Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.     

Ca2+signalling in stomatal guard cells

Ca(2+) is a ubiquitous second messenger in the signal transduction pathway(s) by which stomatal guard cells respond to external stimuli. Increases in guard-cell cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) have been observed in response to stimuli that cause both stomatal opening and closure. In addition, several important components of Ca(2+)-based signalling pathways have been identified in guard cells, including the cADP-ribose and phospholipase C/Ins(1, 4,5)P(3)-mediated Ca(2+)-mobilizing pathways. The central role of stimulus-induced increases in [Ca(2+)](cyt) in guard-cell signal transduction has been clearly demonstrated in experiments examining the effects of modulating increases in [Ca(2+)](cyt) on alterations in guard-cell turgor or the activity of ion channels that act as effectors in the guard-cell turgor response. In addition, the paradox that Ca(2+) is involved in the transduction of signals that result in opposite end responses (stomatal opening and closure) might be accounted for by the generation of stimulus-specific Ca(2+) signatures, such that increases in [Ca(2+)](cyt) exhibit unique spatial and temporal characteristics.     

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.     

Involvement of endogenous abscisic acid in methyl jasmonate-induced stomatal closure in Arabidopsis

In this study, we examined the involvement of endogenous abscisic acid (ABA) in methyl jasmonate (MeJA)-induced stomatal closure using an inhibitor of ABA biosynthesis, fluridon (FLU), and an ABA-deficient Arabidopsis (Arabidopsis thaliana) mutant, aba2-2. We found that pretreatment with FLU inhibited MeJA-induced stomatal closure but not ABA-induced stomatal closure in wild-type plants. The aba2-2 mutation impaired MeJA-induced stomatal closure but not ABA-induced stomatal closure. We also investigated the effects of FLU and the aba2-2 mutation on cytosolic free calcium concentration ([Ca(2+)](cyt)) in guard cells using a Ca(2+)-reporter fluorescent protein, Yellow Cameleon 3.6. In wild-type guard cells, FLU inhibited MeJA-induced [Ca(2+)](cyt) elevation but not ABA-induced [Ca(2+)](cyt) elevation. The aba2-2 mutation did not affect ABA-elicited [Ca(2+)](cyt) elevation but suppressed MeJA-induced [Ca(2+)](cyt) elevation. We also tested the effects of the aba2-2 mutation and FLU on the expression of MeJA-inducible VEGETATIVE STORAGE PROTEIN1 (VSP1). In the aba2-2 mutant, MeJA did not induce VSP1 expression. In wild-type leaves, FLU inhibited MeJA-induced VSP1 expression. Pretreatment with ABA at 0.1 μm, which is not enough concentration to evoke ABA responses in the wild type, rescued the observed phenotypes of the aba2-2 mutant. Finally, we found that in wild-type leaves, MeJA stimulates the expression of 9-CIS-EPOXYCAROTENOID DIOXYGENASE3, which encodes a crucial enzyme in ABA biosynthesis. These results suggest that endogenous ABA could be involved in MeJA signal transduction and lead to stomatal closure in Arabidopsis guard cells.     

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

The characterization of differential calcium signalling in tobacco guard cells

Two novel approaches for the study of Ca2+-mediated signal transduction in stomatal guard cells are described. Stimulus-induced changes in guard-cell cytosolic Ca2+ ([Ca2+]cyt) were monitored using viable stomata in epidermal strips of a transgenic line of Nicotiana plumbaginifolia expressing aequorin (the proteinous luminescent reporter of Ca2+) and in a new transgenic line in which aequorin expression was targeted specifically to the guard cells. The results indicated that abscisic acid (ABA)-induced stomatal closure was accompanied by increases in [Ca2+]cyt in epidermal strips. In addition to ABA, mechanical and low-temperature signals directly affected stomatal behaviour, promoting rapid closure. Elevations of guard-cell [Ca2+]cyt play a key role in the transduction of all three stimuli. However, there were striking differences in the magnitude and kinetics of the three responses. Studies using Ca2+ channel blockers and the Ca2+ chelator EGTA further suggested that mechanical and ABA signals primarily mobilize Ca2+ from intracellular store(s), whereas the influx of extracellular Ca2+ is a key component in the transduction of low-temperature signals. These results illustrate an aspect of Ca2+ signalling whereby the specificity of the response is encoded by different spatial or kinetic Ca2+ elevations.     

Distinct light and clock modulation of cytosolic free Ca2+ oscillations and rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 promoter activity in Arabidopsis

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt). Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca(2+)](cyt), and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca(2+)](cyt) rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca(2+)](cyt). Oscillations of [Ca(2+)](cyt) and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca(2+)](cyt) and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca(2+)](cyt) and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.     

