Plasma Membrane Of Guard Cells Research Articles

A novel semi-dominant mutation in brassinosteroid signaling kinase1 increases stomatal density.

Stomata play a pivotal role in balancing CO2 uptake for photosynthesis and water loss via transpiration. Thus, appropriate regulation of stomatal movement and its formation are crucial for plant growth and survival. Red and blue light induce phosphorylation of the C-terminal residue of the plasma membrane (PM) H+-ATPase, threonine, in guard cells, generating the driving force for stomatal opening. While significant progress has been made in understanding the regulatory mechanism of PM H+-ATPase in guard cells, the regulatory components for the phosphorylation of PM H+-ATPase have not been fully elucidated. Recently, we established a new immunohistochemical technique for detecting guard-cell PM H+-ATPase phosphorylation using leaves, which was expected to facilitate investigations with a single leaf. In this study, we applied the technique to genetic screening experiment to explore novel regulators for the phosphorylation of PM H+-ATPase in guard cells, as well as stomatal development. We successfully performed phenotyping using a single leaf. During the experiment, we identified a mutant exhibiting high stomatal density, jozetsu (jzt), named after a Japanese word meaning 'talkative'. We found that a novel semi-dominant mutation in BRASSINOSTEROID SIGNALING KINASE1 (BSK1) is responsible for the phenotype in jzt mutant. The present results demonstrate that the new immunohistochemical technique has a wide range of applications, and the novel mutation would provide genetic tool to expand our understanding of plant development mediated by brassinosteroid signaling.

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

PH-dependent redox properties of epigallocatechin gallate (EGCG) and its effect on respiration, photosynthesis and cell death in pea

The redox properties of the green tea component epigallocatechin gallate (EGCG) in vitro were studied and its effect on plant (pea) cells was tested. EGCG exhibited both pro- and antioxidant properties. In solutions, EGCG was oxidized by oxygen at physiological (slightly alkaline) pH values. Lowering the pH slowed down this process. Oxidation of EGCG was accompanied by the formation of O2-• and H2O2. In addition, EGCG functioned as an electron donor for peroxidase and, in combination with it, utilized H2O2. Exposure of EGCG to pea cells (leaf cuts or epidermis) suppressed respiration, reduced the transmembrane electric potential difference (Δψ) in mitochondria, and inhibited electron transfer in the photosynthetic electron transfer chain. Among the sites of the photosynthetic redox chain, Photosystem II was the least sensitive to the action of EGCG. EGCG reduced the rate of formation of reactive oxygen species in the epidermis, which was induced by NADH treatment and was registered using 2′,7′-dichlorofluorescine diacetate. In vivo, EGCG at concentrations from 10 µM to 1 mM suppressed KCN-induced death of guard cells in the epidermis, which was determined from the destruction of cell nuclei. EGCG at a concentration of 10 mM disrupted the barrier function of the guard cell plasma membrane, increasing its permeability to propidium iodide.

Epigallocatechin Gallate: pH-Dependent Redox Properties and Effect on Respiration, Photosynthesis, and Cell Death in Pea Plants.

In vitro redox properties of the green tea component epigallocatechin gallate (EGCG) and its effect on pea plant cells were investigated. EGCG was found to exhibit both pro- and antioxidant properties. In solutions, EGCG was oxidized by oxygen at physiological (slightly alkaline) pH values with the generation of O2-• and H2O2, the reaction being slowed down by a decrease in the medium pH. On the other hand, EGCG functioned as an electron donor for peroxidase, resulting in the H2O2 utilization. EGCG suppressed respiration, reduced mitochondrial transmembrane potential difference and inhibited electron transfer in the photosynthetic electron transport chain in pea leaf cells (leaf cuttings and epidermis). Among components of the photosynthetic redox chain, Photosystem II was the least sensitive to the EGCG action. In the epidermis, EGCG reduced the rate of reactive oxygen species formation that was induced by NADH. EGCG at the concentrations from 10 μM to 1 mM suppressed the KCN-induced death of guard cells in the epidermis, which was determined from the destruction of cell nuclei. EGCG at a concentration of 10 mM disrupted the barrier function of the guard cell plasma membrane, increasing its permeability to propidium iodide.

