【作者】 张伟；

【导师】 马引利；

【作者基本信息】 山西师范大学 ， 生物学， 2019， 硕士

【摘要】 硫化氢（hydrogen sulfide,H₂S）和过氧化氢（hydrogen peroxide,H₂O₂）均对植物的生长发育有着极为重要的作用,它们都是调节植物体内信号转导途径的重要成分,但是H₂S在BR诱导的气孔运动过程中的作用及其与H₂O₂之间的关系尚不明确。本文以蚕豆（Vicia faba L.）、拟南芥（Arabidopsis thaliana L.）野生型（Col-0）、H₂S合成突变体（Atl-cdes、Atd-cdes）和H₂O₂合成突变体（AtrbohD、AtrbohF、AtrbohD/F）为实验材料,采用药理学试验、荧光显微技术以及分光光度检测法,研究了H₂S在BR调节的气孔关闭中的效应及其与H₂O₂的关系。所得实验结果主要如下:用不同浓度的H₂S供体NaHS处理蚕豆叶片的表皮条,观测到一定浓度的NaHS可以显著引起蚕豆气孔关闭,并且表现出明显的浓度依赖效应,其中表皮条经0.05mmol·L^-1和0.1 mmol·L^-1的NaHS溶液洗脱实验处理1.5 h后,其气孔开度还能恢复到正常水平。以上结果初步证明,外源H₂S可以诱导蚕豆气孔关闭。L-/D-半胱氨酸脱巯基酶（L-/D-CDes）是植物生成H₂S的主要来源,研究结果显示,HT（H₂S的清除剂）,AOA和NH₂OH（L-/D-CDes的抑制剂）以及C₃H₃KO₃+NH₃（L-/D-CDes反应的产物）均能显著抑制BR诱导的蚕豆和拟南芥野生型（Col-0）气孔关闭、叶片H₂S含量以及L-/D-CDes活性增加;此外,BR能诱导拟南芥野生型（Col-0）气孔的关闭,但对H₂S合成突变体Atl-cdes、Atd-cdes气孔开度没有明显影响。上述结果证明,H₂S参与了BR诱导的蚕豆和拟南芥气孔的关闭,并且H₂S的产生依赖于L-/D-CDes。将蚕豆叶片表皮条用100、200、300、400、500μmol·L^-1的H₂O₂在光下处理3 h,观测到≥100μmol·L^-11 H₂O₂能显著诱导蚕豆气孔关闭,并且表现出明显的浓度依赖效应。这一结果表明,外源H₂O₂能调控蚕豆气孔关闭。SHAM（细胞壁过氧化物酶抑制剂）、ASA（H₂O₂清除剂）、CAT（H₂O₂清除酶）、DPI（H₂O₂生成酶NADPH氧化酶抑制剂）均能够显著抑制BR诱导的蚕豆气孔的关闭以及内源H₂O₂水平提高;BR诱导的拟南芥野生型（Col-0）气孔关闭也能被SHAM、ASA、CAT、DPI所抑制;此外,BR能够诱导野生型（Col-0）及单突变体AtrbohD（NOX基因功能损失）气孔关闭,然而,对单突变体AtrbohF和双突变体AtrbohD/F气孔没有显著的影响,上述结果表明,BR促进蚕豆和拟南芥气孔关闭的效应与H₂O₂有关,并且H₂O₂的产生依赖于NOX途径。SHAM、ASA、CAT、DPI能够明显抑制BR诱导的蚕豆和拟南芥野生型（Col-0）叶片H₂S含量升高以及L-/D-CDes活性的增加,HT、AOA、NH₂OH以及C₃H₃KO₃+NH₃对BR引起的蚕豆和拟南芥野生型（Col-0）保卫细胞H₂O₂的水平升高没有显著影响;另外,BR对AtrbohF和AtrbohD/F叶片中H₂S的含量以及L-/D-CDes的活性没有明显影响;并且BR能够显著提高Atl-cdes和Atd-cdes保卫细胞中H₂O₂的水平。以上结果表明,H₂S位于H₂O₂下游调控BR诱导的气孔关闭过程。综上所述,本文研究结果表明,H₂S参与了BR调控的蚕豆以及拟南芥野生型（Col-0）气孔的关闭,并且在该过程中H₂S是位于H₂O₂下游发挥作用的。此外,BR调控的气孔关闭中H₂O₂的产生依赖于NOX途径,H₂S的产生依赖于L-/D-CDes。更多还原

