Guard Cell Signal Transduction Research Articles

Identification of a Novel Stomatal Opening Chemical, PP242, That Inhibits Early Abscisic Acid Signal Transduction in Guard Cells.

Plants control their stomatal apertures to optimize carbon dioxide uptake and water loss. Stomata open in response to light through the phosphorylation of the penultimate residue, Thr, of plasma membrane (PM) H+-ATPase in guard cells. Stomata close in response to drought and the phytohormone abscisic acid (ABA), and ABA suppresses the light-induced activation of PM H+-ATPase. However, the signaling pathways that regulate the stomatal aperture remain unclear. Previously, we identified a target of rapamycin (TOR) inhibitor, temsirolimus, to induce stomatal opening through chemical screening. In the present study, we further investigated other TOR inhibitors and identified PP242 as a novel stomatal opening chemical. PP242 induced stomatal opening even in the dark, as well as phosphorylation of the penultimate Thr of PM H+-ATPase in guard cells. Interestingly, PP242 completely suppressed ABA-induced stomatal closure, and inhibited ABA-induced activation of SNF1-related protein kinase 2s (SnRK2s), which are essential kinases for ABA signal transduction in guard cells. In vitro biochemical analysis revealed that PP242 did not directly inhibit SnRK2 but rather inhibited upstream ABA signaling components, specifically B3 clade Raf-like kinases. A quadruple mutant of B3 clade Raf-like kinases exhibited an open stoma phenotype that resembled the effect of PP242. However, PP242 still induced stomatal opening in this mutant, suggesting that PP242 also targets other guard cell components. Together, these results reveal that PP242 induces stomatal opening partly by inhibiting steady-state ABA signal transduction.

Starch metabolism in guard cells: At the intersection of environmental stimuli and stomatal movement.

Starch metabolism in guard cells plays a central role in regulating stomatal movement in response to light, elevated ambient CO2 and potentially other abiotic and biotic factors. Here, we discuss how various guard cell signal transduction pathways converge to promote rearrangements in guard cell starch metabolism for efficient stomatal responses, an essential physiological process that sustains plant productivity and stress tolerance. We suggest manipulation of guard cell starch dynamics as a previously overlooked strategy to improve stomatal behavior under changing environmental conditions.

Martin McAinsh.

Martin McAinsh.

Guard cell redox proteomics reveals a role of lipid transfer protein in plant defense

Redox-based post-translational modifications (PTMs) involving protein cysteine residues as redox sensors are important to various physiological processes. However, little is known about redox-sensitive proteins in guard cells and their functions in stomatal immunity. In this study, we applied an integrative protein labeling method cysTMTRAQ, and identified guard cell proteins that were altered by thiol redox PTMs in response to a bacterial flagellin peptide flg22. In total, eight, seven and 20 potential redox-responsive proteins were identified in guard cells treated with flg22 for 15, 30 and 60 min, respectively. The proteins fall into several functional groups including photosynthesis, lipid binding, oxidation-reduction, and defense. Among the proteins, a lipid transfer protein (LTP)-II was confirmed to be redox-responsive and involved in plant resistance to Pseudomonas syringe pv. tomato DC3000. This study not only creates an inventory of potential redox-sensitive proteins in flg22 signal transduction in guard cells, but also highlights the biological relevance of the lipid transfer protein in plant defense against bacterial pathogens. SignificanceProtein redox modifications play important roles in many physiological processes. However, redox proteomics has rarely been studied in plant single cell-types. In this study, isobaric tandem mass tag-based redox proteomics technology was applied to discover redox-sensitive proteins and corresponding cysteine residues in guard cell response to a bacterial flagellin peptide flg22. Many redox-responsive proteins related to photosynthesis, lipid binding, oxidation-reduction, and defense were identified. Using reverse genetics and biochemical analyses, a lipid transfer protein was functionally characterized to be involved in plant defense against pathogens. The study highlights the utility of redox proteomics in discovering new proteins and redox modifications in important stomatal guard cell functions. Furthermore, detailed functional characterization demonstrates the biological relevance of the redox-responsive lipid transfer protein in plant pathogen defense.

Mitogen-activated protein kinases MPK4 and MPK12 are key components mediating CO2 -induced stomatal movements.

Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2 -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2 , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen-activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2 -induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double-mutants), completely lack CO2 -induced stomatal responses and have impaired activation of guard cell S-type anion channels in response to elevated CO2 /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activation and ABA-induced marker gene expression remain intact in the mpk12 mpk4GC double-mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2 /bicarbonate invitro, suggesting that they are not direct CO2 /bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction.

Redox regulation of a guard cell SNF1-related protein kinase in Brassica napus, an oilseed crop.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn2+). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H2O2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H2O2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

Jasmonate-mediated stomatal closure under elevated CO2 revealed by time-resolved metabolomics.

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO2 ) levels are known to induce stomatal closure. However, the current knowledge on CO2 signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO2 using three hyphenated metabolomics platforms: gas chromatography-mass spectrometry (MS); liquid chromatography (LC)-multiple reaction monitoring-MS; and ultra-high-performance LC-quadrupole time-of-flight-MS. A total of 358 metabolites from guard cells were quantified in a time-course response to elevated CO2 level. Most metabolites increased under elevated CO2 , showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO2 treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO2 signaling mutants, we discovered that CO2 -induced stomatal closure is mediated by JA signaling.

MAPK Cascades in Guard Cell Signal Transduction.

Guard cells form stomata on the epidermis and continuously respond to endogenous and environmental stimuli to fine-tune the gas exchange and transpirational water loss, processes which involve mitogen-activated protein kinase (MAPK) cascades. MAPKs form three-tiered kinase cascades with MAPK kinases and MAPK kinase kinases, by which signals are transduced to the target proteins. MAPK cascade genes are highly conserved in all eukaryotes, and they play crucial roles in myriad developmental and physiological processes. MAPK cascades function during biotic and abiotic stress responses by linking extracellular signals received by receptors to cytosolic events and gene expression. In this review, we highlight recent findings and insights into MAPK-mediated guard cell signaling, including the specificity of MAPK cascades and the remaining questions.

Regulated deficit irrigation for crop production under drought stress. A review

Agriculture consumes more than two thirds of the total freshwater of the planet. This issue causes substantial conflict in freshwater allocation between agriculture and other economic sectors. Regulated deficit irrigation (RDI) is key technology because it helps to improve water use efficiency. Nonetheless, there is a lack of understanding of the mechanisms with which plants respond to RDI. In particular, little is known about how RDI might increase crop production while reducing the amount of irrigation water in real-world agriculture. In this review, we found that RDI is largely implemented through three approaches: (1) growth stage-based deficit irrigation, (2) partial root-zone irrigation, and (3) subsurface dripper irrigation. Among these, partial root-zone irrigation is the most popular and effective because many field crops and some woody crops can save irrigation water up to 20 to 30 % without or with a minimal impact on crop yield. Improved water use efficiency with RDI is mainly due to the following: (1) enhanced guard cell signal transduction network that decreases transpiration water loss, (2) optimized stomatal control that improves the photosynthesis to transpiration ratio, and (3) decreased evaporative surface areas with partial root-zone irrigation that reduces soil evaporation. The mechanisms involved in the plant response to RDI-induced water stress include the morphological traits, e.g., increased root to shoot ratio and improved nutrient uptake and recovery; physiological traits, e.g., stomatal closure, decreased leaf respiration, and maintained photosynthesis; and biochemical traits, e.g., increased signaling molecules and enhanced antioxidation enzymatic activity.

Multi-level modeling of light-induced stomatal opening offers new insights into its regulation by drought.

