What do guard cells do

Stomatal aperture regulates plant CO 2 intake and water loss, thus critically influencing growth and water stress responsiveness Pei et al. This allows for molecular genetic, cell biological, biophysical, physiological, and functional genomics analyses of single cell signaling responses Pei et al. Stomatal movements are regulated by turgor pressure leading to water flux. Stomatal opening is achieved by uptake of solutes into the cytosol and vacuoles resulting in water uptake into the guard cells and, thus, turgor increase drives opening of the stomatal pore.

Moreover, malate is produced from starch and functions as one of the major osmotica causing an increase in turgor pressure in guard cells. Anions including Cl - and NO 3 - are transported into the guard cell to further increase turgor pressure. Figure 1 shows an extension of early models of the roles of ion channels and transporters in stomatal opening and closing Schroeder and Hedrich, ; McAinsh et al.

Furthermore, ABA-induced stomatal closure was impaired by the tpk1 mutation Gobert et al. It has been proposed that calcium release is induced by inositol hexakisphosphate Lemtiri-Chlieh et al. SV channels were first thought to mediate anion transport Hedrich et al. SV channels are ubiquitous and have been found in all plant vacuoles, but in spite of their broad distribution in plant vacuoles, their physiological functions are not yet understood. Further studies are required to address the physiological role for this SV channel protein in plants.

CLC family genes function as vacuolar anion nitrate uptake transporters De Angeli et al. For the majority of signals the molecular identity of the sensors is not known, with the notable exception of blue light: The phototropins PHOT1 and PHOT2 were shown to function as blue light receptors in Arabidopsis guard cells Kinoshita et al.

Thus, it would be interesting to test whether or not PHOT1 and PHOT2 receptors lead to an increase in cytosolic calcium in guard cells in response to blue light Figure 2. The RPT2 Root Phototropism 2 protein is a plant specific protein containing a putative protein-protein interaction domain Sakai et al.

A protein was also shown to bind to light-activated, autophosphorylated phototropins in Vicia guard cells Kinoshita et al. In addition, another protein, VfPIP, interacting with Vicia phototropin was identified from a yeast two-hybrid screen Emi et al.

This protein shares a high similarity with the dynein light chain, yet its role remains to be determined. Recently, type 1 protein phosphatase PP1 genes that are expressed in Vicia faba guard cells have been identified as positive regulators of blue light-induced stomatal opening Takemiya et al.

Introduction of a dominant non-functional mutant PP1 into guard cells inhibited light-induced stomatal opening whereas wild type PP1 had no effect. Recently it was shown that blue light inhibits the S-type anion channels that are activated by ppm CO 2 Marten et al.

Furthermore, blue light inhibition of anion channels was not seen in guard cells of the phot1phot2 double mutants, indicating that phototropins also function in this response Marten et al. Compared to wild type, cry1cry2 , and phot1phot2 double mutant guard cells are less responsive to blue light, whereas guard cells of a cry1cry2phot1phot2 quadruple mutant did not respond to blue light at all Mao et al.

Guard cells of COP1 over-expressing plants are hypersensitive to blue light Mao et al. KAT1 is expressed in guard cells Nakamura et al. KAT2 is also expressed in guard cells Pilot et al. In addition, spermidine was shown to inhibit stomatal opening and induce stomatal closure Liu et al. Spermidine is a polyamine that functions as a growth signal in both eukaryotes and prokaryotes. The levels of polyamines increase under stress conditions, suggesting their roles in stress responses Slocum and Flores, Phopholipid kinases also modulate stomatal opening.

PI 4,5-bisphosphate was also shown to inhibit S-type anion channel current in guard cells Lee et al. Furthermore, a mutation in the Arabidopsis PIP5 kinase 4 causes reduced stomatal opening in the light Lee et al. Stomatal opening of the pip5k4 mutant was rescued by exogenous PI 4,5-bisphosphate, indicating that PIP5K4 is a positive regulator of light-induced stomatal opening Lee et al. The Arabidopsis genome encodes 11 ROP-type small G proteins that function as a molecular switch in various cellular responses in plants.

