【作者】 张莉；

【导师】 姚雅琴；

【作者基本信息】 西北农林科技大学 ， 植物学， 2015， 硕士

【摘要】 气孔是植物对外进行气体交换的“窗口”,它的开关不仅与光合和蒸腾有关,而且与植物的生物与非生物胁迫等的抗逆性密切相关。到目前为止,有关气孔运动机理的研究主要局限于保卫细胞,而副卫细胞方面的研究很少,关于气孔运动过程副卫细胞中H2O2、Ca2+、K+的调节关系方面的研究未见报道。我们前期的研究发现玉米叶片气孔开关不仅与保卫细胞中的H2O2有关,而且也与副卫细胞中的H2O2有关。本文以玉米(郑单958)为材料进行了两部分的研究。第一部分是结合药理学处理及细胞化学定位等研究手段,研究玉米叶片副卫细胞中H2O2、Ca2+、K+在气孔开关过程中相互调节的关系。实验通过外加H2O2及NADPH氧化酶抑制剂(DPI)、外加K+及K+通道抑制剂(BaCl2)和Ca2+及胞外Ca2+通道抑制剂(LaCl3),来观察气孔运动过程中保卫细胞与副卫细胞中H2O2、Ca2+、K+的含量及分布变化,确定在两种细胞中它们之间的调节关系。第二部分,为了观察K+的分布,根据光学显微镜和电子显微镜的成像原理,利用多年来分析化学常用的微量钾定性检测试剂亚硝酸钴铅钠(NaPbCo(NO2)6)能特异地与K+结合,形成KPbCo(NO2)6沉淀颗粒的机制,探索出了用光学显微镜和电子显微镜观察K+在细胞水平和亚细胞水平分布的方法。本研究第一部分所取得的结果是:1.外加H2O2,能使保卫细胞中H2O2和Ca2+的含量增加,K+的含量减少;能使副卫细胞中H2O2、Ca2+和K+三者的含量均增加。经DPI处理,能使保卫细胞中H2O2、Ca2+的含量减少,K+的含量增加;能使副卫细胞中H2O2、Ca2+和K+的含量均减少。推断在副卫细胞和保卫细胞中H2O2是Ca2+和K+的上游信号。2.外加K+,能使保卫细胞和副卫细胞中K+增加,会使H2O2的含量减少,可能是过量K+会对H2O2的增加有抑制作用,而Ca2+的变化却不明显;经BaCl2处理,能使保卫细胞或副卫细胞中的K+减少,而H2O2与Ca2+的变化均不明显。推断在副卫细胞和保卫细胞中K+是H2O2和Ca2+的下游信号。3.外加Ca2+,能使保卫细胞中Ca2+和H2O2的含量增加,使K+的含量减少;使副卫细胞中Ca2+、H2O2和K+的含量均增加。经LaCl3处理,能使保卫细胞中Ca2+和H2O2含量减少,K+的含量增加;使副卫细胞中Ca2+、H2O2和K+的含量均减少。推断在副卫细胞和保卫细胞中Ca2+是H2O2和K+的上游信号。综合上述研究结果得出的结论是:保卫细胞中H2O2、Ca2+、K+三者的相互关系符合研究现状,并且根据保卫细胞中三者已有的调节关系:Ca2+和H2O2是K+的上游信号,并且双方是正反馈的调节关系,同时与K+是负相关的调节关系。推测副卫细胞中三者的调节关系与保卫细胞中的相同,即Ca2+和H2O2也是K+的上游信号,两者属于正反馈调节,但是与K+是正相关的调节关系。本研究第二部分所取得的结果是:经过多种实验方案的比较,探索出了亚显微结构中K+的原位检测方法。经此法处理的植物组织,细胞结构保持比较完好,显色特异性强,灵敏度高,经其检测,K+主要分布在维管束,细胞膜,叶绿体,细胞核及核仁内部,与K+分布的研究现状高度吻合,印证了该方法可以作为植物亚显微结构中K+原位检测的新技术。实现了植物亚显微结构中K+的原位检测,可以帮助我们了解K+在亚显微结构中分布位置和相对含量的差异。更多还原

