保卫细胞的ABA信号转导

植物激素脱落酸(ABA)调节植物体多种生理过程,尤其在一些逆境条件下,植物体中ABA大量合成,诱导气孔关闭,从而有效地调控植物体内的水分平衡.尽管人们对ABA诱导气孔关闭作用已得到共识,但有关信号转导的细节还很不清楚.该文简要介绍了研究气孔保卫细胞信号转导途径的相关技术以及与ABA信号转导直接相关的ABA受体、第二信使、蛋白质磷酸化和离子通道调节等方面的最新妍究进展.并在前人研究工作的基础上,勾画出气孔保卫细胞ABA、H2O2的信号转导模式图.     

Early ABA Signaling Events in Guard Cells

The plant hormone abscisic acid (ABA) regulates a wide variety of plant physiological and developmental processes, particularly responses to environmental stress, such as drought. In response to water deficiency, plants redistribute foliar ABA and/or upregulate ABA synthesis in roots, leading to roughly a 30-fold increase in ABA concentration in the apoplast of stomatal guard cells. The elevated ABA triggers a chain of events in guard cells, causing stomatal closure and thus preventing water loss. Although the molecular nature of ABA receptor(s) remains unknown, considerable progress in the identification and characterization of its downstream signaling elements has been made by using combined physiological, biochemical, biophysical, molecular, and genetic approaches. The measurable events associated with ABA-induced stomatal closure in guard cells include, sequentially, the production of reactive oxygen species (ROS), increases in cytosolic free Ca²⁺ levels ([Ca²⁺]_i), activation of anion channels, membrane potential depolarization, cytosolic alkalinization, inhibition of K⁺ influx channels, and promotion of K⁺ efflux channels. This review provides an overview of the cellular and molecular mechanisms underlying these ABA-evoked signaling events, with particular emphasis on how ABA triggers an “electronic circuitry” involving these ionic components.     

Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging

Guard cells, which form stomata on the leaf epidermis, play important roles in plant gas exchange and defense against pathogens. Abscisic acid (ABA) is a phytohormone that can be induced by drought and leads to stomatal closure. Guard cells have been a premier model system for studying ABA signal transduction. Despite significant progress on the identification of molecular components in the ABA signaling pathway, our knowledge of the protein components is very limited. Here, we employ a recently developed multiplexed isobaric tagging technology to identify ABA-responsive proteins in Brassica napus guard cells. A total of 431 unique proteins were identified with relative quantitative information in control and ABA-treated samples. Proteins involved in stress and defense constituted a major group among the 66 proteins with increased abundance. Thirty-eight proteins were decreased in abundance and fell into several functional groups including metabolism and protein synthesis. Many of the proteins have not been reported as being ABA responsive or involved in stomatal movement. A large percentage of the protein-coding genes contained ABA-responsive elements. This study not only established a comprehensive inventory of ABA-responsive proteins, but also identified new proteins for further investigation of their functions in guard cell ABA signaling.     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis

Cytokinins and auxins are major phytohormones involved in various aspects of plant growth and development. These phytohormones are also known to antagonize the effects of abscisic acid (ABA) on stomatal movement, and to affect ethylene biosynthesis. As ethylene has an antagonistic effect on ABA-induced stomatal closure, the possibility that the antagonistic effects of these phytohormones on ABA were mediated through ethylene biosynthesis was investigated. Both the cytokinin, 6-benzyladenine (BA), and the auxin, 1-naphthaleneacetic acid (NAA), antagonized ABA-induced stomatal closure in a manner similar to that following application of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). However, these effects were negated when ethylene signalling, perception, or biosynthesis were blocked. As stomatal aperture is regulated by changes in guard cell volume, ABA application was found to reduce the volume of the guard cell protoplasts (GCP). It was found that BA, NAA, or ACC application compensated perfectly for the reduction in GCP volume by ABA application in WT plants. The above observations suggest that cytokinins and auxins inhibit ABA-induced stomatal closure through the modulation of ethylene biosynthesis, and that ethylene inhibits the ABA-induced reduction of osmotic pressure in the guard cells.     

