Ca2+参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3AM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;叶照和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Ca~(2 )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诱导气孔关闭的趋势,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。     

保卫细胞钙信号的研究进展

钙(Ca^2 )是多种信号途径的第二信使。Ca^2 成像技术的成熟和发展为显示保卫细胞胞质Ca^2 浓度([Ca^2 ]cyt)的分布及外界刺激引起[Ca^2 ]cyt的变化模式提供了很好的研究工具,关于细胞内外Ca^2 库释放Ca^2 的机制也有了较清楚的认识。拟南芥突变体的研究使Ca^2 信号上游分子及其排序更加明确,[Ca^2 ]cyt增加下游的磷酸化和去磷酸化过程也是气孔关闭必需的生理过程。     

NO可能作为Ca^2＋的下游信号介导乙烯诱导的蚕豆气孔关闭

以蚕豆（Vicia faba L．）为材料,利用药理学实验,结合激光共聚焦显微技术和分光光度法．探讨Ca^2＋和一氧化氮（nitric oxide,NO）在乙烯（ethylene,Eth）调控气孔运动信号转导中的作用。结果表明．光下乙烯利（0．004％,0．04％,0．4％）可诱导蚕豆叶片气孔关闭,且具有时间和剂量效应：NO清除剂cPTIO、硝酸还原酶（nitrate reductase,NR）抑制剂NaN3及胞外Ca^2＋螯合剂EGTA可部分逆转乙烯诱导的气孔关闭;乙烯能够明显增加气孔保卫细胞NO水平;提高蚕豆叶片NO含量和NR活性．并且NO的含量变化与NR活性的变化趋势基本一致;NR抑制剂NaN3可抑制乙烯诱导的气孔保卫细胞和叶片NO含量的增加;清除胞外Ca^2＋可减弱乙烯对NO含量和NR活性的诱导效应。说明Ca^2＋和NO均参与乙烯诱导的蚕豆气孔关闭,且NO（主要由NR途径合成）可能位于Ca^2＋下游参与调控这一信号转导过程。     

Ca2+参与水杨酸诱导蚕豆气孔运动时的信号转导

在一定条件下,外源水杨酸(SA)可以诱导蚕豆(Vicia faba L.)气孔关闭,阻止气孔张开。以Fluo-3—AM作为Ca^2 的荧光探针,利用激光共聚焦扫描显微技术,对水杨酸调控气孔运动中保卫细胞胞质Ca^2 的变化趋势及Ca^2 的来源进行了研究。结果表明,水杨酸可引起胞质Ca^2 增加,这种变化发生在气孔开度改变之前。Ca^2 螯合剂BAPTA（1,2-bis(2-amino phenox-y)ethane-N,N,N′,N′-tetraacetic acid,1mmol/L)几乎可以完全抑制水杨酸诱导气孔开度减小的作用;胞外Ca^2 螯合剂EGTA(2mmol/L)、质膜Ca^2 通道抑制剂尼群地平(nifedipine,NIF,1μmol/L)和LaCl3(1mmol／L)可不同程度地减弱水杨酸诱导气孔关闭的效应。BAPTA(1mmol／L)预处理后,水杨酸不再引起胞质Ca^2 含量改变;尼群地平能够降低水杨酸引起的胞质Ca^2 增加的幅度。说明Ca^2 可能参与水杨酸诱导气孔运动的信号转导。水杨酸引起胞内升高的Ca^2 可能既来自胞外又来自胞内,胞内Ca^2 库可能是其主要来源。     

H+参与茉莉酸调控蚕豆气孔运动的信号转导

以BCECF-AM为pH的荧光探针,结合激光共聚焦扫描显微技术,研究H 可能参与茉莉酸(JA)调控气孔运动信号转导途径的结果表明,0.1~100μmol·L~(-1)浓度的(-)JA可诱导蚕豆气孔关闭,在引起气孔孔径改变之前,(-)JA能引起蚕豆保卫细胞胞质的碱化;而(±)JA可诱导气孔适当开放,它未引起蚕豆保卫细胞胞质中pH的明显改变。药理学实验证明,质膜上质子泵的抑制剂矾酸钠能减弱(-)JA诱导气孔关闭的作用;而质膜上质子泵的激活剂壳梭孢菌素(fusicoccin)基本上未改变(±)JA的作用趋势。(-)JA和(±)JA刺激保卫细胞胞质Ca2 变化则表现出不同趋势。说明不同异构体形式的JA在调节气孔运动中的作用和信号转导途径有所不同。     

超氧阴离子通过提高[Ca2+]cyt调节保卫细胞K+通道和气孔运动

氧化信号参与了许多生理过程的调控。用膜片钳和激光共聚焦显微镜,采用可以产生O2^ 的甲基紫精处理蚕豆(Vicia faba L)保卫细胞,测定了O2^ 对气孔运动调节过程中胞质Ca^2 离子浓度和细胞质膜K^ 通道活性的变化,结果表明甲基紫精可以促进气孔的关闭,乙二醇四乙酸酯(Ethylene glycol bis(2-aminoethyl)tetra-acetic acid,EGTA)、抗坏血酸(Ascorbic acid,AsA)和过氧化物酶(Catalase,CAT)可以消除小于10^-5mol／L甲基紫精对气孔运动的影响;10^-2和10^-5mol/L的甲基紫精可使保卫细胞胞质Ca^2 浓度有不同程度提高,并伴随有钙震荡。蚕豆气孔保卫细胞质膜内向K^ 通道可被咆外甲基紫精抑制,而这种抑制和[Ca^2 ]cyt有关。推测甲基紫精产生的O2^-对蚕豆气孔运动的调节,主要是通过O2^ 诱导的胞内游离Ca^2 浓度的升高,从而抑制了通过保卫细胞质膜K^ 内向电流。     

