【作者】 侯宁宁；

【导师】 杨振明；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 本文以大豆耐铝基因型吉育70为材料,采用液体培养的方法,研究铝胁迫下脱落酸（ABA）对大豆耐铝性的调控,首次采用^<sup>3H-ABA和分根实验探讨ABA在大豆体内的运转、分布和再分配等问题,并从有机酸分泌,抗氧化系统及信号分子NO等角度探讨ABA如何调控大豆耐铝性等问题。外源ABA和ABA合成抑制剂可分别缓解和加重铝对大豆根伸长的抑制作用;铝诱导提高大豆叶片与根部的内源ABA的含量,而镧提高大豆根部的内源ABA含量;在分根实验中,同一株大豆幼苗的侧根分为part A和part B两个部分进行不同处理,发现只有与铝直接接触的part A根伸长受抑,part A根部内源ABA含量较对照提高,但part B根部内源ABA含量较对照减少。³H-ABA的示踪实验表明,ABA以大于3.2 cm·min^-1)的速率从根部运输到叶部或从叶部运输到根部,铝胁迫加快了ABA在大豆体内的运输,改变了³H-ABA在大豆幼苗体内的再分配,并使³H-ABA倾向于分配在受铝胁迫的根部。通过以上实验表明,ABA可能作为信号分子调控大豆耐铝性。外源ABA对铝胁迫下大豆根系柠檬酸分泌量、根系内的柠檬酸含量和柠檬酸合成酶的活性均无影响,且利用不同基因型和时间段的实验表明,内源ABA水平与大豆根系柠檬酸的分泌没有直接的响应关系。因此推测,ABA可能不是通过增加柠檬酸分泌从而调控大豆耐铝性。外源ABA处理抑制了铝胁迫诱导的大豆根和叶片MDA含量,O₂^·–产生速率、H₂O₂含量的增加,且进一步提高了铝对POD活性的提高,因而推测,ABA通过提高POD活性而提高大豆耐铝的。铝胁迫提高内源NO水平,NO供体SNP能够缓解铝对根生长的抑制作用,且缓解铝胁迫下FLU对根伸长的抑制作用,NO供体SNP和NO清除剂cPTIO分别显著增加和减少铝诱导提高的内源ABA含量,外源ABA和FLU分别抑制和增加Al胁迫下大豆根尖NO含量的提高。因而推测,NO是调控大豆耐铝毒重要信号分子之一,且在缓解铝毒害的信号通路中,NO对内源ABA的水平变化起着重要的正向调控作用, ABA对内源NO的水平变化起着反馈抑制的调控作用。更多还原

【Abstract】 Aluminum （Al） toxicity is one of the most significant constaints limiting plant growth in acid soils. The primary symptom of Al toxicity is a rapid inhibition of root growth, resulting in a reduced and damaged root system and limited water and mineral nutrient uptake. Abscisic acid （ABA） considered as an important endogenous plant hormone, ABA accumulates to high concentrations in organs and tissues of dormancy plants. It appeared that ABA was a general endogenous inducer of tolerance to environment stresses and significantly increased drought,salt and chilling tolerance by activating the anti-adverse stress system. In the present study, with solution culture, Jiyu70 was selected as the Al-tolerant cultivars for the research of effect of ABA involved in Al tolerance in soybean. By the split-root experiment and ³H-ABA radioisotope technique to explore the transport and distribution of ABA in soybean, and from aspects of organic acid secretion, antioxidant systems and signaling molecule NO to approach how ABA regulated Al tolerance .The results were as follows:1. ABA was speculated to involve in modulating Al tolerance in soybeanThe major Al toxicity symptom observed in plants is inhibition of root elongation. The root elongation of soybean was inhibited under Al stress, with the increasing of Al concentration, the degree of inhibition enhanced; Al induced root elongation inhibition was respectively alleviated by exogenous ABA and aggravated by ABA synthesis inhibitor-fluridone. In addition, Al-treatment increased the endogenous level of abscisic acid （ABA） in soybean roots in a dose- and time-dependent manners, Al increased the ABA content in both roots and leaves. With similar chemical properities, La only increased the ABA content in roots. Split-root experiments （Part A and Part B）show that in the same soybean seedings, inhibition of root growth when the roots of Part A exposed to Al, the growth of roots of Part B without Al did not inhibited. Endogenous ABA in the roots of Part A elevated significantly compared with control, endogenous ABA in the roots of Part B reduced compared with control. We found that ABA inclined to redistribute in the root part under Al stress. By the whole-root and split-root experiment, binding with ³H-ABA radioisotope technique, ABA could be transported up or down in soybean seedling. Calculated transport rate was 3.2 cm·min^-1 or more. Al accelerated ABA transport and ABA inclined to distribute in the root part under Al stress. More ABA was accumulated in the roots with direct contact of Al.Above all, ABA is probably a signal molecule transmission between aboveground and underground under Al stress to involve in modulating Al tolerance in soybean2. ABA and Al induced citrate exudation of soybeanAl induced organic acid secretion is one of the most important Al tolerance mechanism in plants. The focus of our research is to examine whether ABA is involved in the Al-induced secretion of citrate. our research to identify the relationship of ABA and Al induced citrate exudation in soybean roots : 1)Split-root experiments （Part A and Part B） show that endogenous ABA in the roots of Part A exposed to Al elevated and Al induced citrate exudation in soybean roots, endogenous ABA in the roots of Part B without Al reduced and no citrate exudation in soybean roots. 