【作者】 李慧；

【导师】 谭金芳； 韩燕来； 赵会杰；

【作者基本信息】 河南农业大学 ， 作物营养生理， 2009， 博士

【摘要】 小麦是我国第二大主要作物,其产量与品质直接关系到国计民生。随着全球气候变暖,干旱是限制农业生产的最主要因子之一,其中小麦抗旱生理生化与分子生物学研究将为解决上述问题提供有力的理论支撑。如何通过外源物质的调控提高小麦的抗旱性,是目前小麦抗旱机制研究的热点。本文采用15%聚乙二醇-6000 (PEG-6000)对小麦幼苗进行轻度干旱胁迫处理,并通过添加不同浓度的一氧化氮(nitric oxide, NO)供体硝普钠(sodium nitroprusside, SNP),研究外源NO对提高小麦抗旱性的机理及其效应转导机制,为干旱地区和半干旱地区的粮食安全生产提供理论依据与技术支撑。主要研究结果如下:1.干旱胁迫下小麦幼苗叶中H+-ATP酶和Ca2+-ATP酶活性显著升高后迅速下降,丙二醛(MDA)和质量膜透性增加,株高、鲜重、干重以及相对含水量(RWC)降低;0.1 mmol?L-1 SNP和0.5 mmol?L-1 SNP可提高干旱胁迫下小麦幼苗叶中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,降低超氧阴离子(O2-.)和过氧化氢(H2O2)水平,降低膜脂过氧化,稳定生物膜的结构和功能,H+-ATP酶和Ca2+-ATP酶也以可保持更高的活性;而1.0 mmol?L-1 SNP则加剧膜质过氧化,产生负效应。2.干旱胁迫下小麦幼苗叶片一氧化氮合酶(NOS)活性显著增加,且钙依赖型cNOS (CaM的组成型NOS)快速调控NO产生,但是随着胁迫时间的延长,不依赖钙iNOS (CaM的诱导型NOS)的活性在NOS活性比例缓慢增加,而硝酸还原酶(NR)产生NO的能力只占总NR提取物活性的很小一部分;0.1 mmol?L-1 SNP处理可显著提高干旱胁迫下小麦幼苗叶片NOS和NR活性,诱导NO水平提高,显著缓解膜脂过氧化;随着SNP浓度的提高,0.5 mmol?L-1 SNP和1.0 mmol?L-1 SNP逐渐出现抑制NO合成。用质膜Ca2+通道抑制剂LaCl3与SNP共处理,显著减弱或抵消SNP促进NO合成作用。SNP显著提高干旱胁迫下小麦幼苗叶片NO合成酶活性和NO含量,有效缓解膜的氧化损伤,而Ca2+参与SNP对干旱胁迫下小麦幼苗叶片NO水平的调控。3.干旱胁迫引起小麦幼苗叶片的光合色素含量增加,光合速率(Pn)则随胁迫时间的延长不断降低,而细胞间隙CO2浓度(Ci)随气孔导度(Gs)的下降而减少,处理12h后开始缓慢回升,证实胁迫初期气孔限制是光合速率下降的主要原因,后期则以非气孔限制为主。0.1 mmol?L-1 SNP可提高干旱胁迫下小麦幼苗叶片叶绿素a和叶绿素b含量和Pn,加剧Gs降低,进一步诱导气孔关闭,降低蒸腾速率(E)来延缓干旱胁迫下的水分蒸发;还能增加受旱小麦PsⅡ开放反应中心的比例(qp),使更多的光能传递给PsⅡ反应中心,减少激发能用于非光化学反应的热耗散(NPQ),从而促使更多的激发能用于光化学反应(ΦPSⅡ)。另外,叶片的气体交换参数(Pn、Gs)和叶绿素荧光参数(qp、NPQ )对SNP的反应也表现出明显的浓度效应。低浓度SNP处理(0.1 mmol?L-1 SNP和0.5 mmol?L-1 SNP)明显改善了小麦叶片的光合性能从而促进干旱胁迫下小麦幼苗叶片游离氨基酸总量(TFA)、脯氨酸(Pro)和可溶性糖(SS)等渗透调节物质的积累,且增加了叶片的水势和渗透势。4.干旱胁迫下小麦幼苗叶片诱导蛋白含量明显增多,并于胁迫处理3 h出现一个26 kD蛋白,0.1 mmol?L-1 SNP不但在数量上诱导干旱胁迫蛋白增加(如分子量26kD、31kD和37kD等),还可在时间上把蛋白诱导过程提前,如26 kD蛋白在胁迫1 h就急剧增加。NO专一清除剂血红蛋白的添加则清除了0.1 mmol?L-1SNP对干旱胁迫下小麦幼苗的诱导效应。同时,Ca2+还参与了SNP对干旱胁迫下小麦幼苗叶片诱导蛋白的调控,LaC13添加后小麦幼苗叶片的胁迫诱导蛋白则在蛋白含量上明显减少,同时在时间上推迟蛋白诱导过程。综上所述,0.1 mmol?L-1 SNP可显著缓解干旱胁迫造成的氧化损伤;随着浓度的提高,即0.5 mmol?L-1 SNP的缓解作用逐渐降低,首先NO合成受到抑制,其次是气孔导度(Gs)升高,气孔关闭受阻,进而影响其他的调控机制;1.0 mmol?L-1 SNP则加剧小麦幼苗生理代谢紊乱。因此,外源NO对小麦幼苗生长生理的调控具有一定的剂量效应,0.1 mmol?L-1 SNP对小麦幼苗生长生理的调控作用最明显。更多还原

