SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone--an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.     

蓝光调节气孔运动的信号途径及其与脱落酸相互关系研究

气孔运动与植物水分代谢密切相关.保卫细胞可有效感知和整合多种环境信号,通过控制离子进出调节其膨压,影响气孔开与闭.诸多研究表明,蓝光信号诱导气孔开放和逆境信号脱落酸(ABA)促进气孔关闭构成了气孔运动的两大研究领域.该文就保卫细胞中蓝光信号传递及与ABA信号交叉控制气孔开闭的研究进展进行综述,以了解气孔对蓝光和ABA反应的最新进展,为发展耐旱与提高作物水分利用效率生物技术的改进提供理论支持.     

A defined range of guard cell calcium oscillation parameters encodes stomatal movements.

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a 'calcium clamp' and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term 'calcium-reactive' closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was 'calcium programmed' by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.     

The HIC signalling pathway links CO2 perception to stomatal development

Stomatal pores on the leaf surface control both the uptake of CO2 for photosynthesis and the loss of water during transpiration. Since the industrial revolution, decreases in stomatal numbers in parallel with increases in atmospheric CO2 concentration have provided evidence of plant responses to changes in CO2 levels caused by human activity. This inverse correlation between stomatal density and CO2 concentration also holds for fossil material from the past 400 million years and has provided clues to the causes of global extinction events. Here we report the identification of the Arabidopsis gene HIC (for high carbon dioxide), which encodes a negative regulator of stomatal development that responds to CO2 concentration. This gene encodes a putative 3-keto acyl coenzyme A synthase--an enzyme involved in the synthesis of very-long-chain fatty acids. Mutant hic plants exhibit up to a 42% increase in stomatal density in response to a doubling of CO2. Our results identify a gene involved in the signal transduction pathway responsible for controlling stomatal numbers at elevated CO2.     

CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells

The continuing rise in atmospheric [CO2] is predicted to have diverse and dramatic effects on the productivity of agriculture, plant ecosystems and gas exchange. Stomatal pores in the epidermis provide gates for the exchange of CO2 and water between plants and the atmosphere, processes vital to plant life. Increased [CO2] has been shown to enhance anion channel activity proposed to mediate efflux of osmoregulatory anions (Cl- and malate(2-)) from guard cells during stomatal closure. However, the genes encoding anion efflux channels in plant plasma membranes remain unknown. Here we report the isolation of an Arabidopsis gene, SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1, At1g12480), which mediates CO2 sensitivity in regulation of plant gas exchange. The SLAC1 protein is a distant homologue of bacterial and fungal C4-dicarboxylate transporters, and is localized specifically to the plasma membrane of guard cells. It belongs to a protein family that in Arabidopsis consists of four structurally related members that are common in their plasma membrane localization, but show distinct tissue-specific expression patterns. The loss-of-function mutation in SLAC1 was accompanied by an over-accumulation of the osmoregulatory anions in guard cell protoplasts. Guard-cell-specific expression of SLAC1 or its family members resulted in restoration of the wild-type stomatal responses, including CO2 sensitivity, and also in the dissipation of the over-accumulated anions. These results suggest that SLAC1-family proteins have an evolutionarily conserved function that is required for the maintenance of organic/inorganic anion homeostasis on the cellular level.     

Nitric oxide involved in signal transduction of Jasmonic acid-induced stomatal closure of Vicia faba L.

Nitric oxide (NO) and Jasmonic acid (JA) are two key signaling molecules involved in many and diverse biological pathways in plants. Growing evidence suggested that NO signaling interacts with JA signaling. In this work, Our experiment showed that NO exists in guard cell of Vicia faba L., and NO is involved in signal transduction of JAinduced stomata closuring: ( i ) JA enhances NO synthesis in guard cell; ( ii ) both JA and NO induced stomatal closure, and had dose response to their effects; ( iU ) there are synergetic correlation between JA and lower NO concentration in regulation of stomatal movement; (iV) JA-induced stomatal closure was largely prevented by 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO), a specific NO scavenger. An inhibitor of NO synthase (NOS) in mammalian cells, N^G-nitro-L-Arg-methyl eater (L-NAME) also inhibits plant NOS, repressing JA-induced NO generation and JA-induced stomatal closure. We presumed that NO mainly comes from NOS after JA treatment.     