2)Exogenous ABA application had no effect on Al induced citrate exudation, and didn’t affect the Al induced increase of citrate content nor citrate synthase activity. 3)Al-tolerant cultivar secreted more citrate than Al-sensitive cultivar while the ABA content in the roots of both cultivars had no differences under same Al stress. 4)By time course experiment, both citrate secretion rate and endogenous ABA content increased with increasing of Al treatment time during 0-12 h. When endogenous ABA content dropped during the next 6 h pulse （12-18 h） free of Al, citrate secretion rate continued to increase. These results suggested that ABA increased the Al tolerance in soybean, but not by increasing the Al induced citrate exudation from soybean roots. 3. ABA and system of antioxidation under Al stressAn important response to stress by plant is the production of reactive oxygen free radicals, these reactive oxygen species can directly or indirectly activated membrane lipid peroxidation, causing membrane damage and destruction. ABA involved in biotic and abiotic stress caused by oxidative stress, but the effect of ABA on oxygen species generation, lipid peroxidation and antioxidant enzyme activity on soybean roots under Al stress remains unclear, the results showed that Al stress increased the MDA content、O₂^·– generated rate、the content of H₂O₂ in roots and leaves of soybean. Exogenous ABA treatment inhibited the increase in MDA content、O₂^·– generated rate、the content of H₂O₂ induced by Al in roots and leaves of soybean. ABA inhibitors fluridone enhanced the O₂^·–generated rate、H₂O₂ and MDA content in roots and leaves of soybean under Al stress, Al induced the increasing of the activity of POD、SOD、CAT activity. ABA increased the activity of POD; reduced SOD、CAT activity; ABA inhibitors fluridone increased the antioxidant protective enzymes SOD、CAT activity, reduced POD activity. Because of the site of Al toxicity is the roots, ABA is probably a signal molecule transmission between aboveground and underground under Al stress and ABA blocked activities of CAT、SOD and elevated activities of POD. ABA might activate an H₂O₂-mediated pathway, a POD-dependent antioxidative mechanism to protecting the plant against Al-induced oxidative stress.4. the role of NO in alleviating Al toxicity and the relationship between signaling molecule ABAAl induced increase in the NO content in soybean roots. NO donor SNP exhibited significantly greater root elongation and reversed the inhibition of fluridone in root elongation under Al stress. Exogenous SNP enhanced the Al-induced increase in the ABA content in soybean roots, the NO scavenger cPTIO completely reversed the effect of NO on the ABA content in the presence of Al. In the signaling pathway of alleviating Al toxicity, NO plays an positive role in internal source level of ABA, NO alleviated the inhibition of root elongation under Al stress may be due to the arising endogenous ABA content. NO promoted the endogenous ABA content and redued the poisoning effect of Al. However, ABA content improve inhibited NO synthesis. Exogenous ABA and fluridone respectively inhibited and raised NO generation induced by Al. Thus, in the signaling pathway of alleviating Al toxicity, ABA plays a regulatory role of feedback inhibition in the level of the source of NO.更多还原