H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

H2S位于SOS上游参与盐胁迫诱导的拟南芥气孔关闭

以拟南芥野生型、SOS突变体(Atsos1、Atsos2和Atsos3)、H2S合成相关酶L-/D-半胱氨酸脱巯基酶(L-/D-CDes)基因缺失突变体(Atl-cdes和Atd-cdes)和过表达株系(OEL-CDes和OED-CDes)为材料研究了H2S和SOS信号转导途径在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片H2S含量、L-/D-CDes活性及其基因表达量显著升高,诱导野生型拟南芥和OEL-CDes和OED-CDes叶片气孔关闭,但对Atl-cdes和Atd-cdes气孔开度无显著影响;而H2S清除剂次牛磺酸(hypotaurine,HT)可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明H2S参与盐胁迫诱导的拟南芥气孔关闭过程。外源H2S诱导野生型拟南芥气孔关闭,但对SOS突变体气孔开度无显著影响;同时盐胁迫下Atsos1、Atsos2和At-sos3亦表现出H2S含量及L-/D-CDes活性显著升高,且与野生型相比,盐胁迫对Atl-cdes和Atd-cdes叶片AtSOS基因表达量无显著影响。表明盐胁迫诱导气孔关闭过程中H2S位于SOS上游。     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO₂浓度升高可以诱导植物叶片气孔关闭, 提高植物对高浓度CO₂的适应性。但植物如何感知CO₂浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚。利用高通量、非侵入的远红外成像技术, 建立了拟南芥(Arabidopsis thaliana)气孔对CO₂浓度变化反应相关的突变体筛选技术, 筛选出对环境CO₂浓度敏感的拟南芥突变体ecs1。遗传学分析表明, ecs1为单基因隐性突变体, 突变基因ECS1编码一个跨膜钙离子转运蛋白。与野生型拟南芥相比, 360 μL·L^–1CO₂可引起ecs1突变体叶片温度上升和气孔关闭, ecs1突变体对900 μL·L^–1CO₂长时间处理具有较强的适应性。进一步的实验表明, 360μL·L^–1CO₂即可诱导ecs1突变体叶片积累较高浓度的H₂O₂, 而900 μL·L^–1CO₂才能够诱导野生型拟南芥叶片积累H₂O₂。因此, ECS1可能参与调节高浓度CO₂诱导的拟南芥气孔关闭和H₂O₂产生, H₂O₂可能作为第二信号分子介导CO₂诱导拟南芥气孔关闭的反应。     

H2S位于SOS上游参与盐胁迫诱导的拟南芥气孔关闭

以拟南芥野生型、SOS突变体印tsosl、Atsos2和Atsos3）、H2S合成相关酶L-／D-半胱氨酸脱巯基酶（L-／D-CDes）基因缺失突变体（Atl-cdes和Atd-cdes）和过表达株系（OEL—CDes和OED-CDes）为材料研究了H,s和SOS信号转导途径在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片H,S含量、L-／D-CDes活性及其基因表达量显著升高,诱导野生型拟南芥和OEL—CDes和OED．CDes叶片气孔关闭,但对Atl-cdes和Atd-cdes气孔开度无显著影响;而H2S清除剂次牛磺酸（hypotaurine,HT）可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明H2S参与盐胁迫诱导的拟南芥气孔关闭过程。外源H2S诱导野生型拟南芥气孔关闭,但对SOS突变体气孔开度无显著影响;同时盐胁迫下Atsosl、Atsos2和Atsos3亦表现出H2S含量及L-／D-CDes活性显著升高,且与野生型相比,盐胁迫对Atl-cdes和Atd-cdes叶片AtSOS基因表达量无显著影响。表明盐胁迫诱导气孔关闭过程中H2S位于SOS上游。     

ABC转运体位于H2S上游参与盐胁迫诱导的拟南芥气孔关闭

本文以拟南芥野生型、ABC转运体缺失突变体（Atmrp4、Atmrp5和Atmrp4/5）为材料研究了硫化氢（hydrogen sulfide,H₂S）和ABC转运体在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片AtMRP4及AtMRP5表达量显著升高,诱导野生型拟南芥叶片气孔关闭,但对Atmrp4、Atmrp5及Atmrp4/5气孔开度无显著影响;而ABC转运体抑制剂格列本脲（glibenclamide,Gli）可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明ABC转运体参与盐胁迫诱导的拟南芥气孔关闭过程。盐胁迫能够引起野生型拟南芥H₂S合成相关酶L-/D-半胱氨酸脱巯基酶（L-/D-CDes）活性及H₂S含量显著升高,而ABC转运体抑制剂格列本脲处理后则没有这种变化,同时盐胁迫也不能引起Atmrp4、Atmrp5及Atmrp4/5的L-/D-CDes活性及H₂S含量显著升高,表明ABC转运体位于H₂S上游参与盐胁迫诱导气孔关闭过程。     

