NO和H2O2在光/暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H2O2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索.结果显示,光下外源NO供体硝普钠(SNP)和H2O2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂NG-氮-L-精氨酸-甲酯(L-NAME)和H2O2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H2O2水平比暗中明显降低.上述结果表明,光/暗通过影响保卫细胞NO和H2O2的水平调控气孔运动.研究还发现,光下H2O2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H2O2的这些效应;光下SNP既诱导H2O2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转.这些结果表明,NO和H2O2在生成及效应上均存在明显的相互作用.另外,L-NAME显著逆转暗和光下H2O2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H2O2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭.     

UV-B辐射对蚕豆叶片气孔运动的间接效应与NO和H2O2有关

0.2 W.m-2的UV-B辐射不仅能诱导整体蚕豆叶片气孔导度和开度的显著降低,而且能明显降低蚕豆叶肉光合活性,但该强度的UV-B辐射却不能明显影响离体表皮条的气孔开度.说明0.2W.m-2的UV-B主要通过间接途径调控了蚕豆叶片气孔运动.借助药理学试验和激光扫描共聚焦显微镜技术,进一步对该间接效应过程中是否有NO和H2O2的参与进行了探讨.结果显示:NO专一性清除剂cPT IO和一氧化氮合酶(NO S)抑制剂L-NAM E均能有效地抑制UV-B辐射诱导的叶片气孔关闭和保卫细胞内源NO水平的升高;H2O2清除剂抗坏血酸(A SC)和过氧化氢酶(CAT)也能有效地逆转UV-B辐射诱导的气孔关闭和保卫细胞内源H2O2含量的升高.另外,外源NO或H2O2处理也能有效地诱导叶片气孔关闭.结果说明0.2W.m-2的UV-B辐射对蚕豆叶片气孔关闭的间接诱导与NO和H2O2有关.     

外源NO、H2O2和ABA对鸡蛋花花冠裂片上气孔关闭的影响

以鸡蛋花花冠裂片下表皮为材料,研究不同浓度及不同处理时间的外源NO、H2O2和ABA对鸡蛋花花冠裂片下表皮上气孔关闭的影响,以及NO、H2O2和ABA在调节花冠上气孔关闭中的相互作用。结果表明：单独施用NO、H2O2和ABA明显诱导气孔关闭,并有浓度效应和时间效应;NO、H2O2和ABA两两混合或三者混合施用所诱导气孔关闭的效应大于其单独施用的。说明在诱导气孔关闭时,NO、H2O2和ABA之间可能有协同效应。     

外源NO和H2O2对洋葱鳞片外表皮气孔开度的调控

以洋葱(Allium cepa L.)肉质鳞片外表皮为材料,研究不同浓度及不同处理时间的外源NO和H2O2对洋葱鳞片外表皮上气孔开度的调节作用,并结合NO清除剂血红蛋白(Hb)和H2O2清除剂过氧化氢酶(CAT)研究调控过程中NO和H2O2的相互关系.结果显示:单独施用不同浓度的NO和H2O2均可诱导洋葱鳞片外表皮气孔不同程度关闭,并且浓度越大时间越长,其诱导气孔关闭效应越明显;NO和H2O2共同施用所诱导气孔关闭的效应大于其单独施用效应;Hb和CAT能明显减弱NO和H2O2诱导的气孔关闭.研究表明,NO和H2O2能有效诱导洋葱鳞片上气孔关闭,存在明显的浓度效应和时间效应,且两者可能互相依赖,具有协同效应.     

