【作者】 姜玉萍；

【导师】 喻景权；

【作者基本信息】 浙江大学 ， 蔬菜学， 2010， 博士

【摘要】 黄瓜是设施栽培的主要蔬菜之一,在生产上和消费上占有主导地位。黄瓜生长发育对环境条件的要求相对较高。然而由于我国农业基础设施相对简陋和生产管理水平相对落后,黄瓜的产量和品质常常受到各种胁迫的影响。油菜素内酯(BRs)具有提高作物光合作用及增加抗逆性的效果,但具体机理还不是十分清楚,因此探明BRs提高光合作用的机理在科学上及实践上具有十分重要的意义。本文以具有不同BRs水平的黄瓜(Cucumis sativus L.)为材料,研究了利用化学遗传学、生理学、分子生物学等手段从光合相关基因表达、H2O2及redox信号的角度探讨了BRs调控作物光合作用的机理,探讨了redox在BR调控光合作用中的信号作用。所得主要结果如下：1.研究了EBR、H2O2对光合参数、Rubisco酶的影响。通过处理24-表油菜素内酯(EBR)和H2O2能直接有效改变黄瓜植株表观形态,并引起CO2同化能力和光化学量子效率(ΦPSII)的变化。EBR、H2O2处理后,PSII电子传递量子效率(ΦPSII)明显增加,这主要是由于光化学猝灭(qP)增加所引起。研究发现,EBR、H2O2均可以调控Rubisco羧化速率(Vc,max)、RuBP再生速率(Jmax)及Rubisco初始活力,以及改变植株的光合作用。此外,EBR和H202导致Rubisco初始活性、蔗糖合成酶(SS)和酸性转化酶(AI)活性的提高,以及可溶性总糖和淀粉含量的累积。总之,EBR、H2O2明显提高了卡尔文循环中的C02同化能力,而其中主要原因是由于EBR、H2O2对Rubisco初始活性有明显促进作用。2.研究了H2O2在BR调控碳水化合物代谢中的作用。通过预处理H202的清除剂DMTU和NADPH氧化酶抑制剂(DPI)进一步分析H2O2在EBR调控碳水化合物积累中的作用。研究发现,外源EBR能够改变碳水化合物的含量而H2O2的清除剂DMTU和NADPH氧化酶抑制剂(DPI)却能够阻断BRs对碳水化合物的诱导作用,这表明BRs改变碳水化合物的含量主要通过激活NADPH氧化酶诱导产生的H2O2而介导的。DMTU和DPI预处理能够抑制EBR对光合作用,碳水化合物含量的调控及相关酶活和基因的表达,这表明由NADPH氧化酶产生的H2O2在EBR调控碳水化合物过程中发挥重要作用。3.研究了H2O2信号介导BRs对作物光合作用的调控。研究发现,外源EBR能够诱导而H2O2的清除剂DMTU和NADPH氧化酶抑制剂(DPI)能够阻断BRs对H2O2的诱导,这表明BRs主要通过激活NADPH氧化酶诱导H2O2的产生。并且BRs诱导的H2O2主要分布于叶肉细胞细胞壁的背壁,而叶绿体、线粒体、细胞核及液泡等组织结构内均无H202产生。DMTU和DPI预处理能够抑制EBR对光合作用,卡尔文循环关键酶活性及相关基因表达的调控,这表明由NADPH氧化酶产生的H2O2在EBR调控光合作用中发挥重要作用。研究发现EBR诱导的RCA活性的增加及蛋白水平表达的提高均能被DMTU和DPI所抑制。这些结果表明,H2O2对于EBR诱导的光合作用的调节具有重要作用。4.研究了氧化还原信号对BRs提高光合作用的调节。进一步分析了氧化还原信号对光合作用的影响。研究发现外源适当浓度的EBR、H2O2处理能够提高GSH/GSSG的比率,但是对谷胱甘肽的总量无显著影响。高浓度的EBR对光合作用无明显作用但可以使谷胱甘肽的总量增加,同时降低了GSH/GSSG的比率。EBR、H2O2提高光合作用的效应可以被GSH合成抑制剂BSO和磷酸戊糖途径的NADPH合成抑制剂6-AN所抵消。外源GSH跟EBR, H2O2一样可以通过提高卡尔文循环相关酶的活力及基因表达从而提高光合作用。这些结果表明,EBR、H2O2诱导的卡尔文循环关键酶活力及基因表达均被BSO和6-AN所抑制,充分证明GSH/GSSG作为一种氧化还原信号对光合作用的调节具有重要作用。5.研究了依赖于BRs水平的谷胱甘肽氧化还原状态对Rubisco活化酶的影响。分析了离体条件下GSH/GSSG的改变对光合作用的调控。研究发现植株的叶绿体中谷胱甘肽对不同BRs水平是有响应的及环境中不同的GSH/GSSG对提取纯化的RCA是有影响的。此外,植物体内的叶绿体中GSH/GSSG随BRs水平的提高而提高,而GSH+GSSG的总量则无明显变化；随着GSH/GSSG的提高,纯化的RCA活力逐渐升高,即水解的时间也随之延长。这些研究结果表明,BRs水平影响了叶绿体内谷胱甘肽的水平及其氧化还原状态；RCA的稳定性受GSH/GSSG的调控。6.研究了EBR在正常和加富CO2条件下,对光合作用的影响。研究发现了在CO2加富条件下,EBR对光合作用的提高效果更为明显,对生长量、光合荧光参数、碳水化合物的含量,及卡尔文循环相关的基因表达都有显著的作用。这些研究结果表明,EBR无论在正常还是加富CO2条件下对黄瓜光合作用都有明显的促进作用。更多还原