ABC转运体位于H2S上游参与盐胁迫诱导的拟南芥气孔关闭

本文以拟南芥野生型、ABC转运体缺失突变体（Atmrp4、Atmrp5和Atmrp4／5）为材料研究了硫化氢（hydrogensulfide,H2S）和ABC转运体在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片AtMRP4及AtMRP5表达量显著升高,诱导野生型拟南芥叶片气孔关闭,但对Atmrp4、Atmrp5及Atmrp4／5气孔开度无显著影响;而ABC转运体抑制剂格列本脲（glibenclamide,Gli）可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明ABC转运体参与盐胁迫诱导的拟南芥气孔关闭过程。盐胁迫能够引起野生型拟南芥H,s合成相关酶L-／D-半胱氨酸脱巯基酶（L-／D-CDes）活性及H2S含量显著升高,而ABc转运体抑制剂格列本脲处理后则没有这种变化,同时盐胁迫也不能引起Atmrp4、Atmrp5及Atmrp4／5的L-／19-CDes活性及H2S含量显著升高,表明ABC转运体位于H2s上游参与盐胁迫诱导气孔关闭过程。     

磷脂酶Dα1与H2S参与冬凌草甲素对拟南芥的化感作用

以拟南芥哥伦比亚野生型(WT)、磷脂酶Dα1(PLDα1)缺失型突变体pldα1、D-/L-半胱氨酸脱巯基酶(D-/L-CDes)缺失型突变体d-cdes和l-cdes幼苗为试验材料,60 μmol·L^-1冬凌草甲素为处理浓度,研究了拟南芥响应二萜类化合物冬凌草甲素的化感作用中磷脂酶Dα1(PLDα1)与气体信号分子硫化氢(H₂S)的信号关系。结果表明: 冬凌草甲素显著提高了野生型拟南芥幼苗H₂S含量、PLD和D-/L-CDes酶的活性及其基因表达;冬凌草甲素处理下,pldα1突变体幼苗的D-CDes和L-CDes活性明显低于WT,外源添加磷脂酸(PA)后D-CDes和L-CDes活性显著提高,并高于WT;冬凌草甲素显著抑制4种株系根的生长,其中d-cdes和l-cdes对冬凌草甲素更加敏感,外施NaHS可以促进冬凌草甲素处理下4种株系根的生长及内源H₂S产生,外施PA只对冬凌草甲素处理下的WT、pldα1和l-cdes株系根的生长及内源H₂S产生有促进作用,而对d-cdes株系没有明显作用。说明PLDα1和H₂S在拟南芥响应冬凌草甲素过程中发挥作用,且PLDα1/PA位于D-CDes上游,参与调控拟南芥幼苗H₂S的产生及根生长的信号过程。     

PI3P在暗诱导的蚕豆气孔关闭中的作用及与H2O2和NO的关系

以蚕豆(Vicia faba)表皮条为材料, 利用磷脂酰肌醇3-激酶(PI3K)的抑制剂沃曼青霉素(WM)和LY294002 (LY)抑制磷脂酰肌醇3-磷酸(PI3P)的形成, 并结合气孔开度分析及激光扫描共聚焦显微镜技术, 探讨暗诱导蚕豆气孔关闭过程中PI3P与过氧化氢(H₂O₂)和一氧化氮(NO)之间的相互关系。结果表明, WM和LY显著抑制暗诱导的保卫细胞H₂O₂和NO的形成以及气孔的关闭, 但不能抑制外源H₂O₂和NO诱导的气孔关闭, 外源H₂O₂和NO处理能完全逆转WM和LY对暗诱导的气孔关闭的抑制效应。实验结果暗示, 在暗诱导的气孔关闭的信号转导途径中PI3P在信号分子H₂O₂和NO的上游起作用。     

eATP位于H2S下游参与乙烯诱导的拟南芥气孔关闭过程

以拟南芥(Arabidopsis thaliana)野生型、H₂S合成突变体(Atl-cdes和Atd-cdes)和ABC转运体突变体(Atmrp4、Atmrp5 和Atmrp4/5)为材料, 探讨乙烯诱导气孔关闭过程中eATP与H₂S之间的关系。结果显示, ABC转运体阻断剂格列本脲(Gli)、P2受体抑制剂磷酸吡哆醛-6-偶氮苯基-2',4'-二硫酸(PPADS)和三磷酸腺苷双磷酸酶(Apyrase)可抑制乙烯诱导的气孔关闭乙烯可提高拟南芥幼苗叶片eATP含量及AtMRP4和AtMRP5相对表达量, 但对Atmrp4、Atmrp5和Atmrp4/5突变体幼苗叶片eATP含量和气孔运动没有显著作用。实验结果表明, eATP是乙烯诱导拟南芥气孔关闭过程的重要信号分子, AtMRP4和AtMRP5参与胞内ATP的分泌H₂S清除剂次牛磺酸(HT)能阻遏乙烯诱导的拟南芥幼苗叶片eATP含量的升高乙烯对Atl-cdes、Atd-cdes幼苗叶片eATP含量及AtMRP4和AtMRP5相对表达量无显著影响。据此推测eATP位于H₂S下游参与乙烯诱导的拟南芥气孔关闭过程。     

