Role In Stomatal Closure Research Articles

Gaining or cutting SLAC: the evolution of plant guard cell signalling pathways.

The evolution of adjustable stomatal pores, enabling CO2 acquisition, was one of the most significant events in the development of life on land. Here, we investigate how the guard cell signalling pathways that regulate stomatal movements evolved. We compare fern and angiosperm guard cell transcriptomes and physiological responses, and examine the functionality of ion channels from diverse plant species. We find that, despite conserved expression in guard cells, fern anion channels from the SLAC/SLAH family are not activated by the same abscisic acid (ABA) pathways that provoke stomatal closure in angiosperms. Accordingly, we find an insensitivity of fern stomata to ABA. Moreover, our analysis points to a complex evolutionary history, featuring multiple gains and/or losses of SLAC activation mechanisms, as these channels were recruited to a role in stomatal closure. Our results show that the guard cells of flowering and nonflowering plants share similar core features, with lineage-specific and ecological niche-related adaptations, likely underlying differences in behaviour.

Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure.

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor.

Stomata are epidermal pores that facilitate plant gas exchange. Grasses have fast stomatal movements, likely due to their dumbbell-shaped guard cells and lateral subsidiary cells. Subsidiary cells reciprocally exchange water and ions with guard cells. However, the relative contribution of subsidiary cells during stomatal closure is unresolved. We compared stomatal gas exchange and stomatal aperture dynamics in wild-type and pan1, pan2, and pan1;pan2 Zea mays (L.) (maize) mutants, which have varying percentages of aberrantly formed subsidiary cells. Stomata with 1 or 2 defective subsidiary cells cannot close properly, indicating that subsidiary cells are essential for stomatal function. Even though the percentage of aberrant stomata is similar in pan1 and pan2, pan2 showed a more severe defect in stomatal closure. In pan1, only stomata with abnormal subsidiary cells fail to close normally. In pan2, all stomata have stomatal closure defects, indicating that PAN2 has an additional role in stomatal closure. Maize Pan2 is orthologous to Arabidopsis GUARD CELL HYDROGEN PEROXIDE-RESISANT1 (GHR1), which is also required for stomatal closure. PAN2 acts downstream of Ca2+ in maize to promote stomatal closure. This is in contrast to GHR1, which acts upstream of Ca2+ , and suggests the pathways could be differently wired.

A New Player in Jasmonate-Mediated Stomatal Closure: The Arabidopsis thaliana Copper Amine Oxidase β.

Plant defence responses to adverse environmental conditions include different stress signalling, allowing plant acclimation and survival. Among these responses one of the most common, immediate, and effective is the modulation of the stomatal aperture, which integrates different transduction pathways involving hydrogen peroxide (H2O2), calcium (Ca2+), nitric oxide (NO), phytohormones and other signalling components. The Arabidopsis thaliana copper amine oxidases β (AtCuAOβ) encodes an apoplastic CuAO expressed in guard cells and root protoxylem tissues which oxidizes polyamines to aminoaldehydes with the production of H2O2 and ammonia. Here, its role in stomatal closure, signalled by the wound-associated phytohormone methyl-jasmonate (MeJA) was explored by pharmacological and genetic approaches. Obtained data show that AtCuAOβ tissue-specific expression is induced by MeJA, especially in stomata guard cells. Interestingly, two Atcuaoβ T-DNA insertional mutants are unresponsive to this hormone, showing a compromised MeJA-mediated stomatal closure compared to the wild-type (WT) plants. Coherently, Atcuaoβ mutants also show compromised H2O2-production in guard cells upon MeJA treatment. Furthermore, the H2O2 scavenger N,N1-dimethylthiourea (DMTU) and the CuAO-specific inhibitor 2-bromoethylamine (2-BrEtA) both reversed the MeJA-induced stomatal closure and the H2O2 production in WT plants. Our data suggest that AtCuAOβ is involved in the H2O2 production implicated in MeJA-induced stomatal closure.

Two calcium-dependent protein kinases enhance maize drought tolerance by activating anion channel ZmSLAC1 in guard cells.

SummaryStomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S‐type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S‐type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca2+‐dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought‐sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA‐activated S‐type anion currents in maize guard cells, while the S‐type anion currents were increased in the guard cells of ZmCPK35‐ and ZmCPK37‐overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1‐mediated Cl‐ and NO3 ‐ currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.