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。     

Ca~(2+)参与NO对蚕豆气孔运动的调控

观察了Ca2 + 、Ca2 + 的螯合剂和Ca2 + 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～ 10 0 μmol/LSNP (sodiumnitroprusside ,硝普纳 )可诱导气孔关闭 ;除去表皮条缓冲液中的Ca2 + 后 ,NO不再影响气孔的运动 ;Ca2 + 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Ca2 + 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Ca2 + 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Ca2 + 的参与 ,并且胞外Ca2 + 更为重要。     

一氧化氮是脱落酸诱导杨树叶片气孔关闭的信号分子

研究了外源NO和ABA对杨树气孔运动调节作用．结果表明,外源NO和ABA都能诱导杨树离体叶片气孔关闭,且具有剂量效应,NO可加强ABA诱导气孔关闭的作用．NO清除剂(c—PTIO)可大大减弱NO和ABA对气孔关闭的诱导作用．证实了NO参与ABA调控气孔开闭运动过程,不同浓度NO供体SNP和ABA处理杨树离体叶片,SOD活性变化不明显,POD活性受到显著抑制．杨树叶片粗酶液的体外实验表明,不同浓度SNP对POD活性的抑制呈明显的浓度及时间效应;而ABA对POD活性则几乎没有影响．本研究证明,NO调节ABA诱导的树木气孔关闭作用,是ABA诱导树木气孔关闭的一种重要信号分子．     

共聚焦显微技术研究SA对蚕豆气孔保卫细胞的影响

水杨酸(salicylic acid,SA)作为植物体内一种内源性的信号分子,具有多种生理功能.实验表明水杨酸以浓度依赖的方式诱导气孔关闭,抑制气孔张开.20U/mL的CAT与SA共同处理时可逆转SA诱导气孔关闭作用的83%~90%.以H2O2荧光探针H2DCFDA结合激光扫描共聚焦显微术直接检测到SA处理可引起保卫细胞内H2O2的产生;在保卫细胞内显微注射CAT可完全阻止SA导致的DCF荧光增强.表明SA诱导的气孔关闭可能与H2O2的产生有关.     

一氧化氮在乙烯诱导蚕豆气孔关闭中的作用

以蚕豆为材料研究了一氧化氮(nitric oxide,NO)和乙烯对气孔运动的影响。结果表明,10μmol/L的NO供体硝普钠(sodium nitroprusside,SNP)以及0.04%的乙烯能明显诱导蚕豆气孔关闭,并且二者共同处理后,能够增强其促进气孔关闭的作用。乙烯合成抑制剂AVG可以减弱NO诱导气孔关闭的程度,NO清除剂c-PTIO和NR抑制剂NaN3也可减弱乙烯诱导气孔关闭的程度,而一氧化氮合酶(nitric oxide synthase,NOS)抑制剂L-NAME对乙烯诱导气孔关闭的作用不明显。推测,在调控蚕豆气孔关闭过程中,NO可能主要通过NR途径参与乙烯调控气孔关闭过程。     

NO和H2O2在光／暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H_2O_2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索。结果显示,光下外源NO供体硝普钠(SNP)和H_2O_2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂N~G-氮-L-精氨酸-甲酯(L-NAME)和H_2O_2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H_2O_2水平比暗中明显降低。上述结果表明,光/暗通过影响保卫细胞NO和H_2O_2的水平调控气孔运动。研究还发现,光下H_2O_2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H_2O_2的这些效应;光下SNP既诱导H_2O_2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转。这些结果表明,NO和H_2O_2在生成及效应上均存在明显的相互作用。另外,L-NAME显著逆转暗和光下H_2O_2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H_2O_2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭。     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H2O2的产生（简报）

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.     

Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba

One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H(2)O(2)), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H(2)O(2) may function as an intermediate in ABA signaling in Vicia faba guard cells. H(2)O(2) inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10(-5) M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H(2)O(2) scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H(2)O(2) was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H(2)O(2) production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H(2)O(2) production involved, and H(2)O(2) may be an intermediate in ABA signaling.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Nitric oxide inhibits blue light-specific stomatal opening via abscisic acid signaling pathways in Vicia guard cells

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO's effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H(+) pumping and H(+)-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H(2)O(2)) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H(2)O(2). ABA, H(2)O(2) and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H(+) pumping in guard cells nor H(+) transport by H(+)-ATPase in the isolated membranes. From these results, we conclude that both NO and H(2)O(2) inhibit blue light-induced activation of H(+)-ATPase by inhibiting the component(s) between phototropins and H(+)-ATPase in guard cells and stimulate stomatal closure by ABA.     

ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.