It was recently shown that ROP2 is expressed in guard cells and translocates to the plasma membrane upon illumination Jeon et al. Expression of a constitutively active form of ROP2 negatively modulated stomatal opening in both Vicia and Arabidopsis Jeon et al. Transcriptional modulation contributes to the regulation of guard cell signaling cascades, positively or negatively.

Two MYB transcription factors have been identified as regulators of stomatal opening. A null mutation in MYB60 leads to a reduction in light-induced stomatal opening and to an enhanced drought tolerance, without affecting ABA sensitivity of guard cells Cominelli et al. Furthermore, only 36 genes were shown to be down- or up-regulated in the atmyb60 mutant, suggesting that MYB60 specifically regulates a limited number of genes Cominelli et al.

Overexpression of MYB61 results in a higher leaf surface temperature indicative of smaller stomatal apertures , whereas atmyb61 null mutants have a lower leaf surface temperature larger stomatal apertures , compared to wild type plants Liang et al. Similar to the situation with atmyb60 , neither overexpression or knockout of MYB61 appears to alter ABA sensitivity of the plants in stomatal movements Liang et al.

However, dark-induced stomatal closure is partially impaired in atmyb61 Liang et al. AtCHX20, a proton antiporter that localizes to endomembranes, was reported to positively regulate light-induced stomatal opening Padmanaban et al.

The CHX family is comprised of 28 members, and their functions are largely unknown Sze et al. Because nitrate is also abundant in soil and transported to plant cells by the nitrate transporter AtNRT1. Figure 2 illustrates the signaling cascade from activation of PHOT1, 2 to stomatal opening in terms of known genes and their relative position in the signal transduction pathway. The figure is clickable and linked to an updated version of Schroeder et al.

The continual rise in atmospheric pCO 2 causes an increase in leaf CO 2 and a reduction of stomatal apertures in many plant species on a global scale Medlyn et al. However, the mechanisms mediating CO 2 -induced stomatal closing are still less understood than those triggered by ABA, and until recently no genetic mutants had been identified that robustly impaired high pCO 2 -induced stomatal closing.

Moreover, the ABA-insensitive mutant gca2 was identified as a first recessive mutation that shows a strong impairment in high CO 2 -induced stomatal closing Young et al. Mutations in the SLAC1 gene encoding a plasma membrane protein with similarity to a bacterial malate transporter exhibit impairment in CO 2 -induced stomatal closing Negi et al.

SLAC1 is highly expressed in guard cells and localized at the plasma membrane Negi et al. SLAC1 shows homology to malate transporters and S-type anion currents are strongly impaired in slac1 alleles Vahisalu et al.

Thus gca2 and slac1 mutants represent first recessive CO 2 -insensitive mutants that are strongly impaired in stomatal closing Young et al. These mutants are also impaired in their ABA response, suggesting that the encoded proteins function downstream of a convergence point between CO 2 - and ABA-induced stomatal closing Figure 3. AtABCB14 is predominantly expressed in guard cells and localized to the plasma membrane. Atabcb14 mutant plants show slightly more rapid high CO 2 -induced reduction in stomatal conductance Lee et al.

This study suggests that AtABCB14 functions during stomatal opening in mediating malate uptake into guard cells, which was released via anion channels during stomatal closing responses and possibly also from mesophyll cells.

AtABCB14 is proposed to osmotically enhance stomatal opening and to remove extracellular malate which is known to enhance activation of anion channels Lee et al. The HT1 protein kinase was identified as a signaling component that mediates a strong negative regulation of CO 2 -induced stomatal closing Hashimoto et al. Thus ht1 mutant alleles that show impaired HT1 kinase activity cause a constitutive high CO 2 stomatal closing response, even when CO 2 concentrations are clamped to low levels Hashimoto et al.