Plant guard cells gate CO2 uptake and transpirational water loss through stomatal pores. As a result of decades of experimental investigation, there is an abundance of information on the involvement of specific proteins and secondary messengers in the regulation of stomatal movements and on the pairwise relationships between guard cell components. We constructed a multi-level dynamic model of guard cell signal transduction during light-induced stomatal opening and of the effect of the plant hormone abscisic acid (ABA) on this process. The model integrates into a coherent network the direct and indirect biological evidence regarding the regulation of seventy components implicated in stomatal opening. Analysis of this signal transduction network identified robust cross-talk between blue light and ABA, in which [Ca2+]c plays a key role, and indicated an absence of cross-talk between red light and ABA. The dynamic model captured more than 1031 distinct states for the system and yielded outcomes that were in qualitative agreement with a wide variety of previous experimental results. We obtained novel model predictions by simulating single component knockout phenotypes. We found that under white light or blue light, over 60%, and under red light, over 90% of all simulated knockouts had similar opening responses as wild type, showing that the system is robust against single node loss. The model revealed an open question concerning the effect of ABA on red light-induced stomatal opening. We experimentally showed that ABA is able to inhibit red light-induced stomatal opening, and our model offers possible hypotheses for the underlying mechanism, which point to potential future experiments. Our modelling methodology combines simplicity and flexibility with dynamic richness, making it well suited for a wide class of biological regulatory systems.

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.

A role for ABA in the plants response to chitosan

A role for ABA in the plants response to chitosan

Nitric oxide in guard cells as an important secondary messenger during stomatal closure.

The modulation of guard cell function is the basis of stomatal closure, essential for optimizing water use and CO2 uptake by leaves. Nitric oxide (NO) in guard cells plays a very important role as a secondary messenger during stomatal closure induced by effectors, including hormones. For example, exposure to abscisic acid (ABA) triggers a marked increase in NO of guard cells, well before stomatal closure. In guard cells of multiple species, like Arabidopsis, Vicia and pea, exposure to ABA or methyl jasmonate or even microbial elicitors (e.g., chitosan) induces production of NO as well as reactive oxygen species (ROS). The role of NO in stomatal closure has been confirmed by using NO donors (e.g., SNP) and NO scavengers (like cPTIO) and inhibitors of NOS (L-NAME) or NR (tungstate). Two enzymes: a L-NAME-sensitive, nitric oxide synthase (NOS)-like enzyme and a tungstate-sensitive nitrate reductase (NR), can mediate ABA-induced NO rise in guard cells. However, the existence of true NOS in plant tissues and its role in guard cell NO-production are still a matter of intense debate. Guard cell signal transduction leading to stomatal closure involves the participation of several components, besides NO, such as cytosolic pH, ROS, free Ca2+, and phospholipids. Use of fluorescent dyes has revealed that the rise in NO of guard cells occurs after the increase in cytoplasmic pH and ROS. The rise in NO causes an elevation in cytosolic free Ca2+ and promotes the efflux of cations as well as anions from guard cells. Stomatal guard cells have become a model system to study the signaling cascade mechanisms in plants, particularly with NO as a dominant component. The interrelationships and interactions of NO with cytosolic pH, ROS, and free Ca2+ are quite complex and need further detailed examination. While assessing critically the available literature, the present review projects possible areas of further work related to NO-action in stomatal guard cells.

The stomata frontline of plant interaction with the environment-perspectives from hormone regulation

Plants have evolved elaborate mechanisms to perceive and integrate signals from various environmental conditions. On leaf surface, stomata formed by pairs of guard cells mediate gas exchange, water transpiration as well as function in response to abiotic and biotic stresses. Stomatal closure could be induced by drought, salt, pathogen and other adverse conditions. This constitutes an instant defense response to prevent further damage to plants. Abscisic acid (ABA) is a major plant hormone involved in stress responses. Stress-activated ABA synthesis causes stomatal closure and prevents opening to reduce water loss and cell dehydration. Key regulatory receptor complex and other important components in the ABA signaling pathway have been identified. However, our knowledge of ABA signal transduction in guard cells is far from complete. Jasmonates are a group of phytohormones generally known to be important for plant defense against insects and necrotrophic pathogens. The increased levels of methyl jasmonate (MeJA) induced by herbivory and pathogen invasion show a similar effect on stomatal movement associated with ROS production as ABA. Investigation of guard cell signaling networks involving the two important phytohormones is significant and exciting. Information about protein and metabolite components and how they interact in guard cells is lacking. Here we review recent advances on hormone signaling networks in guard cells and how the networks integrate environmental signals to plant physiological output.

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.

Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca 2+ affinities

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca(2+)-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca(2+)-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca(2+)-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.

The Clickable Guard Cell, Version II: Interactive Model of Guard Cell Signal Transduction Mechanisms and Pathways.

