G蛋白可能参与细胞外钙调素促进蚕豆气孔关闭的过程

存在于蚕豆保卫细胞质外体的细胞外钙调素(CaM)具有调控气孔运动的重要作用.以蚕豆表皮条为材料,利用激光共聚焦显微技术以及表皮条的生物分析方法研究了细胞外CaM促进气孔关闭的机理.实验结果表明,细胞外CaM能诱导保卫细胞[Ca 2+ ] cyt 升高,且升高程度随细胞外Ca 2+ 浓度的升高而增加;使用Ca 2+ 通道抑制剂的结果表明细胞外CaM诱导保卫细胞[Ca 2+ ] cyt 升高的过程以细胞外Ca 2+ 内流为主.利用G蛋白抑制剂PTX和激活剂CTX后发现,PTX能抑制细胞外CaM诱导的保卫细胞[Ca 2+ ] cyt 升高和气孔关闭,CTX也能通过诱导保卫细胞[Ca 2+ ] cyt 升高来促进气孔关闭,且Ca 2+ 主要来源于细胞外.说明G蛋白可能参与了细胞外CaM促进气孔关闭的过程.根据以上结果我们提出了细胞外CaM调控气孔运动的可能模式:细胞外CaM可能通过激活保卫.     

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。     

一氧化氮可能参与细胞外钙调素诱导蚕豆气孔关闭的过程

细胞外钙调素(CaM)被发现能够调节蚕豆及拟南芥气孔运动,而且G蛋白、H_2O_2及Ca~(2 )参与这一过程．从接受刺激到引起气孔关闭保卫细胞要经历复杂的信号转导,因此细胞外CaM调控气孔运动的信号转导途径还需要深入探讨．研究中以蚕豆下表皮条为材料,用表皮条生物分析和荧光显微技术研究了一氧化氮(NO)在细胞外CaM促进气孔关闭过程中的作用．结果表明:细胞外CaM能诱导保卫细胞内NO升高,一氧化氮合酶(NOS)抑制剂N~G-氮-L-精氨酸-甲酯(L-NAME)和NO清除剂2-苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(PTIO)抑制了这一过程,而硝酸还原酶(NR)抑制剂NaN_3则没有抑制作用;L-NAME和PTIO也抑制细胞外CaM诱导的气孔关闭过程,而NaN_3不能抑制这一过程．说明细胞外CaM主要通过NOS途径诱导NO合成,从而诱导气孔关闭．质膜Ca~(2 )通道抑制剂及Ca~(2 )螯合剂处理实验结果表明,细胞外CaM对保卫细胞内NO的诱导过程依赖Ca~(2 )内流．文中结果为NO参与细胞外CaM调节气孔运动的过程提供了直接证据．     

ABA和H2 O2在NaCl诱导的气孔关闭中的作用

以拟南芥野生型（wt）和ABA合成缺失突变体（aba3）为材料，采用表皮生物分析法研究了NaCl胁迫条件下，ABA和H2O2在气孔关闭中的作用。结果表明：100mmol/L的NaCl可有效诱导wt气孔关闭，而对aba3的气孔运动无明显影响；10^-2mmol/L的H2O2处理或100mmol/L的NaCl与10^-2mmol/L ABA共处理可有效诱导wt与aba3的气孔关闭，且幅度类似；H2O2的性清除剂CAT可部分逆转NaCl处理及NaCl与ABA共处理引起的气孔关闭，因此推测，NaCl胁迫条件下，植物保卫细胞内ABA浓度升高，诱导H2O2的产生，进而诱导了气孔关闭。     

外源NO诱导的拟南芥保卫细胞胞质pH升高先于气孔关闭

以pH敏感的绿色荧光蛋白拟南芥转基因植物为材料,研究了保卫细胞胞质pH变化与外源一氧化氮（nitric oxide,NO）诱导气孔关闭的关系,结果发现,与对照相比,100μmol／LNO供体硝普钠（sodium nitroprusside,SNP）可显著增加胞质的pH（增加约0．29个pH单位）,并诱导气孔关闭．NO清除剂c—PTIO、弱酸丁酸以及质膜H^＋-ATP酶的抑制剂钒酸钠均有效抑制了SNP诱导的保卫细胞胞质碱化和气孔关闭,而弱碱苄胺的作用与丁酸相反,说明保卫细胞胞质碱化介导了外源NO诱导的气孔关闭．另外,钙离子的螯合剂EGTA、钙调蛋白激酶抑制剂ML-7、钙调素的拮抗剂W7以及激酶抑制剂K-252a均可明显抑制外源NO诱导的胞质pH升高和气孔关闭,说明钙离子及磷酸化在NO诱导的保卫细胞胞质碱化和气孔关闭中发挥重要作用．     