Local endocytosis of sucrose transporter 2 in duckweed reveals the role of sucrose transporter 2 in guard cells.

The local endocytosis of membrane proteins is critical for many physiological processes in plants, including the regulation of growth, development, nutrient absorption, and osmotic stress response. Much of our knowledge on the local endocytosis of plasma membrane (PM) protein only focuses on the polar growth of pollen tubes in plants and neuronal axon in animals. However, the role of local endocytosis of PM proteins in guard cells has not yet been researched. Here, we first cloned duckweed SUT2 (sucrose transporter 2) protein and then conducted subcellular and histological localization of the protein. Our results indicated that LpSUT2 (Landoltia punctata 0202 SUT2) is a PM protein highly expressed on guard cells. In vitro experiments on WT (wild type) lines treated with high sucrose concentration showed that the content of ROS (reactive oxygen species) in guard cells increased and stomatal conductance decreased. We observed the same results in the lines after overexpression of the LpSUT2 gene with newfound local endocytosis of LpSUT2. The local endocytosis mainly showed that LpSUT2 was uniformly distributed on the PM of guard cells in the early stage of development, and was only distributed in the endomembrane of guard cells in the mature stage. Therefore, we found the phenomenon of guard cell LpSUT2 local endocytosis through the changes of duckweed stomata and concluded that LpSUT2 local endocytosis might be dependent on ROS accumulation in the development of duckweed guard cells. This paper might provide future references for the genetic improvement and water-use efficiency in other crops.

Elevated CO2 induces rapid dephosphorylation of plasma membrane H+ -ATPase in guard cells.

Light induces stomatal opening, which is driven by plasma membrane (PM) H+ -ATPase in guard cells. The activation of guard-cell PM H+ -ATPase is mediated by phosphorylation of the penultimate C-terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard-cell PM H+ -ATPase in Arabidopsis thaliana. Immunodetection of guard-cell PM H+ -ATPase, time-resolved leaf gas-exchange analyses and stomatal aperture measurements were carried out. We found that light-dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post-transition. Gas-exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard-cell PM H+ -ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse-genetic analyses using guard-cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light-dark transition. Our results indicate that CO2 -mediated dephosphorylation of guard-cell PM H+ -ATPase underlies stomatal closure.

Multiple cyclic nucleotide-gated channels function as ABA-activated Ca2+ channels required for ABA-induced stomatal closure in Arabidopsis.

Abscisic acid (ABA)-activated inward Ca2+-permeable channels in the plasma membrane (PM) of guard cells are required for the initiation and regulation of ABA-specific cytosolic Ca2+ signaling and stomatal closure in plants. But the identities of the PM Ca2+ channels are still unknown. We hypothesized that the ABA-activated Ca2+ channels consist of multiple CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) proteins from the CNGC family, which is known as a Ca2+-permeable channel family in Arabidopsis (Arabidopsis thaliana). In this research, we observed high expression of multiple CNGC genes in Arabidopsis guard cells, namely CNGC5, CNGC6, CNGC9, and CNGC12. The T-DNA insertional loss-of-function quadruple mutant cngc5-1 cngc6-2 cngc9-1 cngc12-1 (hereafter c5/6/9/12) showed a strong ABA-insensitive phenotype of stomatal closure. Further analysis revealed that ABA-activated Ca2+ channel currents were impaired, and ABA-specific cytosolic Ca2+ oscillation patterns were disrupted in c5/6/9/12 guard cells compared with in wild-type guard cells. All ABA-related phenotypes of the c5/6/9/12 mutant were successfully rescued by the expression of a single gene out of the four CNGCs under the respective native promoter. Thus, our findings reveal a type of ABA-activated PM Ca2+ channel comprising multiple CNGCs, which is essential for ABA-specific Ca2+ signaling of guard cells and ABA-induced stomatal closure in Arabidopsis.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

Combined action of guard cell plasma membrane rapid- and slow-type anion channels in stomatal regulation

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and β-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

PAMP-induced peptide 1 cooperates with salicylic acid to regulate stomatal immunity in Arabidopsis thaliana