Ca^2＋参与NO对蚕豆气孔运动的调控

观察了Ca^2 、Ca^2 的螯合剂和Ca^2 通道抑制剂对NO调控的蚕豆气孔运动的影响。结果表明,NO的供体1～100μmol／L SNP(sodium nitroprusside,硝普纳)可诱导气孔关闭;除去表皮条缓冲液中的Ca^2 后,NO不再影响气孔的运动;Ca^2 的螯合剂EGTA和BAPTA几乎可以完全抑制NO诱导的气孔关闭作用;胞内钙通道抑制剂钌红(rutheniumred)和L型Ca^2 通道阻断剂硝苯吡啶(nifedipine)能够减弱SNP诱导气孔运动的关闭趋势;加入Ca^2 通道抑制剂LaCl3,则外源NO失去其诱导气孔关闭的作用。说明在NO调控的气孔运动中,在NO信号途径的下游可能涉及来自胞内和胞外Ca^2 的参与,并且胞外Ca^2 更为重要。     

激光扫描共聚焦显微镜观察胰蛋白酶对肺上皮细胞游离钙离子的影响

目的：研究PAR-2激动剂SLIGKV和tc-LIGRLO、胰蛋白酶及其抑制剂对H292肺上皮细胞[Ca^2＋]i的影响.方法：应用Fluo-3/AM 荧光标记技术和激光扫描共聚焦显微镜（LSCM） 检测不同因素处理的H292肺上皮细胞[Ca^2＋]i.结果：胰蛋白酶、SLIGKV、tc-LIGRLO均能引发H292细胞[Ca^2＋]i的增加,平均荧光强度分别比加入药物前增加267%,60%和37%.胰蛋白酶抑制剂大豆胰蛋白酶抑制剂（SBTI）和α1-抗胰蛋白酶（α1-AT）可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.结论：PAR-2可以介导H292肺上皮细胞[Ca^2＋]i的释放增加,胰蛋白酶抑制剂可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.     

3-羰基辛酰基高丝氨酸内酯诱导拟南芥根细胞Ca~（2＋）内流

N-酰基高丝氨酸内酯（AHLs）是革兰氏阴性细菌群体感应系统（QS）中的胞间通讯信号分子。近年的研究表明AHLs可以调控植物生长发育及防卫反应,但其调控机制尚不清楚。本研究以拟南芥为材料,采用3-羰基辛酰基高丝氨酸内酯（3OC8-HSL）处理转水母发光蛋白基因的拟南芥幼根细胞,利用冷光仪检测3OC8-HSL对拟南芥根细胞中胞质游离Ca2＋浓度（[Ca2＋]cyt）变化的影响,同时采用Ca2＋专一性螯合剂EGTA和Ca2＋通道抑制剂预处理转基因拟南芥根细胞,用全细胞膜片钳技术分析3OC8-HSL诱导拟南芥根细胞中[Ca2＋]cyt升高的Ca2＋来源。结果表明,3OC8-HSL可诱导拟南芥根细胞中[Ca2＋]cyt瞬时升高。这种诱导效应可被EGTA、异搏定（verapamil）、LaCl3所抑制,但LiCl预处理对这种诱导效应无影响。膜片钳分析结果显示,3OC8-HSL可激活质膜Ca2＋通道,增加胞外Ca2＋内流。说明细菌AHLs可诱导植物Ca2＋信号产生,且这种Ca2＋信号主要源于胞外Ca2＋内流,暗示Ca2＋信使系统参与植物对细菌QS信号的响应。     

小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca^2＋]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca^2＋荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca^2＋之间的内在联系。试验结果表明,[Ca^2＋]cyt的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca^2＋内流,进而造成[Ca^2＋]cyt的升高。     

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…     

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.     

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.     

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.     

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter-Specific Cytosolic Calcium Oscillations

Earlier studies have shown that various stimuli can induce specific cytosolic calcium （[Ca^2＋]cyt） oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca^2＋]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean （Vicia faba L.） was used and a rapid ion-exchange treatment with two shifting buffers differing in K^＋ and Ca^2＋ concentrations was applied. The treatment for fivetransients at a 10-min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some Irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation Is encoded by parameter-specific [Ca^2＋]cyt oscillation： the parameters of [Ca^2＋]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely Inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β-mercaptoethanol （an agent capable of reversing water channel inhibition by HgCl2）. Other Inhibitory treatments against Ion transport （i.e. the application of LaCIs, EGTA, or tetraethylammonlum chloride （TEACI）） weakly impaired stomatal oscillation when the compounds were added after rapid ion-exchange treatment. If these compounds were added before rapid-ion exchange treatment, the inhibitory effect was much more apparent （except In the case of TEACI）. The results of the present study suggest that water channels are involved In stomatal oscillation as a downstream element of [Ca^2＋]cyt oscillation signaling.     

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.

Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).     

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.