Guard cells are located in the leaf epidermis and pairs of guard cells surround and form stomatal pores, which regulate CO(2) influx from the atmosphere into leaves for photosynthetic carbon fixation. Stomatal guard cells also regulate water loss of plants via transpiration to the atmosphere. Signal transduction mechanisms in guard cells integrate a multitude of different stimuli to modulate stomatal apertures. Stomata open in response to light. Stomata close in response to drought stress, elevated CO(2), ozone and low humidity. In response to drought, plants synthesize the hormone abscisic acid (ABA) that triggers closing of stomatal pores. Guard cells have become a highly developed model system for dissecting signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single cell. Many new findings have been made in the last few years. This chapter is an update of an electronic interactive chapter in the previous edition of The Arabidopsis Book (Mäser et al. 2003). Here we focus on mechanisms for which genes and mutations have been characterized, including signaling components for which there is substantial signaling, biochemical and genetic evidence. Ion channels have been shown to represent targets of early signal transduction mechanisms and provide functional signaling and quantitative analysis points to determine where and how mutations affect branches within the guard cell signaling network. Although a substantial number of genes and proteins that function in guard cell signaling have been identified in recent years, there are many more left to be identified and the protein-protein interactions within this network will be an important subject of future research. A fully interactive clickable electronic version of this publication can be accessed at the following web site: http://www-biology.ucsd.edu/labs/schroeder/clickablegc2/. The interactive clickable version includes the following features: Figure 1. Model for the roles of ion channels in ABA signaling.Figure 2. Blue light signaling pathways in guard cells.Figure 3. ABA signaling pathways in guard cells.Figure 1 is linked to explanations that appear upon mouse-over. Figure 2 and Figure 3 are clickable and linked to info boxes, which in turn are linked to TAIR, to relevant abstracts in PubMed, and to updated background explanations from Schroeder et al (2001), used with permission of Annual Reviews of Plant Biology.

Roles of ion channels and transporters in guard cell signal transduction

Stomatal complexes consist of pairs of guard cells and the pore they enclose. Reversible changes in guard cell volume alter the aperture of the pore and provide the major regulatory mechanism for control of gas exchange between the plant and the environment. Stomatal movement is facilitated by the activity of ion channels and ion transporters found in the plasma membrane and vacuolar membrane of guard cells. Progress in recent years has elucidated the molecular identities of many guard cell transport proteins, and described their modulation by various cellular signal transduction components during stomatal opening and closure prompted by environmental and endogenous stimuli.

The ATP Binding Cassette Transporter AtMRP5 Modulates Anion and Calcium Channel Activities in Arabidopsis Guard Cells

Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.

Predicting Essential Components of Signal Transduction Networks: A Dynamic Model of Guard Cell Abscisic Acid Signaling

Plants both lose water and take in carbon dioxide through microscopic stomatal pores, each of which is regulated by a surrounding pair of guard cells. During drought, the plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure, thereby promoting water conservation. Dozens of cellular components have been identified to function in ABA regulation of guard cell volume and thus of stomatal aperture, but a dynamic description is still not available for this complex process. Here we synthesize experimental results into a consistent guard cell signal transduction network for ABA-induced stomatal closure, and develop a dynamic model of this process. Our model captures the regulation of more than 40 identified network components, and accords well with previous experimental results at both the pathway and whole-cell physiological level. By simulating gene disruptions and pharmacological interventions we find that the network is robust against a significant fraction of possible perturbations. Our analysis reveals the novel predictions that the disruption of membrane depolarizability, anion efflux, actin cytoskeleton reorganization, cytosolic pH increase, the phosphatidic acid pathway, or K+ efflux through slowly activating K+ channels at the plasma membrane lead to the strongest reduction in ABA responsiveness. Initial experimental analysis assessing ABA-induced stomatal closure in the presence of cytosolic pH clamp imposed by the weak acid butyrate is consistent with model prediction. Simulations of stomatal response as derived from our model provide an efficient tool for the identification of candidate manipulations that have the best chance of conferring increased drought stress tolerance and for the prioritization of future wet bench analyses. Our method can be readily applied to other biological signaling networks to identify key regulatory components in systems where quantitative information is limited.