【Abstract】 Triticum aestivum is the second most important crop in China and its production and quality are directly linked with both international and domestic affairs. Drought is the one of the most important factor to limit the agricultural production are complicated and the study of anti-drought physiology, biochemistry, and molecular biology of T. aestivum will play an important role in providing powerful theoretical guidelines for coping with this problem. The mechanism study is a research focus in enhancing the drought resistance of wheat by exogenous substance. When the wheat seedling (with three fully expanded leaves) were treated with 15% PEG-6000 in combination with different concentrations (0.1, 0.5 and 1.0 mmol?L-1) of exogenous nitric oxide donor sodium nitroprusside (SNP), are studied the drought-resistance and its transduction mechanism to provide a theoretical basis and technical support in food safety production on arid and semi-arid areas. The main research results are as follows:1. The 15% PEG-6000 treatment apparently enhanced the H+-ATPase and Ca2+-ATPase activities before period of deterioration, accelerated the accumulation of malondialdehyde (MDA) and membrane permeability, lowered height, fresh weight dry weight and relative water content (RWC) in wheat seedling leaves under drought stress. 0.1 mmol?L-1 SNP and 0.5 mmol?L-1 SNP could enhance the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), lower O2-.production rate, H2O2 content and membrane lipid peroxidation, stabilize the structure and function of biomembranes, increase the activities H+-ATPase and Ca2+-ATPase. But it is the negative effects to decrease the membranaceous peroxide by 1.0 mmol?L-1 SNP.2. The 15% PEG-6000 could apparently enhance NOS activity in wheat seedling, NO was quickly produced by cNOS, and the proportion of iNOS in NOS was increased slowly after 6 h treatments. NO was also induced by NR, which was a little part of total NR activity. The 0.1 mmol?L-1 SNP treatment could apparently alleviate the drought-induced membrane lipid peroxidation, which consistent with the up-regulation of NOS, NR and NO. With increasing SNP concentration, it had less effect on NO content by 0.5 mmol?L-1 SNP and 1.0 mmol?L-1 SNP. The effect of SNP was decreased after treatment of blocker of plasma membrane Ca2+ channels LaCl3. The above results indicated that exogenous nitric oxide donor (SNP) could apparently increase nitric oxide-synthesis enzyme activities and NO content, and Ca2+ was involved in regulation of NO level in wheat seedling leaves by SNP under drought stress.3. The content of photosynthetic pigment increased, the photosynthetic rate (Pn) decreased with the time under drought stress, While intercellular CO2 concentration (Ci) decreased first with stomatal conductance (Gs) reducing, then slow increased after 12h treatment. Stomatal limitation is the main reason for the decline in Pn at the early stress, the latter are mainly non-stomatal limitation.is the one at the late stress. 0.1 mmol?L-1 SNP could help to increase chlorophyll a, chlorophyll b and Pn, decrease Gs to induced stomatal closure and transpiration rate (E) to relieve evaporation under drought stress. Furthermore, 0.1 mmol?L-1 SNP increased the proportion of opened PSⅡreaction centers (indicated by qp), leading to transference of the more excited light-energy to PSⅡfunction center, decreased the dissipation of excited energy in antenna pigments(NPQ), which increased the more absorbed light to participate in photochemical reaction(ΦPSⅡ). In addition, the effects of NO on the gas exchanges and chlorophyll fluorescence parameters were concentration dependent. At low concentrations (0.1 mmol?L-1 and 0.5 mmol?L-1) SNP significantly increased the Pn and decreased the Gs of the leaves so that they accumulated more osmotic substances (TFA, Pro and SS), and increased their water potential and osmotic potential.4. The stress-induced protein content was increased significantly in wheat seedling leaves under drought stress. The 26 kD protein was induced at the 3h treatment under drought stress. 0.1 mmol?L-1 SNP could not only increase the protein content (such as 26kD, 31kD and 37kD, etc.), but also advance the process of protein induced, such as the 26 kD protein increased significantly at the 1h treatment. Hemoglobin, a specific scavenger of NO, partly reversed the inductive effect. At the same time, LaC13 inhibited significantly the protein synthesis in wheat seedling leaves and postponed the process of protein induction, Ca2+ was involved in regulation of stress-induced protein in wheat seedling leaves by SNP under drought stress.The results suggest that 0.1 mmol?L-1 SNP could significantly alleviate the oxidative damage caused by drought stress. With the increased concentration, 0.5 mmol?L-1 SNP, decreased mitigation role, NO synthesis is inhibited, then Gs increased and stomatal closure blocked to impact of other regulatory mechanism.1.0 mmol?L-1 SNP was aggravated the physiological metabolic disorder of wheat seedlings under drought stress. Therefore, SNP significantly promoted the growth and regulated the physiology of wheat seedlings at 0.1 mmol?L-1 SNP.更多还原