ABSTRACTVarious pathogenic species are capable of penetrating plant leaves through stomata on the leaf surface for propagation by absorbing nutrients in plant interiors. Plants have evolved abilities to close stomata to restrict pathogen infections. The model plant Arabidopsis (Arabidopsis thaliana) closes stomata when FLAGELLIN SENSING2 (FLS2), a receptor protein localized in the plasma membrane (PM) of stomatal guard cells, detects flagellin, a pathogen-associated molecular pattern (PAMP) derived from the bacterial pathogen Pseudomonas syringae. It currently remains largely unknown how flagellin-FLS2 signaling initiates stomatal closure. Our previous studies showed that PAMP-INDUCED PEPTIDE1 (PIP1), an Arabidopsis endogenous peptide, activates immune responses through a PM-localized receptor, RECEPTOR-LIKE KINASE7 (RLK7). Here, we demonstrate that PIP1-RLK7 act downstream of FLS2 to activate stomatal immunity against the bacterial strain Pseudomonas syringe pv. tomato (Pst) DC3118. PIP1 promotes the expression of genes involved in salicylic acid (SA) biosynthesis. SA contributes to the expression of PIP1 preligand prePIP1 and the PIP1-induced stomatal closure. In contrast, methl jasmonate (MJ) and a pathogen-derived jasmonate mimic coronatine (COR) performs an opposite function of SA. SA also promotes the PIP1-induced production of reactive oxygen species (ROS) which is required for PIP1-induced stomatal closure. Overall, PIP1 and SA may form a positive feedback loop to regulate ROS-mediated stomatal immunity in Arabidopsis.

Permeability of the Plasma Membrane for Propidium Iodide and Destruction of Cell Nuclei in the Epidermis of Pea Leaves: The Effect of Polyelectrolytes and Detergents

Damage to the plasma membrane (PM) of cells in pea leaf epidermis determined by its permeability to propidium iodide (PI), which binds to DNA of cell nuclei, and programmed cell death (PCD) detected by the destruction of cell nuclei were investigated. PM of the epidermal cells in the isolated epidermis was permeable to PI (it stained their nuclei). PM of the guard cells did not allow PI to pass through. KCN, an inducer of PCD, caused the destruction of both epidermal and guard cell nuclei. KCN-induced destruction of guard cell nuclei was accompanied by the penetration of PI into the cells. The polycation chitosan at a concentration of 0.1 mg/mL caused the destruction of the epidermal cell nuclei, but the permeability of the guard cell PM for PI staining their nuclei was induced at a concentration of 1 mg/mL. Other polycations (cytochrome c, polylysine, polyethylenimine, and protamine) also caused staining of the guard cell nuclei by PI. Polyanions (polyacrylic acid, dextran, and heparin) initiated the destruction of cell nuclei, which was accompanied by the penetration of PI into cells. Detergents Triton X-100 and lauryldimethylamine-N-oxide produced the permeability of the guard cell PM for PI and prevented the destruction of the nuclei that was induced by KCN. Treatment of the epidermis with Triton X-100 (for 2 h with its subsequent washing) increased the destruction of the guard cell nuclei that was caused by KCN. Polycations polyethyleneimine and protamine were prevented, while chitosan, cytochrome c, and polylysine, on the contrary, enhanced KCN-induced destruction of the guard cell nuclei. The obtained data shows that the destruction of cell nuclei upon induction of cell death with KCN or polyanions is accompanied by damage to PM (producing its permeability for PI). Damage to PM caused by detergents or polycations prior to cell treatment by KCN can prevent or, on the contrary, intensify the destruction of cell nuclei.

Electrophysiological Identification and Activity Analyses of Plasma Membrane K+ Channels in Maize Guard Cells.