In this respect, it is interesting to note that the CO 2 -hypersensitive response in ht1 mutant alleles, continued to show blue light-induced stomatal opening and ABA-induced stomatal closing, even though the degree of stomatal responses differed quantitatively from wild type Hashimoto et al. Note that cross talk occurs among stomatal movement responses and therefore any mutation that greatly impairs one pathway is expected to have a secondary effect on other responses.

For example a blue-light insensitive mutant will show reduced stomatal opening under many conditions and therefore may be expected to quantitatively affect the degree of ABA-induced stomatal closing. Thus, the blue light receptors are presently not added as mechanisms to the primary ABA signaling cascade. Several genes that affect CO 2 -regulated stomatal movements have been identified, but the CO 2 sensing mechanisms remain unknown, in spite of their importance for global plant gas exchange regulation in light of the continuing increase in atmospheric CO 2 Medlyn et al.

The humidity response of stomata is interesting for a number of reasons: i Stomatal closure occurs very rapidly in response to a reduction in relative humidity. Despite its role in stomatal movements, molecular components of the humidity signaling cascade largely remain unknown. Thermal imaging of leaf temperatures allows one to directly measure leaf surface temperature and thus identify mutants with altered stomatal behavior because plants with larger stomatal apertures have a lower leaf surface temperature due to a higher transpiration rate Mustilli et al.

In a forward genetic screen, mutants were screened for reduced stomatal closing in response to stepwise reductions in relative humidity of the air surrounding plants Xie et al. This result suggests, in addition to a proposed ABA-independent pathway Assmann et al. Further research on this response should be interesting to understand how plants sense humidity changes and then turn on the above signaling mechanisms.

Furthermore, it was recently shown that Arabidopsis slac1 mutants are impaired in rapid stomatal closure when induced by low humidity, suggesting an important role of the SLAC1-associated anion channel in low humidity-induced stomatal closing Vahisalu et al. The residual and slowed stomatal closing response in slac1 plants may be due to R-type anion channels, which remained intact in slac1 guard cells Vahisalu et al.

Thus initial insights have been obtained into genetic and physiological mechanisms that mediate low-humidity induced stomatal closing, but more information regarding this important response is needed and early humidity sensing mechanisms remain unknown. As shown in Figure 3 , the ion channels which control this response and many genes encoding positive as well as negative regulators of guard cell ABA signaling have been identified in Arabidopsis. In addition, a weak mutant allele in the Mg--chelatase subunit, cch1 Harper et al.

It is therefore surprising that mutations in this gene have not been identified in conventional forward genetic screens Koornneef et al. In contrast to the cch1 allele, known gun5 mutant alleles in the same Mg-chelatase subunit gene did not affect ABA signaling Shen et al. It is interesting to note, in contrast to other plant hormone receptors, that these three proposed ABA receptors do not share any primary structural features.

Both mediate anion release from guard cells, causing depolarization Figure 1. Essential Function of Anion Channels in Stomatal Closing Anion channels in the plasma membrane of guard cells were proposed to provide a central control mechanism for stomatal closing Schroeder and Hagiwara, Genetic evidence for this model was recently obtained. The SLAC1 protein has distant similarity to a bacterial malate transporter. Since S-type anion channels have a permeability to malate anions Schmidt and Schroeder, , and slac1 mutants lack S-type anion currents in guard cells Vahisalu et al.

ABC transporters have been suggested to regulate S-type anion channels or to serve as the channel themselves Leonhardt et al. Null mutations in AtMRP4 resulted in enhanced stomatal opening in response to light, increased transpirational water loss, and enhanced drought sensitivity, indicating that AtMRP4 is a negative regulator of stomatal opening Klein et al.

Atmrp knockout mutants show reduced stomatal opening, in response to light, when compared to wild type plant responses Klein et al. Atmrp mutants also display other phenotypes consistent with the observed stomatal behavior, including a reduced transpiration rate, reduced waters loss in detached leaves, enhanced drought tolerance, indicating that AtMRP5 positively regulates stomatal opening Klein et al.

Furthermore, the atmrp5 null mutant shows reduced sensitivity to ABA in stomatal closing Gaedeke et al. Whether AtMRP5 directly interacts with ion channel proteins remains presently unknown.