H2S可能作为H2O2的下游信号介导茉莉酸诱导的蚕豆气孔关闭

以蚕豆（Vicia faba）为材料,利用激光共聚焦显微技术和分光光度技术,结合药理学实验,探讨硫化氢（hydrogen sulphide,H2S）和过氧化氢（hydrogen peroxide,H2O2）在茉莉酸（jasmonic acid,JA）调控气孔运动信号转导中的作用。结果表明,H2S合成抑制剂氨氧基乙酸（aminooxy acetic acid,AOA）、羟胺（hydroxylamine,NH2OH）、丙酮酸钾（potasium py-ruvate,C3H3KO3）和氨水（ammonia,NH3）,H2O2清除剂抗坏血酸（ascorbic acid,AsA）,合成抑制剂水杨羟肟酸（salicylhydroxamic acid,SHAM）、二苯基碘（diphenylene iodonium,DPI）均可逆转JA诱导的气孔关闭效应。JA能够明显提高蚕豆叶片及保卫细胞中的H2O2水平、H2S含量和L-/D-半胱氨酸脱巯基酶活性;H2S合成抑制剂可抑制JA引起的叶片H2S含量的增加;而H2O2清除剂则可减弱JA对H2S含量变化和L-/D-半胱氨酸脱巯基酶活性的诱导效应。以上结果表明H2S和H2O2均参与了JA诱导的蚕豆气孔关闭,且H2S（主要由L-/D-半胱氨酸脱巯基酶合成）可能作为H2O2的下游组分参与调控这一信号转导过程。     

H_2O_2介导H_2S诱导的拟南芥气孔关闭

以拟南芥（Arabidopsis thaliana）为材料,研究了过氧化氢（H2O2）在硫化氢（H2S）调控气孔运动信号转导中的作用。结果表明,光下H2S的供体硫氢化钠（NaHS）能够诱导拟南芥气孔关闭;且能够显著提高叶片和保卫细胞胞质H2O2含量;H2O2的清除剂AsA和H2O2合成酶的抑制剂可不同程度地抑制NaHS诱导的拟南芥气孔关闭及叶片和保卫细胞胞质H2O2水平的升高;NaHS对AtrbohD、AtrbohF、Atpao2和Atpao4突变体气孔关闭、叶片和保卫细胞胞质H2O2水平升高的诱导作用要明显的小于野生型,但对AtPAO2和AtPAO4过表达株系叶片和保卫细胞H2O2水平的升高较野生型显著。据此推测,来源于NADPH氧化酶、细胞壁过氧化物酶和多胺氧化酶途径的H2O2参与H2S诱导的拟南芥气孔关闭。     

Chitosan-induced stomatal closure accompanied by peroxidase-mediated reactive oxygen species production in Arabidopsis

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.     

H2S介导ABA诱导蚕豆气孔运动的生理机制研究

以蚕豆为实验材料,利用药理学实验和分光光度法,研究了ABA处理及ABA与H2S合成抑制剂共处理对蚕豆气孔运动的影响,以及体内H2S水平、H2S合成酶L-/D-半胱氨酸脱巯基酶(磷酸吡哆盐依赖性酶)活性变化.结果表明:(1)光下H2S的合成抑制剂羧甲氧基胺半盐酸盐(AOA)、羟氨(NH2OH)、L-/D-半胱氨酸脱巯基酶分解产物C3H3KO3+NH3均明显抑制ABA诱导的蚕豆气孔关闭;(2)外源ABA能够明显提高叶片的H2S水平及L-/D-半胱氨酸脱巯基酶活性;(3)AOA、NH2OH、C3H3KO3和NH3均可以逆转ABA所引起的H2S水平及L-/D-半胱氨酸脱巯基酶活性的升高.研究发现,ABA可通过增强L-/D-半胱氨酸脱巯基酶活性,促进L-/D-半胱氨酸分解生成H2S,进而诱导蚕豆气孔关闭.     

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2?](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2?](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2?](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2?](cyt) oscillations.     

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.     

NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na⁺/K⁺homeostasis in Arabidopsis under salt stress

Maintaining cellular Na(+)/K(+) homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na(+)/K(+) homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na(+) contents, lower K(+) contents, and resultant greater Na(+)/K(+) ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H(2)O(2) can partially reverse the increased effects of NaCl on Na(+)/K(+) ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na(+)/K(+) ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K(+) currents were arrested, and NaCl-promoted increases in cytosolic Ca(2+) and plasma membrane Ca(2+) influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na(+)/K(+) homeostasis, thus improving the salt tolerance of Arabidopsis.     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。