The role of gasotransmitters in movement of stomata: mechanisms of action and importance for plant immunity

Stomatal guard cells are specialized epidermal cells regulating gas exchange. The ability to open or close in response to external and internal cues makes stomata a dynamic and fascinating system. Stomatal closure upon infection ensures restriction of pathogen entry into the plant and forms an essential component of innate immunity. The opening or closure of stomata is dependent on the turgidity or flaccidity of guard cells, respectively, facilitated by several signaling components, including reactive oxygen species, nitric oxide (NO) and Ca2+. Among these, NO is the most extensively studied gasotransmitter. Its pivotal role in stomatal closure by modulating various downstream components as well as regulation of crucial proteins by post-translational modifications makes NO an essential factor. Two more gasotransmitters, carbon monoxide and hydrogen sulfide, also trigger stomatal closure. Other gaseous molecules, like ethylene, methane, sulfur dioxide, ozone, and CO2, can modulate stomatal closure, but they are not considered strictly as gasotransmitters due to specific criteria. We review the signaling events in guard cells triggered by these gasotransmitters leading to stomatal closure. We point out the dual role of NO to promote stomatal closure and stomatal opening. Both NO and H2S help in reinforcing the innate immunity against pathogen attack. Although there is extensive information on the mechanism of NO action on stomata, the enzymatic source of NO or CO is still ambiguous. Similarly, research is warranted to establish the relative importance of and interactions among the three main gasotransmitters. Further studies on gasotransmitters would answer the ambiguity about their functions and confirm if they can act independently.

MEDEA-interacting protein LONG-CHAIN BASE KINASE 1 promotes pattern-triggered immunity in Arabidopsis thaliana.

Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

Metabolic shifts associated with drought-induced senescence in Brachypodium

The metabolic underpinnings of plant survival under severe drought-induced senescence conditions are poorly understood. In this study, we assessed the morphological, physiological and metabolic responses to sustained water deficit in Brachypodium distachyon, a model organism for research on temperate grasses. Relative to control plants, fresh biomass, leaf water potential, and chlorophyll levels decreased rapidly in plants grown under drought conditions, demonstrating an early onset of senescence. The leaf C/N ratio and protein content showed an increase in plants subjected to drought stress. The concentrations of several small molecule carbohydrates and amino acid-derived metabolites previously implicated in osmotic protection increased rapidly in plants experiencing water deficit. Malic acid, a low molecular weight organic acid with demonstrated roles in stomatal closure, also increased rapidly as a response to drought treatment. The concentrations of prenyl lipids, such as phytol and α-tocopherol, increased early during the drought treatment but then dropped dramatically. Surprisingly, continued changes in the quantities of metabolites were observed, even in samples harvested from visibly senesced plants. The data presented here provide insights into the processes underlying persistent metabolic activity during sustained water deficit and can aid in identifying mechanisms of drought tolerance in plants.

MtABCG20 is an ABA exporter influencing root morphology and seed germination of Medicago truncatula.

Summary Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non‐dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP‐binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl–radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild‐type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.

The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure.

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high-throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non-redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image-based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.

Hydraulics Regulate Stomatal Responses to Changes in Leaf Water Status in the Fern Athyrium filix-femina.

Stomatal responses to changes in leaf water status are important for the diurnal regulation of gas exchange and the survival of plants during drought. These stomatal responses in angiosperm species are well characterized, yet in species of nonseed plants, an ongoing debate surrounds the role of metabolism, particularly the role of the hormone abscisic acid (ABA), in functionally regulating stomatal responses to changes in leaf water status. Here, we measured the stomatal response to changes in vapor pressure difference (VPD) in two natural forms of the fern species Athyrium filix-femina, recently suggested to have stomata that are regulated by ABA. The two forms measured had considerable differences in key hydraulic traits, including leaf hydraulic conductance and capacitance, as well as the kinetics of stomatal response to changes in VPD. In both forms, the stomatal responses to VPD could be accurately predicted by a dynamic, mechanistic model that assumes guard cell turgor changes in concert with leaf turgor in the light, and not via metabolic processes including the level of ABA. During drought, endogenous ABA did not play a role in stomatal closure, and exogenous ABA applied to live, intact leaves did not induce stomatal closure. Our results indicate that functional stomatal responses to changes in leaf water status in ferns are regulated by leaf hydraulics and not metabolism. With ferns being sister to seed plants, this result has implications for the evolutionary reconstruction of functional stomatal responses across vascular land plant lineages.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