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.     

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.     

Drought-induced guard cell signal transduction involves sphingosine-1-phosphate

Stomata form pores on leaf surfaces that regulate the uptake of CO2 for photosynthesis and the loss of water vapour during transpiration. An increase in the cytosolic concentration of free calcium ions ([Ca2+]cyt) is a common intermediate in many of the pathways leading to either opening or closure of the stomatal pore. This observation has prompted investigations into how specificity is controlled in calcium-based signalling systems in plants. One possible explanation is that each stimulus generates a unique increase in [Ca2+]cyt, or 'calcium signature', that dictates the outcome of the final response. It has been suggested that the key to generating a calcium signature, and hence to understanding how specificity is controlled, is the ability to access differentially the cellular machinery controlling calcium influx and release from internal stores. Here we report that sphingosine-1-phosphate is a new calcium-mobilizing molecule in plants. We show that after drought treatment sphingosine-1-phosphate levels increase, and we present evidence that this molecule is involved in the signal-transduction pathway linking the perception of abscisic acid to reductions in guard cell turgor.     

丙酮酸转运体AtMPC3介导植物干旱胁迫响应机理研究

在干旱胁迫下,植物通过自身复杂而有效的应对机制减少水分散失,其中关闭植物气孔降低蒸腾作用是一个重要环节。脱落酸(ABA)是植物响应干旱胁迫产生的可以诱导气孔关闭、减少水分散失的重要植物激素。丙酮酸是光合作用糖酵解的产物,需要借助线粒体丙酮酸转运体(MPCs)进入线粒体中进行后续物质和能量代谢。本研究发现拟南芥线粒体丙酮酸转运体AtMPC3参与介导植物干旱胁迫响应,在外源施加ABA条件下,AtMPC3基因缺失突变体的气孔较野生型开度更小,植物失水率更低,表现出更强的抗旱能力。结果说明AtMPC3在脱落酸促进植物气孔关闭及干旱应答过程中的重要作用,对于提高植物抗旱能力以及作物产量提升具有潜在应用价值及重要意义。     

pH induced elastic modulus of guard cell wall in stomatal movement

Guard cell wall properties are important in stomatal movement. Previous research focused on the structure and anatomy of guard cell walls, but little is known about the physical changes that take place within the walls during stomatal opening and closure. In this work, we investigate the volumetric elastic modulus (ε) of the guard cell wall at different pH values during stomatal opening in Vicia faba epidermal strips using a cell pressure probe. The volumetric elastic modulus of the guard cell wall decrease...     

Involvement of nitric oxide in the signal transduction of salicylic acid regulating stomatal movement

The effects and the relationship between sali-cylic acid(SA)and nitric oxide(NO) on Vicia faba L.stomatal movement were studied.The results here showed that exogenous SA and NO induced stomatal closure,100μmol/L SA induced a rapid and striking NO increase in the cytosol of guard cells.This phenomenon was largely prevented by 2000μmol/L 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide(PTIO),a specific NO scavenger,and 25μmol/L N^G-nitro-L-Arg-methyl eater (L-NAME),an inhibitor of NO synthase(NOS) in mammalian cells that also inhibits plant NOS.In addition,SA-induced stomatal closure was largely prevented by PTIO and L-NAME.These results provide evidence that guard cells generate NO in response to SA via NOS-like activity,and that such NO production is required for full stomatal closure in response to SA.H-(1,2,4)-oxadiazole-[4,3-α]quinoxalin-l-one(ODQ),an inhibitor of guanylate cyclase,and nicotinamide,an antagonist of cADPR production,inhibited the effects of SA-and NO-induced stomatal closure.It suggests that both cGMP and cADPR might mediate the signal transduction of SA and NO-induced stomatal closure.     

Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins

In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA1 (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.     

Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate

RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli--including light, osmotic stress and the phytohormone indoleacetic acid--and InsP3 activates Ca2+ channels and Ca2+ flux across plant vacuolar and microsomal membranes. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements may be controlled by cytoplasmic Ca2+. So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-1-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3) reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels. The data are consistent with a transient rise in cytoplasmic free Ca2+ and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.     

An α-expansin, <Emphasis Type="Italic">VfEXPA1</Emphasis>, is involved in regulation of stomatal movement in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

The wall loosening of guard cells differs from other types of plant cells. However, the regulation of wall loosening during stomatal movement is poorly understood. VfEXPA1 is an α-expansin gene cloned from Vicia faba epidermal strips. Expression of VfEXPA1 is regulated by darkness and submergence, and is not affected by light and abscisic acid (ABA). In situ hybridization showed that VfEXPA1 is expressed primarily in the guard cells. Overexpression of VfEXPA1 in transgenic tobacco accelerated light-induced stomatal opening, and increased both transpiration and photosynthetic rates under favorable growth conditions. Our results indicate the guard cell-expressed expansin VfEXPA1 plays an important role in regulation of stomatal opening.     

Localization of muscarinic acetylcholine receptor in plant guard cells

Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. InVicia faba L. andPisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane ofVicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.     

Ca^2＋ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2 with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2 concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2 in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2 or ruthenium red to block Ca^2 released from vacuole respectively, the results showed that the increased cytosolic Ca^2 mainly come from intracellular Ca^2 store. The evidence supports that Ca^2 is involved in guard-cell response to ACh and that Ca^2 sigual is coupled to mAChRs in ACh signal transduction in guard cells.     

MAP kinase specifically mediates the ABA-induced H<Subscript>2</Subscript>O<Subscript>2</Subscript> generation in guard cells of<Emphasis Type="Italic">Vicia faba</Emphasis> L.

The plant hormone abscisic acid (ABA) is involved in regulating adverse physiological processes, including stomatal closure, seed development and germination, and mediating many environmental stress responses, such as drought, salinity and extreme temperatures[1,2]. In re-sponse to various stress stimuli, ABA synthesis is in-creased in plant cells, which triggers a series of physio-logical responses to adapt the stress conditions[1—3]. For example, under water deficit, ABA acts directly on…     

钙依赖型蛋白激酶在ABA调控的杨树气孔运动中的作用

群众杨叶片下表皮经过植物激素脱落酸(ABA)和钙依赖型蛋白激酶抑制剂三氟拉嗪(TFP)处理后,在扫描电镜下观察了气孔开度的变化,并用透射电镜结合X-射线能谱显微分析技术,对保卫细胞内的K~ 、Ca~(2 )含量进行了研究。结果表明:ABA几乎完全抑制光照条件下叶片气孔的开放,而同时加入TFP则显著降低ABA对气孔开放的抑制作用。在ABA作用下,保卫细胞细胞质中的K~ 含量下降但Ca~(2 )含量增加;而同时加入TFP则可逆转ABA对K~ 、Ca~(2 )的作用。研究结果证明,钙依赖型蛋白激酶可能通过对保卫细胞内K~ 、Ca~(2 )的调节作用而介导了ABA调控气孔运动的信号转导过程。     

GPX3参与了H2O2对保卫细胞质膜钾通道的调控

通过表皮条生物分析、失水率测定及膜片钳实验研究谷氨酸过氧化物酶3(GPX3)能够调节保卫细胞质膜钾通道活性,并因此来介导H2O2诱导的拟南芥气孔关闭过程.与野生型Col-0相比,gpx3缺失突变在气孔开度和细胞失水方面均有较大差异.全细胞膜片钳模式下,1mmol/L的H2O2处理使野生型保卫细胞质膜钾离子通道外向电流从50pA左右激增到240pA左右,而gpx3的电流则变化不大.单通道电流的进一步分析表明gpx3不能像野生型一样对1mmol/L的H2O2处理产生明显反应,野生型与突变体的单通道电流差距高达10倍以上.据此推断GPX3是通过特异地调节外向钾通道来介导H2O2的信号传递.