异三聚体G蛋白参与调节花粉细胞内钙离子浓度

利用激光共聚焦显微镜测定了川百合(Lilium daviddi)花粉细胞内游离钙离子浓度,研究了细胞质膜上异三聚体G蛋白激活剂和抑制剂对花粉细胞内钙离子浓度的影响以及G蛋白激活后钙信号产生的可能途径.结果表明异三聚体G蛋白激活剂霍乱毒素(CTX)可以明显提高细胞内钙离子水平,产生带有一定时空特征的钙信号,而其抑制剂百日咳毒素(PTX)则显著降低细胞内钙离子水平;L型钙通道阻断剂异搏定和IP 3 受体抑制剂肝素都可以明显抑制霍乱毒素引起的钙离子水平升高.由此推断,异三聚体G蛋白可能在花粉细胞内钙离子水平调控过程中发挥重要调节作用,它有可能是通过促进细胞外钙离子内流和细胞内钙库释放两条途径起作用的.     

绒毛白蜡营养器官中Na^＋、K^＋、Cl^-的分布

为研究白蜡的抗盐生理机制，对盐渍生境下1—3年生绒毛白蜡（F．velutina）不同营养器官中盐离子的分布进行了分析。研究结果显示，不同器官中Na^＋、K^＋和Cl^-含量存在显著差异。根系，Na^＋、K^＋含量最高，Na^＋平均达0．72％，K^＋平均达13．95％；叶片，Cl^-含量最高，平均达1．04％；Na^＋、Cl^-含量均随树龄的增大而减少，K^＋含量随树龄的增大而增加。结果表明绒毛白蜡的高耐盐特性受营养器官中盐离子分布规律支持，并且绒毛白蜡能够随着树龄增长，逐步适应盐渍生境，耐盐能力逐步提高。     

PI3P和NO在UV-B诱导蚕豆气孔关闭中的关系

以蚕豆(Vicia faba L.)叶片下表皮为材料,结合气孔开度分析和保卫细胞内源一氧化氮(NO)水平的测定,研究了NO和磷脂酰肌醇3-激酶(PI3K)的催化产物磷脂酰肌醇3-磷酸(PI3P)在紫外线B(UV-B)诱导气孔关闭中的关系。结果显示:UV-B辐射诱导蚕豆保卫细胞NO产生和气孔关闭的效应能被PI3K抑制剂沃曼青霉素(WM)和LY294002(LY)显著抑制。同时,外源NO释放剂硝普钠(SNP)处理能完全逆转WM和LY对UV-B诱导气孔关闭的抑制效应,而WM和LY却不能抑制外源SNP诱导蚕豆气孔关闭的效应。结果说明,在UV-B诱导蚕豆气孔关闭的信号转导途径中PI3P的作用在NO上游。     

活性氧与NO在SO2诱导蚕豆气孔运动中的作用

以蚕豆叶表皮为材料,研究SO2胁迫时叶面气孔运动及其调节途径.研究发现,用浓度1～200μmol/L的SO2衍生物(亚硫酸钠与亚硫酸氢钠混合液)处理蚕豆叶下表皮后,气孔开度明显减小,气孔保卫细胞内活性氧(ROS)、一氧化氮(NO)和钙离子(Ca2+)水平显著升高.采用抗氧化剂抗坏血酸和过氧化氢酶,钙离子干扰剂EGTA和LaCl3,以及NO合成抑制剂NaN3与NO清除剂c-PTIO,分别与SO2衍生物同时作用时,SO2诱发的气孔关闭效应得到有效缓解,保卫细胞内ROS、NO和Ca2+水平随之改变.抗氧化剂和NO干扰剂能阻止SO2诱导的胞内ROS、NO和Ca2+水平升高;EGTA和LaCl3能降低SO2诱导的胞内NO和Ca2+升高,但不影响ROS水平.研究结果表明,较高浓度SO2能诱导气孔关闭,SO2胁迫诱导ROS和NO合成增加,ROS和NO通过钙信号系统调节气孔开度.     