【Abstract】 Cucumber (Cucumis sativus) is one of the major corps in horticultural cultivation and has key roles in agricultural production and consumption. The growth and development of cucumber are dependent on the suitable environment conditions. However, the productivity and quality of cucumber are always affected by various stresses due to the simple and crude horticultural facilities. Brassinosteroids (BRs) was applied widely in increasing yields and stress tolerance, however, the mechanism is not very clear. Hence, it is of great importance to study the mechanisms of BRs how to regulate photosynthesis. This study uses cucumber with different brassinosteroid (BRs) levels to study the involvement of redox signal in BRs and H2O2 regulate photosynthesis. Through chemical genetics, physiological and molecular biological experiments, we investigate the effects of redox signal on BRs-regulated photosynthesis. The results are as follows:1. We have examined effects of 24-epibrassinolide (EBR) and H2O2 on gas-exchange, chlorophyll fluorescence characteristics and Rubisco activity. EBR and H2O2 significantly increased the light-saturated net CO2 assimilation rate (Asat). Increased Asat in EBR and H2O2-treated leaves was accompanied by increases in the maximum carboxylation rate of Rubisco (Vc, max) and in the maximum rate of RuBP regeneration (Jmax). EBR and H2O2-treated leaves also had a higher quantum yield of PSII electron transport (ΦpsⅡ) than the controls, which was mainly due to a significant increase in the photochemical quenching (qP). EBR and H2O2 did not influence photorespiration. In addition, significant increases in the initial activity of Rubisco and in the sucrose, soluble sugars, and starch contents were observed followed by substantial increases in sucrose synthase (SS), and acid invertase (AI) activities after EBR or H2O2 treatments. It was concluded that EBR and H2O2 increase the capacity of CO2 assimilation in the Calvin cycle, which was mainly attributed to an increase in the initial activity of Rubisco. 2. We have studied the role of H2O2 signal in BRs regulate carbohydrate metabolism. To determine whether ROS plays a critical role in EBR-regulate carbohydrate, we analyzed the effects of diphenyleneodonium (DPI), an inhibitor of NADPH oxidase and dimethylthiourea (DMTU), an H2O2 scavenger, on EBR-induced changes in carbohydrate and key enzymes. However, pretreatment with DPI or DMTU completely abolished the effects of EBR on carbohydrate content and key enzymes. The results demonstrated that H2O2 plays a critical role in BRs regulate carbohydrate metabolism.3. We have examined the role of H2O2 signal in EBR-induced photosynthesis. To determine whether ROS plays a critical role in EBR-induced photosynthesis, we analyzed the effects of diphenyleneodonium (DPI), an inhibitor of NADPH oxidase and dimethylthiourea (DMTU), an H2O2 scavenger, on EBR-induced photosynthesis and calvin cycle enzymes. However, pretreatment with DPI or DMTU completely abolished the effects of EBR on photosynthesis and Rubisco activase. Immunogold-labeling and Western blotting demonstrated that BRs upregulated the expressions of RCA. The results demonstrated that H2O2 plays a critical role in BRs induced photosynthesis.4. We have examined the involvement of glutathione redox status in BRs-induced changes in photosynthesis. To determine the involvement of altered redox state in EBR-and H2O2-stimulated photosynthesis, we analyzed the effect of GSH, buthionine sulfoximine (BSO) and 6-aminonicotinamide (6-AN) on Asat with or without EBR or H2O2 treatment. In EBR-and H2O2-treated plants, the overall GSH+GSSG contents were not significantly changed but the GSH/GSSG ratios were significantly increased. Pretreatment with BSO or 6-AN blocked EBR-and H2O2-induced increase in the GSH/GSSG ratio mostly by restoring the reduced GSSG contents. However, the enzymes activities of calvin cycle were increased by exogenous GSH application. These results strongly suggested that increased GSH/GSSG ratio in EBR-and H2O2-treated plants is important for increased photosynthesis.5. We have studied the effects of EBR and Brz treatment on the accumulation of GSH and GSSG and the GSH/GSSG ratio of chloroplasts. Neither EBR nor Brz application had significant effects on the total glutathione pool (GSH+GSSG). On the other hand, the GSH/GSSH ratio of chloroplasts was substantially altered by EBR and Brz treatment. Thus, BRs-stimulated CO2 assimilation was associated with a reduced cellular redox state. GSH/GSSG ratio plays an essential role in regulation of CO2 assimilation through regulating Rubisco activase. BRs regulates the GSH/GSSG ratio, expression of chloroplastic gene and changes in Rubsico activation state is associated with amount of Rubisco activase.6. We have studied the effects of EBR on photosynthesis under ambient and elevated CO2 conditions. We found that the effects of EBR on growth mass, photosynthetica and fluorescence parameters or genes expression of Calvin-cycle were more visible under elevated CO2 condition. These results strongly suggested that EBR has effects on photosynthesis both at ambient and elevated CO2 conditions.更多还原