The null rop10 mutant shows enhanced ABA response in stomatal closure, and a dominant-negative mutant of ROP10 partially suppresses abi mutant phenotypes in seed germination and root elongation Zheng et al. CAS encodes a plant-specific protein that has a single transmembrane domain. Phospholipids, phospholipid kinases, and phospholipid lipases function in guard cell ABA signaling. Phosphatidylinositol 3- and 4-phosphate, sphingosinephosphate S1P , and phytosphingosinephosphate phyS1P are additional phospholipids that positively regulate ABA signaling, indicating that PI3 kinase, PI4 kinase, and sphingosine kinase are positive effectors of the ABA signaling cascade Ng et al.

Analysis of ABA responses in seed germination, compared to guard cells, show differential effects of G protein mutants.

Both gpa1 and gcr1 mutants show ABA hypersensitivity in seed germination and early seedling development, indicating that GPA1 negatively regulates this signaling pathway Pandey et al. Reactive oxygen signaling mechanisms in guard cells Reactive oxygen species ROS and the gaseous nitric oxide NO function as second messengers in guard cell ABA signaling.

NADPH oxidases are multi spanning membrane proteins that produce extracellular superoxide Keller et al. The ethylene receptor ETR1 was reported to mediate ethylene- and H 2 O 2 -induced stomatal closure, and AtrbohF functions in this pathway Desikan et al. However, ethylene was also shown to counteract and delay ABA-induced stomatal closure Tanaka et al.

Thus further research would be helpful to illuminate these apparently counter-acting physiological ethylene responses in stomatal regulation. In addition, the redox state of the H 2 O 2 scavenger ascorbate plays a role in stomatal movements. Overexpression of dehydroascorbate reductase results in increased ascorbate reduction, a lower H 2 O 2 level in guard cells, and increased stomatal conductance and water transpiration Chen and Gallie, The dominant ABA insensitive abi and abi loci were isolated Koornneef et al.

Localization of the mutant abi protein into the nucleus is required for the ABA-insensitive response Moes et al. When the nuclear localization sequence in abi is disrupted, the transcription of genes regulated by ABA is rescued to wild-type levels, thus suggesting that abi functions as a hypermorphic allele Moes et al.

ABI2 physically interacts with the SOS2 protein kinase, and this interaction is disrupted by the abi mutation Ohta et al. Note that no pharmacological inhibitors for PP2Cs are known. Therefore, the dominant natures of the abi and abi alleles Koornneef et al. Furthermore, the PP2A inhibitor okadaic acid phenocopies the rcn1 phenotypes in wild type plants Kwak et al.

For reviews on transcription factors that function in ABA signaling, readers are referred to the following reviews Giraudat, ; Finkelstein et al. Further analyses of AtPP2CA transcripts in abh1 showed that this effect can contribute to the abh1 phenotype, but detailed analyses also demonstrated that additional mechanisms are required for generating the ABA hypersensitivity of abh1 Kuhn et al. These results imply that RNA processing and turnover is utilized by plant cells to regulate ABA signaling or that rate-limiting genes that function in ABA signaling are affected in these mRNA processing mutants for more details, see Kuhn and Schroeder, Studies with transgenic plants showed that over-expression of the MYB44 transcription factor enhances ABA sensitivity in stomatal closure Jung et al.

These data indicate that there are signaling branches within ABA signaling, but that there are also essential signaling nodes, including the above mechanisms Hetherington and Woodward, Thus it appears likely that some mechanisms more directly mediate ABA signaling, and other mechanisms may be peripheral modulators. However, more research is needed to distinguish such mechanisms. Moreover, many genes and mechanisms are waiting to be identified, including the mechanisms that mediate CO 2 sensing, humidity sensing as well as understanding the diverse proposed ABA receptors.

The study of guard cell signaling provides insights into how the many cellular processes assemble together to create a quantifiable single cell output. Thus, identification and characterization of genes that are highly and specifically expressed in guard cells using various approaches, including gene trap screens and cell-type specific microarray analyses Leonhardt et al.