Stomatal movement, which plays an essential role in plant transpiration and photosynthesis, is controlled by ion channels that mediate K+ and anion fluxes across the plasma membrane (PM) of guard cells. These channels in dicots are accurately regulated by various physiological factors, such as pH, abscisic acid (ABA) and Ca2+; however, the data in monocots are limited. Here the whole-cell patch-clamping technique was applied to analyze the properties and regulations of PM K+ channels in maize guard cells. The results indicated that the hyperpolarization-activated inward-rectifying channels were highly K+-selective. These inward K+ (Kin) channels were sensitive to extracellular K+. Their slope factor (S) decreased when the apoplastic K+ concentration decline, causing a positive shift of the half-activation potential (V1/2). Their activities were promoted by apoplastic acidification but inhibited by apoplastic and cytosolic alkalization. Nevertheless, the outward K+ (Kout) channel activities were uniquely promoted by cytosolic alkalization. Both apoplastic and cytosolic ABA inhibited Kin channels independent of cytosolic Ca2+ ([Ca2+]cyt). And two Ca2+-dependent mechanisms with different Ca2+ affinities may mediate resting- and high-[Ca2+]cyt-induced inhibition on Kin channels, respectively. However, resting [Ca2+]cyt impaired the inhibition of Kin channels induced by apoplastic ABA, not cytosolic ABA. Furthermore, the result that high [Ca2+]cyt attenuated ABA-induced inhibition highlighted the importance of [Ca2+]cyt for Kin channel regulation. There may exist a Ca2+-dependent regulation of the Ca2+-independent ABA signaling pathways for Kin channel inhibition. These results provided an electrophysiological view of the multiple level regulations of PM K+ channel activities and kinetics in maize guard cells.

Stomatal response to blue light in crassulacean acid metabolism plants Kalanchoe pinnata and Kalanchoe daigremontiana.

Blue light (BL) is a fundamental cue for stomatal opening in both C3 and C4 plants. However, it is unknown whether crassulacean acid metabolism (CAM) plants open their stomata in response to BL. We investigated stomatal BL responses in the obligate CAM plants Kalanchoe pinnata and Kalanchoe daigremontiana that characteristically open their stomata at night and close them for part of the day, as contrasted with C3 and C4 plants. Stomata opened in response to weak BL superimposed on background red light in both intact leaves and detached epidermal peels of K. pinnata and K. daigremontiana. BL-dependent stomatal opening was completely inhibited by tautomycin and vanadate, which repress type 1 protein phosphatase and plasma membrane H+-ATPase, respectively. The plasma membrane H+-ATPase activator fusicoccin induced stomatal opening in the dark. Both BL and fusicoccin induced phosphorylation of the guard cell plasma membrane H+-ATPase in K. pinnata. These results indicate that BL-dependent stomatal opening occurs in the obligate CAM plants K. pinnata and K. daigremontiana independently of photosynthetic CO2 assimilation mode.

Insights into ABA-mediated regulation of guard cell primary metabolism revealed by systems biology approaches

Despite the fact that guard cell abscisic acid (ABA) signalling pathway is well documented, our understanding concerning how and to which extent ABA regulates guard cell metabolism remains fragmentary. Here we have adopted different systems approaches to investigate how ABA modulates guard cell central metabolism by providing genes that are possibly ABA-regulated. By using previous published Arabidopsis guard cell transcript profiling data, we carried out an extensive co-expression network analysis using ABA-related genes and those related to the metabolism and transport of sugars, starch and organic acids. Next, we investigated the presence of ABA responsive elements (ABRE) in the promoter of genes that are highly expressed in guard cells, responsive to ABA and co-expressed with ABA-related genes. Together, these analyses indicated that 44 genes are likely regulated by ABA and 8 of them are highly expressed in guard cells in both the presence and absence of ABA, including genes of the tricarboxylic acid cycle and those related to sucrose and hexose transport and metabolism. It seems likely that ABA may modulate both sucrose transport through guard cell plasma membrane and sucrose metabolism within guard cells. In this context, genes associated with sucrose synthase, sucrose phosphate synthase, trehalose-6-phosphate, invertase, UDP-glucose epimerase/pyrophosphorylase and different sugar transporters contain ABRE in their promoter and are thus possibly ABA regulated. Although validation experiments are required, our study highlights the importance of systems biology approaches to drive new hypothesis and to unravel genes and pathways that are regulated by ABA in guard cells.

