionally three malate transporters have been recently cloned that are responsible for cytosol to vacuole and cytosol to apoplast exchange^22,41,42 and their role is discussed in detail in the section “On the role of organic acids as a component of the stomata signaling cascade.”

CO₂ and Stomatal Development

By adjusting stomatal distribution and density plants can respond to environmental factors such as CO₂ and water and light availability.^7,8,43 Thus, in addition to the control that CO₂ concentrations have upon regulation of stomatal aperture, the mechanisms controlling the abundance of stomatal complexes are also crucial in determining photosynthetic performance and water use efficiency. Correlation of guard cell size, stomatal density and conductance with atmospheric CO₂ has been assessed over millions of years⁴⁴ and it suggests a mechanism connecting long-term CO₂ change with the ecological radiation of land plants and increasing gas-exchange capacity of land plants over geologic time. During their millions-of-years history, plants have been exposed to large variations in environmental conditions that prompted genetic adaptations toward mechanisms that optimize individual fitness. Over this period, plant adaptation to CO₂ is apparent as periods with high CO₂ favored species with few relatively large stomata and low stomatal conductance (g_s), whereas periods with low CO₂ (as at present) favored species with many relatively small stomata and higher g_s.⁴⁴

Changes in stomatal development have been observed under high CO₂ atmospheric conditions, where decreases in stomatal numbers are observed in parallel with increases in atmospheric CO₂ concentration.^8,45 Particularly interesting results have been found for the relationship between stomatal densities and atmospheric CO₂ concentrations. It has been demonstrated that a wide range of species show a reduction in stomatal density with CO₂ enrichment⁴⁶ and that several tree species respond to atmospheric CO₂ concentrations by an alteration in stomatal density.^47,48 Furthermore, responses of stomatal development to light intensity or CO₂ concentration have been shown to involve signaling from mature leaves to the developing leaves.⁴³ Molecular evidences have been provided which corroborate that CO₂ levels are indeed linked to stomatal development.⁴⁹ A high carbon dioxide (HIC) gene has been isolated from Arabidopsis which represents a negative regulator of stomatal development that is responsive to the prevailing CO₂ concentration. This gene encodes a putative 3-keto acyl coenzyme A synthase, which is involved in the synthesis of very-long-chain fatty acids.⁴⁹ Further studies also performed with Arabidopsis thaliana, have revealed that stomatal development and patterning is regulated by responsive mitogen-activated protein kinases (MPK),⁵⁰ and the stress-responsive AtMPK3, along with AtMPK6 was shown to play a key role in regulating stomatal development and patterning in addition to its role in biotic and abiotic stress signaling. Interestingly, recent evidence suggests that density and development of stomata on the Arabidopsis thaliana leaves depend, in part, on the microRNA-mediated regulation of Agamous-like16 (AGL16) gene.⁵¹ AGL16 is a member of the MADS box protein family, which mRNA is targeted for sequence-specific degradation by the microRNA miR824. Additionally several potential quantitative trait loci have been also identified in poplar^52,53 and thus it seems reasonable to assume that leaf traits may be robust enough to be developed as markers for accelerated breeding programs, offering another research approach to increase crop yield.

ABA Sensing and Signaling

ABA is involved in broad of physiological process, such as regulating seed germination and seedling growth and it is also essential in processes that lead to plant resistance upon biotic and abiotic stresses.¹⁸ Under drought and high salinity conditions the levels of ABA increase in the roots which is perceived by the guard cells where it triggers changes in ion fluxes leading to stomata closure and inhibiting stomatal opening.^38,54,55 Altogether these effects minimize water loss and allow plants to cope with drought episodes.

Several efforts have been devoted to identifying ABA receptors in guard cells and the findings of candidates for ABA receptors have been reported. A candidate for ABA-binding protein (ABAR) was identified recently.⁵⁶ In this study the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signaling, was shown to bind ABA and function as a positive regulator in seed germination, post-germination growth and stomatal movement in Arabidopsis. Further analysis revealed that ABAR/CHLH is a ubiquitous protein, which indicates that it might be able to perceive the ABA signal at the whole-plant level.⁵⁶ In another study homologs for a G-protein coupled receptors (GPCRs), known as a conserved mechanism for the extracellular signal perception at the plasma membrane in eukaryotic organisms,⁵⁷ were found Arabidopsis plants. It has been shown that a G-protein coupled receptor 2 (GCR2) encoding a putative GPCR is localized in the plasma membrane and that it interacts with the G protein α-subunit GPA1 to mediate ABA responses. This protein receptor binds ABA with high affinity at physiological concentration.⁵⁸ However, it is important to mention that the characterization of GCR2 as a GPCR and ABA receptor is still a matter of discussion.^59–61 More recently, other GPCR-like proteins (GTG1 and GTG2) were identified and its characterization revealed that the GTG proteins represent a novel class of proteins with topology similar to GPCRs but with classic GTP-binding/GTPase activity.⁶² Further analysis providing biochemical and phenotypic evidences suggest that GTG1 and GTG2 proteins are redundantly involved in G protein-coupled ABA signaling and are, or are parts of, ABA receptor complexes⁶² and their detailed characterization will likely clarify the mechanism underlying the ABA response.