Furthermore, dynamic modeling and computational simulations of ABA signaling as done by Li et al. We also thank Michelle Turek for critical reading of this manuscript and Daren Miller for help with the webpage. Allan, A. Plant Cell 6 , Allen, G. Control of ionic currents guard cell vacuoles by cytosolic and luminal calcium.

Plant J. Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era Plant Cell 14 , Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells.

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Cytoplasmic calcium regulates voltage-dependent ion channels in plant vacuoles. EMBO J. Hetherington, A. The role of stomata in sensing and driving environmental change. Homann, U. Hosy, E. Hugouvieux, V. Cell , Hunt, L. Phospholipase C is required for the control of stomatal aperture by ABA.

In addition, other ITCs, nitriles, and thiocyanates e. Manipulation of glucosinolate metabolic pathways by plant metabolic engineering and breeding approaches may lead to development of crop varieties with combined disease and drought resistance. Recently, another sulfur containing compound, hydrogen sulfide H 2 S , generated by L-cysteine desulfhydrase was shown to act upstream of NO to modulate ABA-dependent stomatal closure Scuffi et al. The plant leaf metabolome can boast as many as 5, different metabolites Bino et al.

Considering the roles of established metabolites in guard cell functions, we have begun the heydays of functional genomics, fluxomics, and systems biology toward understanding of this highly sophisticated single cell type model system. Although studies on guard cell metabolism are highly biased toward ABA and osmolytes owing to their primary importance in stomatal movement, the identification of additional critical metabolites as shown in Figure 1 underlying or correlated with stomatal movement will form a solid foundation toward a broader understanding of optimal plant adaptation to environmental changes.

For example, although progress in the study of stomatal movement in plant immunity has been made Zhang et al. Information currently available has revealed universal and diverse metabolites and pathways leading to stomatal responses. Many years of traditional breeding has unknowingly selected varieties with cool leaf temperature in some species, i. For instance, in Pima cotton Gossypium barbadense L. Furthermore, in cotton, stomatal conductance and leaf cooling were significantly correlated with fruiting prolificacy and yield during the hottest period of the year Radin et al.

Although we did not focus on the roles of stomatal ontogeny, shape, size, and distribution, which can also significantly affect plant water balance, growth and biomass, the engineering of stomatal development and response as a means to improve water use efficiency is an attractive approach to improve drought tolerance in crops Schroeder et al. Guard cell metabolomics and systems biology hold the potential to unravel key molecular networks that control plant productivity and defense in a changing climate.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Akter, N. Negative regulation of methyl jasmonate-induced stomatal closure by glutathione in Arabidopsis.

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Lee, K. Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid. Cell , — Lee, M. Cell Biol. The anatomy of stomata in many monocotyledonous plants differs, but the principle of their movement is much the same Chen et al. In closing the pore, guard cells reverse this process by metabolizing these solutes or releasing them to the apoplast.

Mature guard cells lack plasmodesmata Wille and Lucas, , so all of the inorganic ions, and during closure much of the organic solute that is not metabolized, must be transported across the plasma membrane. Thus, the bulk of solute transported across the plasma membrane also must pass across the tonoplast surrounding the vacuole. E, Single optical section from a Z-stack through two kidney-shaped guard cells surrounding one stoma center , showing the punctate distribution of GORK-GFP around the periphery of the two guard cells.

Z-plane transects taken along the x axis at positions 1 to 5 are shown below. F, The full three-dimensional projection of the Z-stack clearly shows the punctate character of GORK localization and the prevalence of the channel at the junctions between the two guard cells.

Data are from C. Eisenach, Ph. See Eisenach et al. References are as follows: Humble and Raschke ; Raschke et al. GC, Guard cells. Data relate to V. References are as follows: Fischer ; Allaway ; Raschke et al. Values incorporate data from Commelina communis and Arabidopsis guard cells. Guard cells coordinate solute flux actively through a number of major transport pathways at the plasma membrane and the tonoplast during stomatal movements Box 1; Allen et al.