F-box protein DOR functions as a novel inhibitory factor for abscisic acid-induced stomatal closure under drought stress in Arabidopsis,

Guard cells, which form stoma in leaf epidermis, sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. Under drought stress, plants synthesize abscisic acid (ABA), which in turn induces a rapid closing of stoma, to prevent water loss by transpiration. However, many aspects of the molecular mechanism for ABA-mediated stomatal closure are still not understood. Here, we report a novel negative regulator of guard cell ABA signaling, DOR, in Arabidopsis (Arabidopsis thaliana). The DOR gene encodes a putative F-box protein, a member of the S-locus F-box-like family related to AhSLF-S(2) and specifically interacting with ASK14 and CUL1. A null mutation in DOR resulted in a hypersensitive ABA response of stomatal closing and a substantial increase of drought tolerance; in contrast, the transgenic plants overexpressing DOR were more susceptible to the drought stress. DOR is strongly expressed in guard cells and suppressed by ABA treatment, suggesting a negative feedback loop of DOR in ABA responses. Double-mutant analyses of dor with ABA-insensitive mutant abi1-1 showed that abi1-1 is epistatic to dor, but no apparent change of phospholipase Dalpha1 was detected between the wild type and dor. Affymetrix GeneChip analysis showed that DOR likely regulates ABA biosynthesis under drought stress. Taken together, our results demonstrate that DOR acts independent of phospholipase Dalpha1 in an ABA signaling pathway to inhibit the ABA-induced stomatal closure under drought stress.     

Hormone interactions in stomatal function

Research in recent years on the biology of guard cells has shown that these specialized cells integrate both extra- and intra-cellular signals in the control of stomatal apertures. Among the phytohormones, abscisic acid (ABA) is one of the key players regulating stomatal function. In addition, auxin, cytokinin, ethylene, brassinosteroids, jasmonates, and salicylic acid also contribute to stomatal aperture regulation. The interaction of multiple hormones can serve to determine the size of stomatal apertures in a condition-specific manner. Here, we discuss the roles of different phytohormones and the effects of their interactions on guard cell physiology and function.     

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO(2), suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H(2)O(2) or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.     

Reactive oxygen species signaling and stomatal movement in plant responses to drought stress and pathogen attack

Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO_2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO_2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species(ROS). Under abiotic and bioticstress conditions, extracellular ROS are mainly produced by plasma membrane-localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network,primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO_2 signaling, and immunity responses.Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli.     

Signalling drought in guard cells

A number of environmental conditions including drought, low humidity, cold and salinity subject plants to osmotic stress. A rapid plant response to such stress conditions is stomatal closure to reduce water loss from plants. From an external stress signal to stomatal closure, many molecular components constitute a signal transduction network that couples the stimulus to the response. Numerous studies have been directed to resolving the framework and molecular details of stress signalling pathways in plants. In guard cells, studies focus on the regulation of ion channels by abscisic acid (ABA), a chemical messenger for osmotic stress. Calcium, protein kinases and phosphatases, and membrane trafficking components have been shown to play a role in ABA signalling process in guard cells. Studies also implicate ABA-independent regulation of ion channels by osmotic stress. In particular, a direct osmosensing pathway for ion channel regulation in guard cells has been identified. These pathways form a complex signalling web that monitors water status in the environment and initiates responses in stomatal movements.     

Two Arabidopsis guard cell-preferential MAPK genes,MPK9 and MPK12, function in biotic stress response

Abscisic acid (ABA) plays a major role in plant development and adaptation to severe environmental conditions. ABA evokes cellular events to regulate stomatal apertures and thus contributes to the plant’s ability to respond to abiotic stresses. Reactive oxygen species (ROS) are produced in response to ABA and mediate ABA-induced stomatal closure. We have shown that two MAP kinases, MPK9 and MPK12, are highly and preferentially expressed in guard cells and function as positive regulators of ROS-mediated ABA signaling in guard cells. Cell biological and electrophysiological analyses demonstrated that MPK9 and MPK12 act downstream of ROS and cytosolic Ca²⁺ and upstream of anion channels in the guard cell ABA signaling cascade. Plant pathogens use stomata as the primary gateway to enter into their hosts, and previous studies have indicated crosstalk between ABA and defense signaling. Here we show that mpk9-1/12-1 double mutants are highly susceptible to Pseudomonas syringae DC3000 compared to WT plants. These results suggest that the regulation of stomatal apertures by MPK9 and MPK12 contributes to the first line of defense against pathogens.     

Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution

Stomatal pores evolved more than 410 million years ago [1,?2]?and allowed vascular plants to regulate transpirational water loss during the uptake of CO(2) for photosynthesis [3]. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens [2] respond to environmental signals in a similar way to those of flowering plants [4] and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response [5] is?involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) [6], is rescued by substitution with the moss P.?patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P.?patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago [7], prior to the evolution of ferns [8, 9].     

Stomatal deregulation in Plasmopara viticola-infected grapevine leaves

In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA) treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.     

Methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid in guard cells

Plants tightly control stomatal aperture in response to various environmental changes. A drought-inducible phytohormone, abscisic acid (ABA), triggers stomatal closure and ABA signaling pathway in guard cells has been well studied. Similar to ABA, methyl jasmonate (MeJA) induces stomatal closure in various plant species but MeJA signaling pathway is still far from clear. Recently we found that Arabidopsis calcium dependent protein kinase CPK6 functions as a positive regulator in guard cell MeJA signaling and provided new insights into cytosolic Ca²⁺-dependent MeJA signaling. Here we discuss the MeJA signaling and also signal crosstalk between MeJA and ABA pathways in guard cells.Key words: methyl jasmonate, abscisic acid, guard cell, reactive oxygen species, nitric oxide, calciumStomata, which are formed by pairs of specialized cells called guard cells, control gas exchanges and transpirational water loss. Guard cells can shrink and swell in response to various physiological stimuli, resulting in stomatal closing and opening.^1,2 To optimize growth under various environmental conditions, plants have developed fine-tuned signal pathway in guard cells. Abscisic acid (ABA) is synthesized under drought stress and induces stomatal closure to reduce transpirational water loss.² ABA signal transduction in guard cells has been widely studied. ABA induces increases of various second messengers such as cytosolic Ca²⁺, reactive oxygen species (ROS) and nitric oxide (NO) in guard cells. These early signal components finally evoke ion efflux through plasma membrane ion channels, resulting in reduction of guard cell turgor pressure.Jasmonates are plant hormones synthesized via the octadecanoid pathway and regulate various physiological processes in plants such as pollen maturation, tendril coiling, senescence and responses to wounding and pathogen attacks.³ Similar to ABA, jasmonates also trigger stomatal closure and the response is conserved among various plant species including Arabidopsis thaliana,⁴ Hordeum vulgare,⁵ Commelina benghalensis,⁶ Vicia faba,⁷ Nicotiana glauca,⁸ Paphiopedilum Supersuk⁹ and Paphiopedilum tonsum.⁹ A volatile methyl ester of jasmonic acid (JA), methy jasmonate (MeJA), has been widely used for studying jasmonate signaling pathway. To date, pharmacological and reverse genetic approaches have revealed many important signal components involved in MeJA-induced stomatal closure and suggest a signal crosstalk between MeJA and ABA in guard cells. In this review, we mainly focus on the three important second messengers, ROS, NO and cytosolic Ca²⁺ and discuss recent advance about MeJA signaling and signal interaction between MeJA and ABA in guard cells.     

Abscisic acid-induced actin reorganization in guard cells of dayflower is mediated by cytosolic calcium levels and by protein kinase and protein phosphatase activities

In guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore. Abscisic acid (ABA), a signal for stomatal closure, induces rapid depolymerization of cortical actin filaments and the slower formation of a new type of actin that is randomly oriented throughout the cell. This change in actin organization has been suggested to be important in signaling pathways involved in stomatal closing movement, since actin antagonists interfere with normal stomatal closing responses to ABA. Here we present evidence that the actin changes induced by ABA in guard cells of dayflower (Commelina communis) are mediated by cytosolic calcium levels and by protein phosphatase and protein kinase activities. Treatment of guard cells with CaCl2 induced changes in actin organization similar to those induced by ABA. Removal of extracellular calcium with EGTA inhibited ABA-induced actin changes. These results suggest that Ca2+ acts as a signal mediator in actin reorganization during guard cell response to ABA. A protein kinase inhibitor, staurosporine, inhibited actin reorganization in guard cells treated with ABA or CaCl2, and also increased the population of cells with long radial cortical actin filaments in untreated control cells. A protein phosphatase inhibitor, calyculin A, induced fragmentation of actin filaments in ABA- or CaCl2-treated cells and in control cells, and inhibited the formation of randomly oriented long actin filaments induced by ABA or CaCl2. These results suggest that protein kinase(s) and phosphatase(s) participate in actin remodeling in guard cells during ABA-induced stomatal closure.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Main conclusion

Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N’,N’-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.