Additional studies to identify key players in ABA signaling pathway are ongoing. It includes the type 2C protein phosphatases (PP2Cs) ABI1 and ABI2, that act negatively regulating ABA responses.⁶³ In this study, interaction partner of ABI1 and ABI2, named as regulatory components of ABA receptor (RCARs) were identified. RCARs proteins belong to a family with 14 members in Arabidopsis and one of its members, RCAR1, was shown to bind ABA, to mediate ABA-dependent inactivation of ABI1 or ABI2 in vitro, and to antagonize PP2C action in plants.⁶³ Due to the possibility that ABA receptors may be molecular and functionally redundant or required for vital processes⁶⁴ chemical genetic approaches have been also used to identify ABA receptors in plants.⁶⁵ In this study, a synthetic growth inhibitor called pyrabactin that function as a selective ABA agonist and acts through PYRABACTIN RESISTANCE 1 (PYR1), a founding member of START protein family, called PYR/PYLs, is required for both pyrabactin and ABA signaling in vivo. It was shown that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs protein. It is assumed that PYR/PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. In brief, these studies provide in vitro evidence that perception of ABA signaling by the PYR/PYL/RCAR proteins shuts down negative regulation of ABA signaling by PP2Cs.¹

Cross Talk between ABA and the “Clock”

Several studies reported that ABA is regulated by the circadian clock^66,67 and ABA-related genes controlled by the clock have also been reported in references 68–70. Evidence of a feedback loop between cADPR and the circadian oscillator suggests a mechanism by which the circadian coordination of ABA-transducing components might be involved in clock-mediated regulation of plant responses to ABA.⁶⁹ The influence of the clock is not limited to gene expression but is also evidenced in the circadian regulation of physiological processes controlled by ABA such as drought stress where ABA induces the closure of the stomatal pore, and this closure was found to be gated by the clock.^66,67 Evidence of a feedback mechanism that links circadian clock with plant responses to drought was recently provided.⁶⁷ In this study, TOC1 (timing of CAB expression 1), a key clock component binds to the promoter of the ABA-related gene (ABAR/CHLH/GUN5) and controls its circadian expression. In agreement with the importance of both ABA and its regulation by genes related to the clock pathway it is generally assumed that the circadian clock is able to confer an advantage to plants, but the nature of that advantage was until recently unresolved. However, recent reports have demonstrated the importance of circadian transcription translation loops for regulating C assimilation and starch metabolism for optimizing plant growth.^71,72 In addition, it seems that transported product(s) of photosynthesis, such as sugars, are important inputs for regulating circadian transcription translation loops.^73,74 This explains why plants gain advantage from circadian control.

Light Regulation of Stomatal Movement

Plants have evolved multiple photoreceptor systems to monitor light quality, quantity and direction. It is well known that stomatal morphogenesis is controlled by genetic as well as environmental factors and in general, an increase in light intensity results in an increase in stomatal index and a systemic signal from mature leaves determine the response.^43,75–78 Light is perceived by various photoreceptors,^79,80 and stomatal movements are regulated by both blue and red light.¹⁴ The blue-light response of stomata appears to be strongly affected by red light, and the underlying mechanisms in the interaction between blue-light signaling and guard cell chloroplasts were recently reviewed in reference ¹⁴.

It is shown that blue light-induced stomatal opening is mediated by the blue light receptor phototropins (PHOT1 and PHOT2) and cryptochromes (CRY1 and CRY2).⁸¹ Recent findings suggest that the light control of stomatal development is mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.⁸² Blue light is required for the activation of phototropins, plant-specific Ser/Thr autophosphorylating kinases, and the activated phototropins transmit the signal to the plasma membrane H⁺-ATPase for its activation.^80,83 Activation of the H⁺-ATPase is caused by the phosphorylation of a Thr residue in the C terminus with subsequent binding of a 14-3-3 protein to the Thr residue.^84,85 Since phototropins are Ser/Thr protein kinases, it might be possible that phototropins directly phosphorylate the H⁺-ATPase. However, this has been shown not to be the case since it was demonstrated that protein phosphatase 1 (PP1), a major member of the PPP family of Ser/Thr protein phosphatases, mediates the signaling between phototropins and H⁺-ATPase in guard cells.⁸⁶ Therefore, ABA is likely to inhibit the signaling molecule(s), including phototropins, PP1, H⁺-ATPase and other unidentified components.

It has also been suggested that the guard cell response to red light is in part an indirect response to red-light-driven intercellular CO₂ uptake in the mesophyll.⁸⁷ For example, it has been shown that chloroplast-containing guard cells in albino sections of variegated leaves do not respond to photosynthetically active radiation, but are sensitive to blue light and CO₂, bringing into question a direct role of guard cell photosynthesis on red-lightmediated stomatal opening in intact leaves.³⁵

On the Role of Organic Acids as a Component of the Stomata Signaling Cascade

The importance of organic acids such as malate and fumarate on the regulation of stomatal aperture has been recently addressed.^37,88,89 Nevertheless the molecular regulatory hierarchy underlying this highly specialized cell type is as yet not fully understood and is discussed bellow.