Stomatal opening is promoted by light and by the breakdown of starch Horrer et al. Transport at the tonoplast is coordinated with ion flux across the plasma membrane, in part because transporters at both membranes share a common pool of solutes and metabolites in the cytosol. The characteristics of ion transport for solute accumulation are generally shared among plant cells, including guard cells. Where guard cells differ from the norm is their ability to coordinate solute release and close the stomatal pore.

The origins of the changes in pH i observed in the presence of ABA are still to be determined but are most likely an emergent property of interactions between ion transport and metabolism Chen et al. A number of other stimuli promote stomatal closure, including darkness, high CO 2 partial pressures pCO 2 , and several plant pathogens.

The mechanics of the changes in transport in each case are thought to follow a pattern similar to that for ABA. Indeed, the characteristics required for closure generally limit transport changes to those evoked by ABA Chen et al.

Of course, the signal cascades need not be the same, but what detail is available at present is often fragmentary. More recently, the SLAC1 channel, and its activation, has become a focal point for dissecting the mechanisms of CO 2 regulation. The hydration of dissolved CO 2 is very slow in the absence of carbonic anhydrase Gutknecht et al.

So it is no surprise that stomata respond sluggishly to pCO 2 changes in the ca1ca4 double mutant Hu et al. One recent study Wang et al.

The results are also difficult to reconcile with a publication from the same group suggesting that phosphorylation sites on both sides of the membrane are essential for the SLAC1 response to pCO 2 Yamamoto et al.

Thus, the relationships between aquaporins and carbonic anhydrases in water and CO 2 permeability are a matter of debate Grondin et al. Following publication of the Arabidopsis genome in , the list of guard cell transporters, and even more so that of the regulatory proteins functional in guard cells, has expanded rapidly.

Less is known for sugar transport Ritte et al. Finally, the plasma membrane aquaporin PIP2;1 was recently shown to promote ABA -mediated stomatal closure, most likely by enhancing the capacity for water flux Grondin et al. These receptors mark the beginning of a key phosphorylation cascade triggered by ABA. Other transporters undoubtedly remain to be identified, and intermediates such as the gasotransmitter H 2 S Scuffi et al.

Indeed, even without knowledge of individual genes and the proteins they encode, electrophysiological studies provide quantitative information about the kinetic and regulatory properties that are essential to understand the functions of these transporters and their contributions to stomatal movements.

A comprehensive list of these transporters and their functional characteristics at the plasma membrane and tonoplast are included in Tables III to VI , together with the corresponding genes from Arabidopsis where known. Genetic codes relate to Arabidopsis; functional data relate to V.

Shorthand identifiers in parentheses cross-reference to Box 1. References are as follows: A Blatt, a ; Blatt et al. Stoichiometry determined as the minimum thermodynamic requirement to drive net accumulation.

Genetic codes relate to Arabidopsis, functional data relate to V. References are as follows: A Schroeder et al. Equivalent characteristics available in the literature for Nicotiana tabacum , Arabidopsis, and Zea mays.

Calculated from whole-cell and single-channel currents recorded under equivalent conditions. References are as follows: A Bentrup et al.

Channel densities calculated from whole-cell and single-channel currents recorded under equivalent conditions. Characteristics introduced for the purpose of modeling Hills et al. References are as follows: A Fricker and Willmer, a ; Davies et al. Conversely, for stomatal function, knowledge of a gene product based on mutant analysis alone is often uninformative and, without functional information, can be misleading.

Ceramidases are enzymes that cleave phospholipids and, together with sphingosine kinases, are important for sphingosinephosphate S1P synthesis.

The TOD1 promoter is active in guard cells, and its gene product is able to complement a yeast mutant lacking ceramidase activity. However, TOD1 is also present in other cell types, the tod1 mutation affects stomatal aperture in both the presence and absence of ABA , and its phenotype is pleiotropic, affecting stomata, pollen growth rate, and fertility.

