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.     

Guard cell abscisic acid signalling and engineering drought hardiness in plants

Guard cells are located in the epidermis of plant leaves, and in pairs surround stomatal pores. These control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. Guard cells have become a highly developed system for dissecting early signal transduction mechanisms in plants. In response to drought, plants synthesize the hormone abscisic acid, which triggers closing of stomata, thus reducing water loss. Recently, central regulators of guard cell abscisic acid signalling have been discovered. The molecular understanding of the guard cell signal transduction network opens possibilities for engineering stomatal responses to control CO2 intake and plant water loss.     

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.     

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.     

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.     

NO,H2O2介导根系渗透胁迫和脱落酸诱导的气孔关闭

运用表皮实验和激光扫描共聚焦显微镜技术对NO和H2O2在根系渗透胁迫和外源脱落酸（ABA）处理诱导蚕豆气孔关闭中的作用及其相互关系进行了研究．结果表明，渗透胁迫及外源ABA处理既促进保卫细胞内源NO和H2O2形成，也诱导气孔关闭；外源H2O2和SNP可促进气孔关闭，也分别诱导保卫细胞NO和H2O2产生．还对根系渗透胁迫诱导蚕豆气孔关闭中ABA、NO和H2O2的关系进行了讨论，认为渗透胁迫可能通过ABA诱导NO和H2O2产生，促进气孔关闭且NO和H2O2之间存在相互作用．     

ATHK1 acts downstream of hydrogen peroxide to mediate ABA signaling through regulation of calcium channel activity in Arabidopsis guard cells

Plants gradually develop their ability to tolerate environmental water deficit as part of the evolutionary process.Abscisic acid(ABA) plays a critical role during drought and osmotic stress.Several histidine protein kinases are regarded as osmotic sensors or regulators in the adaptive response of plants to water deficit.In this study,we report that ATHK1,which was previously shown to function as an osmotic regulator,is involved in ABA-induced stomatal signaling in Arabidopsis.Mutants null for ATHK1 expression were unable to transmit normal ABA responses in guard cells,including inducing stomatal closure,producing hydrogen peroxide and activating calcium influx.Moreover,patch clamp and confocal analysis demonstrated that ATHK1 may function downstream of hydrogen peroxide in ABA-induced stomatal closure,by regulating calcium channel activity and calcium oscillation in Arabidopsis guard cells.     

Homologue structure of the SLAC1 anion channel for closing stomata in leaves

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 ? resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.     

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.     

Involvement of carbon monoxide produced by heme oxygenase in ABA-induced stomatal closure in Vicia faba and its proposed signal transduction pathway

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Inter- estingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Ad- ditionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream interme- diates in the CO signaling responsible for stomatal closure.     

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.     

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.     

两种MAP激酶共同调节蚕豆气孔保卫细胞中ROS信号途径研究

运用表皮条生物学分析、激光共聚焦扫描及膜片钳技术,在MEK1/2和p38MAP激酶专一性抑制剂PD98059和SB203580处理下,观察促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)家族成员介导蚕豆保卫细胞中氧化信号机制.结果表明,PD98059能阻断并逆转ABA和H2O2抑制质膜内向K 电流、ABA诱导H2O2产生及气孔关闭,SB203580也表现相似的作用.因此,两种MAP激酶可能共同调节ABA诱导ROS产生和气孔关闭.     

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.     

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.     

Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Ethylene-induced nitric oxide production and stomatal closure in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> depending on changes in cytosolic pH

We investigated changes in cytosolic pH and nitric oxide (NO) during ethylene-induced stomatal closure in Arabidopsis thaliana using pharmacological, laser scanning confocal microscopy (LSCM), and spectrophotography techniques. Treatment with ethephon (a direct source of ethylene when applied to plants) and 1-aminocycloaminopropane-1-carboxylic acid (ACC, an ethylene precursor) resulted in a rapid accumulation of NO and cytosolic alkalinization in guard cells. Acetic acid (a weak acid) and sodium orthovanadate (NaVO3; a plasmalemma H+-ATPase inhibitor) reduced stomatal closure induced by ethylene and blocked ethylene-induced activity of nitrate reductase. However, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, had no effect. These results suggest that NO production is downstream of the rise in cytosolic pH in A. thaliana.     

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…