Ca^2＋参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3 AM为Ca^2＋荧光探针,结合激光共聚焦扫描显微技术,观察到在处理后数十秒内,气孔关闭之前,茉莉酸（JA）可引起[Ca^2＋]cyt的迅速上升;对照和JA的前体物亚麻酸（LA）几乎不能引起[Ca^2＋]cyt的明显变化;钙的螯合剂EGTA预处理可完全阻断JA诱导气孔关闭的效应,并且JA不再引起保卫细胞[Ca^2＋]cyt增加;质膜Cah通道的抑制剂硝苯吡啶（nifedipine,NIF）可减弱JA诱导气孔关闭的效应,也使JA诱导保卫细胞[Ca^2＋]cyt增加的幅度有所下降;胞内Ca^2＋释放的抑制剂钌红不能明显改变JA诱导气孔关闲的趋势,但使JA引起的保卫细胞[Ca^2＋]cyt增加有所降低。实验结果表明：Ca^2＋参与JA诱导气孔关闭的信号转导;推测JA引起的[Ca^2＋]cyt升高可能主要来源于胞外,但不能完全排除胞内Ca^2＋的释放。     

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca(2+) mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca(2+)](cyt), production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca(2+) transients mainly due to plasma membrane Ca(2+) influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca(2+)](cyt) elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca(2+) mobilization and may play a crucial role in temporal regulation of [Ca(2+)](cyt) signature.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Extracellular calmodulin(CaM)plays significant roles in many physiological processes,but little is known about its mechanism of regulating stomatal movements.In this paper,whether CaM exists in the guard cell walls of Arabidopsis and whether depolymerization of actin cytoskeleton is involved in extracellular CaM-induced stomatal closing are investigated.It is found that CaM exists in guard cell walls of Arabidopsis,and its molecular weight is about 17 kD.Bioassay using CaM antagonists W7-agarose and anti-CaM serum shows that the endogenous extracellular CaM promotes stomatal closure and delays stomatal opening.The long radial actin filaments in guard cells undergo disruption in a time-dependent manner during exogenous CaM-induced stomatal closing.Pharmacological experiments show that depolymerization of actin cytoskeleton enhances the effect of exogenous CaM-induced stomatal closing and polymerization reduces the effect.We also find that exogenous CaM triggers an increase in [Ca2+]cyt of guard cells.If [Ca2+]cyt increase is blocked with EGTA,exogenous CaM-induced stomatal closure is inhibited.These results indicate that extracellular CaM causes elevation of [Ca2+]cyt in guard cells,subsequently resulting in disruption of actin filaments and finally leading to guard cells closure.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in Arabidopsis

CaM ubiquitously presents inside eukaryotic cells. CaM抯 gene expression and its subcellular localization are regulated by light, osmotic stress, pathogens, plant hormones, etc.[1]. Intracellular CaM of plant displays important functions in pathogenesis and wounding reaction[2] and hypersensitive response[3]. CaM has been found extracellular spaces in many plant species, such as soluble extracts of oat coleoptile cell walls[4], the wheat coleoptile cell walls[5], maize root tips cell walls[6…     

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Sugar-induced increase in cytosolic Ca(2+) in Arabidopsis thaliana whole plants.

Using Ca(2+)-dependent photoprotein aequorin-transformed Arabidopsis thaliana, sugar-induced increase in cytosolic free Ca(2+ )concentration ([Ca(2+)](cyt))( )was investigated by luminescence imaging technique. When 0.1 M sucrose was fed to roots of autotrophically grown intact whole plants whose roots had been incubated overnight with coelenterazine to reconstitute aequorin systemically, strong and transient (within 20 s) luminescence was observed in the roots; that luminescence was followed by weak luminescence moving from the lower leaves to the upper leaves. The moving rate of luminescence was roughly comparable to that of [(14)C]sucrose. Application of 0.1 M glucose or fructose induced transient luminescence in excised leaves. No such luminescence was observed in heterotrophically grown (with sucrose) whole plants or in excised tissues. mRNA levels of sucrose-H(+) symporter genes AtSUC1 and AtSUC2 were higher in autotrophic plants than in heterotrophic plants. These results indicate that influx of transported sucrose together with H(+) into the mesophyll cells of autotrophic plants may depolarize the membrane potential, and subsequently activate a voltage-gated Ca(2+) channel on the plasma membrane, resulting in a [Ca(2+)](cyt) increase. The [Ca(2+)](cyt) increase might initiate Ca(2+ )signaling leading to the expression of genes related to biosynthesis of storage carbohydrates. Hexoses, when applied, might also be involved in the [Ca(2+)](cyt) increase mediated by monosaccharide-H(+) co-transporters.     

Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2

Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca2+ ([Ca2+]cyt) and H2O2 in Vicia faba guard cells combined with epidermal strip bioassay suggests that ExtCaM induces an increase in both H2O2 levels and [Ca2+]cyt, leading to a reduction in stomatal aperture. Pharmacological studies implicate heterotrimeric G protein in transmitting the ExtCaM signal, acting upstream of [Ca2+]cyt elevation, and generating H2O2 in guard cell responses. To further test the role of heterotrimeric G protein in ExtCaM signaling in stomatal closure, we checked guard cell responses in the Arabidopsis (Arabidopsis thaliana) Galpha-subunit-null gpa1 mutants and cGalpha overexpression lines. We found that gpa1 mutants were insensitive to ExtCaM stimulation of stomatal closure, whereas cGalpha overexpression enhanced the guard cell response to ExtCaM. Furthermore, gpa1 mutants are impaired in ExtCaM induction of H2O2 generation in guard cells. Taken together, our results strongly suggest that ExtCaM activates an intracellular signaling pathway involving activation of a heterotrimeric G protein, H2O2 generation, and changes in [Ca2+]cyt in the regulation of stomatal movements.