Or is it part of an assembly necessary for the general integrity of cellular homeostasis, including ABA signaling? A number of protein kinases and phosphatases have been identified to affect stomatal movements, both opening and closing. Initially, much information was drawn from inhibitor studies, their actions on aperture, ion flux, and transport current Macrobbie, ; Blatt, Subsequent work has benefitted from mutational screening and site-directed mutagenesis, and in a handful of cases, we now have knowledge of their phosphorylation targets.

Table IX summarizes the major groups of these kinases and phosphatases, and we direct the reader to several excellent reviews Shimazaki et al. Functions listed relate to the kinase group as a whole, not necessarily to the specific kinase on the same line of text.

References are as follows: A Pei et al. Recent work has taken advantage of the Xenopus oocyte as a platform to reconstitute plausible regulatory cascades with the SLAC1 anion channel and several protein kinases, including the SnRK2-type kinase OST1 originally identified in thermal screening for reduced drought sensitivity Merlot et al.

What they have largely failed to address to date are the connections between protein phosphorylation, whether of SLAC1 or associated targets, and its integration with other signaling intermediates and transporters in vivo.

For example, in analyzing the V. Stange et al. In effect, Chen et al. The findings to date, therefore, beg questions about the target sites, kinase and phosphatase specificities, and their relationships to SLAC1 control in vivo. Nor are the data clear cut or consistent. Subsequent studies focused on other kinases. Other inconsistencies are evident when comparing the reconstituted systems in oocytes with the in vivo characteristics in the guard cell.

The mutant cipk23 showed enhanced sensitivity to ABA and closed the stomata Cheong et al. This latter study does present difficulties for interpretation, notably the dissimilar effects of complementations in two different slac1 mutant lines and a lack of some key controls, but the work appears to highlight differences in SLAC1 activation by elevated pCO 2 and by ABA. At least for the discrepancies between oocytes and guard cells, the most plausible explanation is that a subset, possibly all, of these kinases engage different targets in vivo from those available when reconstituted in oocytes and that their regulation of SLAC1 is normally indirect.

In assessing the actions of the kinase and phosphatase mutants, we need to keep in mind that the guard cells also harbor other anion channels, including ALMT12, which is also affected by the OST1 kinase, but for which less information is available at present Meyer et al.

So, in the absence of supporting data in vivo, the relevance of the studies in oocytes must be interpreted with caution. It is time that experiments move beyond reconstitution studies in oocytes. Further progress now will depend, most importantly, on time-resolved phosphorylation assays carried out in vivo.

Guard cells integrate ion transport with secretory traffic that adds new membrane surface as the cells expand; conversely, rates of endocytosis coordinate with solute export as the cell volume decreases.

While membrane traffic has generally correlated with changes in external osmolality and cell volume Homann and Thiel, ; Shope et al.

One mechanism that surfaced recently follows on the identification of the plasma membrane protein SYP, and its tobacco Nicotiana tabacum homolog NtSYR1, associated with the ABA regulation of guard cell ion channels Leyman et al.

The association with ion transport is well illustrated by Eisenach et al. The mutant mimicked the phenomenon of so-called programmed closure, previously ascribed to a memory of stress that leads stomata to reopen only slowly Allen et al. The syp SNARE mutation slows stomatal reopening and shows a strong growth phenotype at moderate relative humidities. A, Stomatal apertures normalized to values at time zero for stomata from the wild type and the SYPcomplemented syp mutant black circles and the mutants syp white circles and syp black triangles before, during, and after the closing stimulus of elevated CaCl 2 outside gray bar.

This figure was modified from Eisenach et al. The connections of the SNAREs to solute transport go well beyond ion channel traffic, however, as was recognized early on Leyman et al.

Honsbein et al. These VSD s incorporate a series of fixed positive charges that, with a change in voltage, drive the VSD conformation, moving it partway across the membrane and drawing open the channel pore Lai et al. Grefen et al. Images are projections of Arabidopsis roots transiently transformed using the tetracistronic vector pTecG-2in1-CC Karnik et al. Bright-field images are single medial plane images with fluorescence overlaid. VSD structures are shown in the closed, open, and again closed conformations right, top to bottom corresponding to the conditions and VSD constructs used.