【Abstract】 Hydrogen sulfide（H₂S）and Hydrogen peroxide（H₂O₂）play an important role in thegrowth and development of plants.They are important components that regulate the signal transduction pathway in plants.However,the role of H₂S in BR-induced stomatal movement and the relationship between H₂S and H₂O₂ remain unclear.In this paper,Vicia faba L.,Arabidopsis thaliana L.,H₂S synthetic mutants（Atl-cdes,Atd-cdes）and H₂O₂ synthetic mutants（AtrbohD,AtrbohF,AtrbohD/F）were used as experimental materials.Pharmacological test,laser confocal microscopy and spectrophotometry were used.The role of H₂S in BR controlled stomatal closure and its relationship with H₂O₂ were examined.The experimental results are as follows:1.After the epidermal strips of broad bean leaves were treated with different concentrations of H₂S donor NaHS,it was observed that a certain concentration of NaHS could induce the stomatal closure of broad bean significantly,and showed a concentration-dependent effect.The stomatal opening of the epidermal strips could return to normal level after 1.5 h of elution treatment with 0.05 mmol·L^-11 and 0.1 mmol·L^-11 NaHS solution.The above results preliminarily proved that H₂S induced by NaHS could induce stomatal closure in V.faba.2.L-/D-cysteine desulfurase（L-/D-CDes）is the main source of H₂S in plants.The results showed that HT（scavenger of H₂S）,AOA and NH₂OH（inhibitor of L-/D-CDes）,C₃H₃KO₃+NH₃（product of L-/D-CDes reaction）can inhibit BR-induced stomatal closure of broad bean and A.thaliana wild type（Col-0）in varying degrees.In addition,BR could induce stomatal closure in A.thaliana wild-type（Col-0）,but had no significant effect on H₂S synthetic mutants Atl-cdes and Atd-cdes stomatal closure.These results indicated that H₂S was involved in BR-induced stomatal closure in V.faba and A.thaliana,the production of H₂S depends on L-/D-CDes.3.After treated with H₂O₂ of different concentrations for 3 h,it was observed that the stomatal closure of V.faba leaves could be significantly induced by H₂O₂ of more than100μmol·L^-1,and the effect was concentration dependent.The results showed that exogenous H₂O₂ could induce stomatal closure in V.faba.4.ASA（an important reducing substrate for H₂O₂ removal）,CAT（one of the H₂O₂ scavenging enzymes）,DPI（an inhibitor of H₂O₂-generating enzyme NADPH oxidase）and SHAM（an inhibitor of inhibitor of cell wall peroxidase）could effectively inhibit BR-induced stomatal closure and the increase of endogenous H₂O₂ level in V.faba,while BR-induced stomatal closure in A.thaliana wild type（Col-0）could also be inhibited by ASA,CAT,DPI and SHAM.In addition,BR could induce stomatal closure of wild type（Col-0）and single mutant AtrbohD in A.thaliana,but had no significant effect on single mutant AtrbohF and double mutant AtrbohD/F.These results suggest that BR promotes stomatal closure in V.faba and A.thaliana and is related to H₂O₂,and the generation of H₂O₂ depends on the NOX pathway.5.ASA,CAT,DPI and SHAM could decrease the content of H₂S and the activity of L-/D-CDes in BR-induced broad bean and A.thaliana wild type（Col-0）leaves.HT,AOA,NH₂OH,C₃H₃KO₃+NH₃ had no significant effect on the level of H₂O₂ in BR-induced guard cells of broad bean and A.thaliana wild type（Col-0）;BR had no significant effect on the levels of H₂O₂ in AtrbohF and AtrbohD/F leaves.The content of H₂S and the activity of L-/D-CDes were not significantly affected,and the level of H₂O₂ in guard cells of Atl-cdes and Atd-cdes was effectively increased.These results suggest that H₂S is involved in the regulation of BR-induced stomatal closure in the downstream of H₂O₂.In conclusion,H₂S is involved in the closure of BR-induced stomata of V.faba and A.thaliana wild type（Col-0）,and H₂S plays a role downstream of H₂O₂ in the process of H₂S-induced stomatal closure in V.faba and A.thaliana.In addition,the production of H₂O₂in BR-induced stomatal closure was dependent on the NOX pathway,and H₂S production was dependent on L-/D-CDes.更多还原