Malate has long been discussed as an important regulator of stomatal opening and is thought to be an integral part of the mechanism by which guard cells adjust their action in response to external CO₂ concentrations.^{30,34,36,87,90} Recently a plasma membrane ABC malate uptake transporter was identified in guard cells. The gene encoding the ABC transporter family member ABCB14 (ABC transporter B family member 14), was identified and characterized in Arabidopsis plants.²² This study demonstrated that AtABCB14 protein modulates the stomatal closure on transition to higher atmospheric CO₂ concentration environment. High CO₂ levels accelerates stomatal closure in plants lacking AtABCB14 protein and exogenously applied malate mimics the high CO₂ levels responses in AtABCB14 protein deficient plants. Thus it represents a negative regulator of CO₂-induced stomatal closure in Arabidopsis plants and plays its function by transporting malate from apoplast into guard cells elevating the osmotic pressure. It is however clear that many other components of the CO₂ signal transduction pathway still needs to be identified.

Additional evidence for the role of organic acids in the regulation of stomatal movement has been recently provided. The antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase (SDH) in tomato plants resulted in an increased photosynthesis (25%) and biomass at the whole plant level via an organic acid-mediated effect on stomatal aperture.³⁷ Furthermore, measurement of apoplastic and protoplastic organic acid concentrations in these transformants, and also in transformants with deficient stomatal efficiency functioning,⁹¹ revealed a negative correlation between the concentrations of fumarate and gas exchange through the stomata. Altogether, these information alongside that recently obtained for AtABCB²² and potentially also SLAC1,^23,38 coupled with the transport of malate by the vacuolar aluminum-activated malate transporter (ALMT6),⁹² provide strong evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal function. It has also been shown that fumarate is present mainly in photosynthetically active tissue, its content rises with plant age and light intensity with the total fumarase activity remains constant throughout the diurnal cycle⁹³ as well as is present in high levels in phloem exudates⁹⁴ and these results are consistent with the observation that fumarate has similar effects as malate during stomatal closure.²² Given that malate is physiologically present in the apoplast at higher concentrations, it would seem likely that it exerts greater influence on stomatal aperture than fumarate does in vivo. When considered alongside the recent identification of transporters which import malate (or fumarate), into the guard cell,²² or have been assumed to export it,^23,38 these studies thus provide a mechanism by which these organic acids can influence stomatal function. Moreover they provide strong support of the theory of Mott (2009) that guard cells are not autonomously regulated and strongly support the contention of Mott and others that the mesophyll harbors significant control over guard cell function.^6,22,89

Despite the fact that these data clearly document the importance of organic acids in guard cell function, the experiments discussed thus far do not, in their own right, rule out interaction with other well characterized mechanisms of guard cell regulation such as those controlled by ABA, potassium or calcium.^{11,12,28,95,96}

Most plant species (except onion, which does not accumulate starch in guard cells) accumulate malate preferentially over other anions. A close correlation has been obtained between stomatal opening and malate accumulation in guard cells.^27,37 It is well known that malate can be produced through the degradation of starch in guard cells under blue light.¹⁴ Interestingly, the opening of stomata by blue light was severely impaired in Arabidopsis phosphoglucomutase mutant plants, which did not accumulate starch in guard cell chloroplasts.⁹⁷ However, this blue-light response was restored in the presence of high concentrations of Cl⁻, possibly because Cl⁻ replaced malate as a necessary counterion for K⁺ highlighting the complexity of the mechanisms responsible for the stomatal control.

Conclusion and Future Perspective

Stomatal opening is induced by low CO₂ concentrations, high light intensity and high humidity. By contrast closing is promoted by high CO₂ concentrations, darkness, drought and the plant hormone ABA. It is evident that significant progress has been made towards the elucidation of the cellular and molecular processes involved in stomatal patterning and aperture. Nevertheless it is clear that further experimentation is required to tease out the molecular hierarchy shared between the mechanisms involved in stomatal movement. It seems likely that the relative importance of each regulatory mechanism will vary with circumstances; however, a fuller understanding of this is surely required to engineer sustainable increases in crop yield. Finally, it is interesting to note that although neither malate nor fumarate exert their effects on stomata by affecting ABA, the phytohormone could, conditionally, act upstream of the organic acids, given that a recent study in Arabidopsis revealed the SDH2-3 gene to be upregulated by ABA.⁹⁸ It will be important to establish the functional significance of this observation in future studies. Further genetic, molecular biological and biochemical analyses will be required to identify other components of the signal transduction pathway(s) and the corresponding effectors that are involved in the stomatal responses in order to obtain a deeper understanding of the biochemical mechanisms and the precise factors and environmental cues underlying this phenomenon. Finally, from an applied perspective and since water is likely to be at premium in the coming decades⁹⁹ it will be interesting to ascertain whether the increased yield observed in SDH antisense plants can also be obtained under conditions of supra-optimal water supply.