【Abstract】 The stoma is the "window" for the gas exchange in plant, and its movement is not only related to its photosynthesis or transpiration status, but also closely related to the biotic and abiotic stresses. So far, researches about the mechanism of stomatal movement have been concentrated on guard cells rather than the subsidiary cells, and there is no available research being done on the H2O2, Ca2+, K+ regulation relationship among stomatal movement in subsidiary cells. Our previous study has found that the stomata movement is not only related to the H2O2 in guard cells, but also the H2O2 in subsidiary cells.Our research has been designed into two parts. In the first part, we use maize(Zhengdan 958)as materials, combined with pharmacological treatment and cytochemical localization of research methods to investigate the regulatory relationship among H2O2, Ca2+, K+ in the process of stomatal movement in maize leaves. In order to observe H2O2、Ca2+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous H2O2 and NADPH oxidase inhibitor(DPI) were applied to control the content of H2O2. In order to observe Ca2+, K+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous K+ and K+ channel inhibitor(BaCl2) were applied to control the content of K+. In order to observe H2O2、K+ content and distribution variation in guard cells and subsidiary cells during stomatal movement, exogenous Ca2+ and extracellular Ca2+ channel inhibitors(LaCl3) were applied to control the content of Ca2+. In the second part, in order to observe K+ distribution, we used a well developed analytically chemical reagent NaPbCo(NO2)6, which can be used to detect the trace potassium qualitative detection reagent by specifically binding to K+ and forming KPbCo(NO2)6 precipitates. This method has improved the existing K+ optical microscope detection method at a cellular level and created a novel K+ detecting method in electron microscope at a subcellular level based on the imaging principle of optical microscope and electron microscope.The results of the first part of this study are as follows:1. Exogenous H2O2 can elevate the content of Ca2+ and H2O2 and decrease the content of K+ in guard cells, while increase the content of the three elements in the subsidiary cells. Exogenous DPI treatment can decrease the content of Ca2+ and H2O2 and increase the content of K+ in guard cells, while decrease the content of the three elements in the subsidiary cells. This result indicates that H2O2 in guard cells and accessory cells is an upstream signal of Ca2+ and K+.2. Exogenous K+ can elevate the content of K+ and decrease the content of H2O2 in both guard cells and subsidiary cells. No obvious variation was observed in Ca2+ during such process. Exogenous BaCl2 treatment can decrease the content of K+ in guard cells or subsidiary cells. This result indicates that K+ in guard cells and accessory cells is a downstream signal of H2O2 and Ca2+.3. Exogenous Ca2+ can elevate the content of Ca2+ and H2O2 and decrease the content of K+ in guard cells, while increase the content of the three elements in the subsidiary cells. Exogenous LaCl3 treatment can decrease the content of Ca2+ and H2O2 and increase the content of K+ in guard cells, while decrease the content of the three elements in the subsidiary cells. This result indicates that Ca2+ in guard cells and subsidiary cells is an upstream signal of H2O2 and K+.Based on the above research results we reached a conclusion: the relationship among H2O2, Ca2+, K+ in guard cells is consistent with the current opinion. And according to the existing regulation relationship theory in guard cells, which believes Ca2+ and H2O2 is the upstream signal of K+, and their regulation is positively related and negatively related to K+, we speculated that it is the same in subsidiary cells as in guard cells, which is, Ca2+ and H2O2 is the upstream signal of K+, and the two are positive feedback regulation, but K+ is positively regulated by the two elements.The results of the second part of this study are as follows:We created a situ detection of potassium ions in submicroscopic structure by comparison a variety of experimental program. After this treatment, cell structure of plant tissue will remain relatively intact and color will show specificity. The method is highly sensitivite and according to the test result, K+ is mainly distributed in the vascular bundles, cell membrane, chloroplast, the nucleus and nucleolus, which is considerably consistent with current research results. It confirms the feasibility of this method to be used as new technology in detecting the potassium ions in plant submicroscopic structures in situ. This method can help us understand the distribution and relative differences in content of positions in the submicroscopic structure.更多还原