Iron plays a critical role in stomatal closure in cauliflower

Studies have been made of the effects of iron deficiency on water status and transpiration parameters in iron-deficient plants. Cauliflower (Brassica oleracea L. var Botrytis cv Pusa) plants were grown in sand culture with 0.1mM (control) and 0.001mM (deficient) iron supply. Young leaves of Fe-deficient plants showing mild interveinal chlorosis were thin and soft with lesser epicuticular wax than the green leaves of Fe-sufficient plants. Fe deficiency induced increases in specific water content, water potential, stomatal aperture and transpiration and a decrease in diffusive resistance in the young leaves. Although Fe deficiency enhanced stomatal and cuticular transpiration, the greater stomatal opening was mainly responsible for increased transpiration. Fe deficiency stifled diurnal changes in diffusive resistance, ABA accumulation and guard cell K+ content in the leaves. Though Fe deficiency did not affect maximum stomatal opening under favourable conditions, it retarded stomatal closure under conditions of developing water deficit or decreasing light intensity. Higher rates of transpirational water loss in Fe-deficient leaves are appearing to be related to the slow and incomplete stomatal closure, which are attributable to the putative involvement of Fe in the biosyntheses of wax and ABA in Fe-deficient leaves.

WD40-REPEAT 5a functions in drought stress tolerance by regulating nitric oxide accumulation in Arabidopsis.

Nitric oxide (NO) generation by NO synthase (NOS) in guard cells plays a vital role in stomatal closure for adaptive plant response to drought stress. However, the mechanism underlying the regulation of NOS activity in plants is unclear. Here, by screening yeast deletion mutants with decreased NO accumulation and NOS-like activity when subjected to H2 O2 stress, we identified TUP1 as a novel regulator of NOS-like activity in yeast. Arabidopsis WD40-REPEAT 5a (WDR5a), a homolog of yeast TUP1, complemented H2 O2 -induced NO accumulation of a yeast mutant Δtup1, suggesting the conserved role of WDR5a in regulating NO accumulation and NOS-like activity. This note was further confirmed by using an Arabidopsis RNAi line wdr5a-1 and two T-DNA insertion mutants of WDR5a with reduced WDR5a expression, in which both H2 O2 -induced NO accumulation and stomatal closure were repressed. This was because H2 O2 -induced NOS-like activity was inhibited in the mutants compared with that of the wild type. Furthermore, these wdr5a mutants were more sensitive to drought stress as they had reduced stomatal closure and decreased expression of drought-related genes. Together, our results revealed that WDR5a functions as a novel factor to modulate NOS-like activity for changes of NO accumulation and stomatal closure in drought stress tolerance.

Elevated Carbon Dioxide Induces Stomatal Closure of Arabidopsis thaliana (L.) Heynh. Through an Increased Production of Nitric Oxide

Elevated carbon dioxide (CO2) has been shown to enhance the photosynthesis of plants, while inducing stomatal closure in leaves. Stomatal closure is always a key issue regarding plants’ responses against stress under elevated CO2. This paper examined the mechanism underlying CO2 elevation-induced stomatal closure in Arabidopsis. Endogenous nitric oxide (NO) in guard cells was detected by the specific probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. The Arabidopsis wild type, nitric oxide synthase (NOS) mutant atnos1, and nitrate reductase (NR) mutant nia1nia2 were used as experimental materials. The present study shows that elevated CO2-induced NO accumulation in guard cells by 120 % and decreased the aperture of stomata by 32 % compared with ambient CO2. CO2 elevation-induced stomatal closure was reversed by scavenging NO. Here, we suggested that NO plays an important role in stomatal closure of Arabidopsis induced by elevated CO2. The pharmacological and genetic evidence shows that both the NOS inhibitor L-NAME and NR inhibitor tungstate significantly decreased NO accumulation in guard cells and inhibited stomatal closure, whereas the stomatal aperture of both the atnos1 and nia1nia2 mutants decreased slightly under elevated CO2, indicated that both NOS-like and NR were involved in CO2 elevation-induced NO accumulation. Therefore, we conclude from these findings that elevated CO2 increases the level of NO through both NOS-like and NR, which then induces stomatal closure in Arabidopsis under elevated CO2.