Identification of SLAC1 anion channel residues required for CO2/bicarbonate sensing and regulation of stomatal movements

Increases in CO2 concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO2] rise cause closing of stomatal pores, thus affecting plant-water relations globally. However, the underlying CO2/bicarbonate (CO2/HCO3-) sensing mechanisms remain unknown. [CO2] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO2/HCO3- regulation of SLAC1 is relevant for CO2 control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO2/HCO3- in Xenopus oocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO3- regulation of SLAC1. Notably, gas-exchange experiments with slac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO2 regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation of S-type anion channels by CO2/HCO3-, but not by ABA, was impaired, indicating the relevance of R256 for CO2 signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO2/HCO3- sensors in guard cells.

Hydrogen sulfide mediates ion fluxes inducing stomatal closure in response to drought stress in Arabidopsis thaliana

In many plant species, hydrogen sulfide (H2S) triggers stomatal closure, which is produced mainly by two classes of enzymes, cysteine desulfhydrases (CDes) and O-acetyl-L-serine (thiol) lyases (OASTLs). Stomatal movement is accompanied by several ion fluxes across the plasma membranes of guard cells. In this paper, we detected the fluxes of H+, Ca2+, K+ and Cl− in guard cells of wild-type Arabidopsis thaliana and the mutants associated with H2S production (lcd, OE-LCD, des, OE-DES, oastl-a1, oastl-a2, oastl-b and oastl-c), using a non-invasive micro-test technique. The results showed that endogenous H2S induced a transmembrane K+ efflux, and Ca2+ and Cl− influxes, while not affecting the flow of H+. Furthermore, the K+ channel was the main osmolyte responder during the regulation of stomatal movement by H2S in response to drought stress. Finally, the two classes of enzymes produced H2S, CDes and OASTLs, played different roles in regulating stomatal movements. Thus, H2S mediates ion fluxes inducing stomatal closure in response to drought stress in Arabidopsis thaliana.

The Membrane Transport System of the Guard Cell and Its Integration for Stomatal Dynamics.

Stomatal guard cells are widely recognized as the premier plant cell model for membrane transport, signaling, and homeostasis. This recognition is rooted in half a century of research into ion transport across the plasma and vacuolar membranes of guard cells that drive stomatal movements and the signaling mechanisms that regulate them. Stomatal guard cells surround pores in the epidermis of plant leaves, controlling the aperture of the pore to balance CO2 entry into the leaf for photosynthesis with water loss via transpiration. The position of guard cells in the epidermis is ideally suited for cellular and subcellular research, and their sensitivity to endogenous signals and environmental stimuli makes them a primary target for physiological studies. Stomata underpin the challenges of water availability and crop production that are expected to unfold over the next 20 to 30 years. A quantitative understanding of how ion transport is integrated and controlled is key to meeting these challenges and to engineering guard cells for improved water use efficiency and agricultural yields.

Guard cell-specific down-regulation of the sucrose transporter SUT1 leads to improved water use efficiency and reveals the interplay between carbohydrate metabolism and K+ accumulation in the regulation of stomatal opening

Sucrose is a key metabolite in guard cell (GC) regulation and has been proposed to form a key metabolic connection between mesophyll cells and GCs. However, the importance of sucrose transport across the GC plasma membrane in stomatal movements remains to be determined. Here, we characterized transgenic tobacco plants expressing an antisense gene construct for the SUCROSE TRANSPORTER 1 (SUT1) driven by the GC specific promoter KST1. GCs of transgenic plants contained less sucrose, more starch and had increases in K+ content. Lower GC sucrose content was associated with lower stomatal conductance that, in turn, led to a lower transpiration rate and higher carbon isotope composition ratio compared to wild type plants. Although the transgenic plants showed reduced growth under optimal water supply condition, they showed improved growth compared to wild type plants under osmotic stress and demonstrated lower water consumption that was correlated with higher relative water content under water-deficit conditions. This demonstrates the desiccation postponement phenotype of these plants together with an improved tolerance to water deficit. Taken together, our results indicate that SUT1 has an important role in stomatal movements and suggest that genetic manipulation of GC sucrose transport could be used to obtain plants with greater water use efficiency.