For clarity, only water molecules light blue on either side in and out of the membrane are shown. This figure was modified from Grefen et al. It remains to be seen whether these interactions affect water permeability directly in addition to aquaporin traffic Chaumont and Tyerman, , possibly to aquaporin function associated with stomatal closure in ABA Grondin et al. Certainly, there is reason to suspect that water flux, like that of solute transport, may be coordinated directly with membrane traffic as part of a supermolecular response complex.

If this wealth of information on guard cell transport is not daunting enough, it is further compounded by the interactions of solute transport across each bounding membrane.

Separating the intrinsic characteristics of transport interactions from those of extrinsic regulation, such as by protein phosphorylation, is often challenging. Membrane voltage is a major factor determining osmotic solute flux for stomatal movements Tables III — VI , and it is central to understanding these transport interactions. From an enzyme kinetic standpoint, voltage serves as a driving force, an electrical substrate, that acts on each charge-carrying transporter in a manner analogous to the mass action effect of adding a chemical substrate to an enzymatic reaction; voltage is also an electrical product of charge-carrying transporters Blatt, Most important, voltage is a shared intermediate in the charge circuit of each membrane and, therefore, ensures an interdependency between all charge-carrying transporters.

Voltage also determines the activity of several ion channels that contribute to solute flux across both the plasma membrane and the tonoplast. These characteristics also imply a strong kinetic enhancement as the membrane depolarizes toward 0 mV.

So, both the first and third of the three prerequisites are met. It is the second prerequisite that has proven more difficult to establish.

The Glu receptor-like channels GLR3. Indeed, to date, these are the only channels in plants known to exhibit such characteristics. However, stomata of the mutant responded normally to ROS H 2 O 2 and ABA , the channels show no evidence of self-limitation, and their localization to the plasma membrane, based solely on the diffuse distribution of an overexpressed, GFP-tagged construct, is unconvincing. Best estimates Chen et al. These compartments almost certainly include the endoplasmic reticulum Garcia-Mata et al.

Although essential for any rational approach to engineering stomata, relating the transport capacity of guard cells to stomatal movements in quantitative mechanistic terms poses a number of difficulties Buckley, As a consequence, relatively few studies have progressed beyond the qualitative analysis of mutant associations.

The physical requirement for charge to balance means that the transport of each ionic species is necessarily joined to that of all others across the same membrane, unless this connection is bypassed by the circuit of a voltage clamp Blatt, A second difficulty arises from the general finding that the ion fluxes needed for stomatal movements reflect only a small fraction of the maximal capacity of several transporters mediating these fluxes Thiel et al.

As a case in point, during stomatal opening, the solute content of a typical V. For Arabidopsis, the changes are equivalent to 0. Instead, it is the balance between the sum of all transporters at the membrane that limits solute flux.

Again, manipulating solute flux through any one transporter inevitably affects this balance and, thereby, directly affects other transporters at the same membrane. Systems modeling offers one approach to overcoming these difficulties.

It enables the detailed knowledge available for the individual transporters to be reconstructed within the physiological framework of the cell. Effective physiological models are constrained by fundamental physical laws and the known kinetic relationships, ligand binding, and related regulatory properties for each transporter.

Such models address the difficulties inherent to understanding how transport and metabolic activities are temporally connected. Boolean network models Li et al. Of course, the real test of any model is its capacity not only for reproducing experimental observations but for predicting new and unexpected behaviors. In this regard, the development of the OnGuard platform for modeling stomata Chen et al. If we are to design crops with improved water use efficiency and able to cope with reduced water availability, then manipulating stomatal conductance is an obvious target.

Reducing stomatal density has already proven successful in some contexts Condon et al. The challenge, therefore, will be to moderate stomatal conductance without a significant cost in photosynthetic assimilation Lawson and Blatt,