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．     

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...     

肾蕨叶表皮结构和气孔器发育的研究

在光学和电子显微镜下对肾蕨(Nephrolepis auriculata (L.)Trimen)叶表皮结构和气孔器发育进行了观察.肾蕨成熟叶表皮细胞呈不规则形,含有叶绿体.气孔器仅存在于下表皮,每个气孔器有2个保卫细胞,但副卫细胞3～6个不等.仅有2个副卫细胞和保卫细胞同源.保卫细胞和副卫细胞都含有叶绿体.气孔器的发育过程可分为3个主要时期:1)保卫细胞母细胞形成期;2)气孔器幼期;3)气孔器成熟期.在扫描电镜下观察,覆盖在表皮基本细胞和气孔器细胞外壁的角质膜呈波纹状.     

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.     

烟草花药发育过程中钙动态分布的初步观察

在烟草花药发育过程中,用焦锑酸钾沉淀的松弛结合钙显示出了一个与花药发育事态有关的动态:在孢原细胞时期的花药中几乎看不到钙颗粒.但到小孢子母细胞时期,花药中开始有钙颗粒沉淀,而且显示出向花药室中流动的迹象.当花粉母细胞进行减数分裂时,花药绒毡层细胞和花粉母细胞中出现了许多的细小钙颗粒.在小孢子发育时期,在绒毡层细胞解体之前,细胞中的钙被集中到小液泡中.在小孢子细胞质中仍有较多的钙颗粒,同时在花粉壁上也有钙颗粒沉积在外壁的基部.到小孢子分裂形成二胞花粉后,细胞质中的钙明显减少.当二胞花粉中的大液泡消失,细胞质中积累淀粉粒以后,花粉中可看到的钙颗粒很少.同时,在花药维管束周围的薄壁细胞中,又出现了较多的钙颗粒,表明花粉对钙离子的需求可能已降低.烟草花药发育过程中钙的动态分布特征暗示着钙参与了调控花粉发育过程,尤其是与小孢子转变过程中的重要事态有密切关系.     

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.     

人参果试管苗与移栽成活苗气孔行为的比较

通过对人参果（Solanum muricatum Ait.)试管苗与移栽成活苗气孔大小、气孔开度日变化及保卫细胞对碘-硫酸反应的观察比较,研究了试管苗与移栽成活苗气孔行为的差异,结果表明,试管苗与移栽成活苗的气孔大小日变化幅度不大,但两者气孔开度的日变化明显不同;试管苗日变化曲线平稳,无明显波动;移栽成活苗日变化曲线呈明显波峰状,在13：00达到最大值,碘-硫酸反应表明,滴加硫酸后,试管苗与移栽成活苗气孔开度都显著增大,与硫酸作用前相比,移栽成活苗的增加幅度约6倍于试管苗,这可能与两者保卫细胞壁刚性不同有关。     

Localization of NOS-like protein in guard cells of Vicia faba L. and its possible function

Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of ni- tric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS in- hibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO genera- tion from NOS pathway is the main pathway in the stress and JA responses. The pharmacological ex- periment showed that increasing the Ca2 at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2 -dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.     

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.     

Regulation of the inward K+-channels in stomatal guard cells by cytoskeletal microtubules

Patch clamp techniques were applied to investigating the regulation of the inward K⁺-channels inVicia stomatal guard cells by cytoskeletal microtubules. The intracellular addition of either microtubule-disassembling reagent amprophos-methyl (APM) or microtubule-stabilizing reagent taxol resulted in significant inhibition of the inward K⁺-currents across the plasma membranes ofVicia stomatal guard cells. The results suggest that the activation of the inward K⁺-channels in stomatal guard cells requires proper dynamic organization of cytoskeletal microtubules. The regulation of the inward K⁺-channels in guard cells by microtubules may mediate the regulation of stomatal movements by cytoskeleton.