Lipoxygenase 2 functions in exogenous nitric oxide-induced stomatal closure in Arabidopsis thaliana.

Nitric oxide (NO) and lipoxygenase (LOX)-derived oxylipins play important roles in stomatal closure in plants, and LOX-NO crosstalk has been indicated in mesophyll cells. However, whether the crosstalk also exists in guard cells is not clear and the detailed mechanisms remain unknown. Here, we report that exogenous sodium nitroprusside (SNP, a NO donor)-induced stomatal closure was clearly impaired in the AtLOX2 null mutant lox2-1 compared with wild-type (WT) Arabidopsis thaliana (L.) Heynh. Patch clamp analysis showed that the SNP-suppressed activity of inward-rectifying potassium channels in lox2-1 guard cell protoplasts was reduced. Moreover, SNP promoted an increase in cytosolic Ca2+ concentration in guard cells of lox2-1 mutants was inhibited compared with the WT. These results suggest that AtLOX2 plays an important role in NO-induced stomatal closure by affecting the cytosolic Ca2+ concentration increase and the activity of inward-rectifying potassium channels in guard cells. Furthermore, lox2-1 mutants showed a higher rate of leaf water loss and a relatively wider stomatal aperture than the WT under normal growth conditions. These data imply that AtLOX2 might modulate stomatal movement by increasing oxylipin generation in A. thaliana.

Hypoxia induces stem and leaf nitric oxide (NO) emission from poplar seedlings.

Hypoxia leads to NO formation in poplar roots. Additionally, either NO or a NO derivative is transported from the roots to the shoot causing NO emission from aboveground plant organs. Nitric oxide (NO) is involved in the response of plants to various forms of stress including hypoxia. It also seems to play an important role in stomatal closure during stress exposure. In this study, we investigated the formation of NO in roots of intact poplar (Populus×canescens) plants in response to hypoxia, as well as its dependence on nitrate availability. We further addressed the question if root hypoxia triggers NO emission from aboveground plant parts, i.e., stems and leaves of young poplar trees. Our results indicate that NO is formed in poplar roots in response to hypoxia and that this production depends on the availability of nitrate and its conversion product nitrite. As long as nitrate was available in the nutrient solution, NO emission of roots occurred; in the range of the nitrate concentrations (10-100µM) tested, NO emission was widely independent on nitrate concentration. However, the time period in which NO was emitted and the total amount of NO emitted strongly depended on the nitrate concentration of the solution. Hypoxia also led to increased NO emissions from the leaves and stems of the trees. There was a tight correlation between leaf and stem NO emission of hypoxia-treated plants. We propose that NO is produced by nitrate reductase in the roots and either NO itself, a metabolic NO precursor, or a NO derivative is transported in the xylem sap of the trees from the roots to the shoot thereby mediating NO emission from aboveground parts of the plant.

ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity.

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1-mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA-dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA-dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahGsiz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA-responsive genes, such as PR1 and PR2. Furthermore, other SA-accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahGsiz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.

Role of polyamines and phospholipase D in maize (Zea mays L.) response to drought stress

An in vitro experiment was conducted to elucidate the role of polyamines and phospholipase D (PLD) in maize (Zea mays L.) response to drought stress simulated by PEG-6000. During the early stage of drought stress, an increase of PLD activity played a major role in stomatal closure as an adaptive response to drought stress and this process was independent of polyamine synthesis. Under prolonged drought stress, the total content of polyamines including putrescine (Put), spermidine (Spd) and spermine (Spm) increased at 120min after drought stress treatment while the ratio of Spd+Spm/Put decreased, which caused an increase in PLD activity, relative membrane permeability and malondialdehyde content (MDA). Exogenous polyamines enhanced PLD activity, and the greatest effect was induced by Put. Therefore, we concluded that PLD played an important role in mitigation of drought stress damage in the early stage of drought stress which was independent of polyamine accumulation. However, in the later stage of drought stress, the obvious elevation of PLD activity led to serious membrane damage regulated by the ratio of Spd+Spm/Put.