论文标题:高温锻炼和水杨酸诱导葡萄幼苗耐热性的细胞学机制研究
Study of Cellular Mechanism of Thermotolerance Induced by Heat Acclimation or Salicylic Acid in Young Grape Plants
论文作者 刘悦萍
论文导师 黄卫东,论文学位 博士,论文专业 果树学
论文单位 中国农业大学,点击次数 160,论文页数 89页File Size9011k
2003-05-01论文网 http://www.lw23.com/lunwen_3068072/ 葡萄幼苗,高温锻炼,SA,耐热性,细胞学机制
Grape plant,Salicylic acid,Heat acclimation,Thermotolerance,Cellular mechanism
以葡萄(Vitis vinifera×V．labrussa L．cv．Jingxiu)幼苗为试材，从细胞学水平比较研究了高温锻炼和SA诱导葡萄耐热性的机制，并初步探讨了SA的信号转导途径，结果如下。 利用透射电镜技术对高温热激下细胞超微结构进行了观察，细胞结构在高温热激下明显被破坏，高温锻炼预处理会对叶肉细胞造成轻微的伤害，但延缓了随后的高温热激对叶肉细胞结构的损伤，主要表现在质膜、液泡膜、细胞核和叶绿体等部位。SA预处理对叶肉细胞结构无明显影响，但在随后的高温热激下，SA预处理细胞结构的受伤程度远低于H_2O处理。 通过两相分配法纯化幼苗叶片的质膜，以及采用氯化铈沉淀的细胞化学方法，分别从生化水平和组织化学水平研究了质膜的H~+-ATP酶和Ca~(2+)-ATP酶活性，最后得到一致的结果。即高温锻炼和SA预处理提高了膜上的H~+-ATP酶和Ca~(2+)-ATP酶活性，并且在随后的高温热激下，保持了质膜上两种酶的稳定性。采用45±0.5℃热激6h时，高温锻炼和SA预处理幼苗质膜的H~+-ATP酶活性分别为各自对照的7.1和3.7倍，而Ca~(2+)-ATP酶活性分别为各自对照的4.4和4.6倍。在热激6h时，电镜观察结果为高温锻炼和SA预处理的叶片，其细胞质膜H~+-ATP酶和Ca~(2+)-ATP酶仍有一定的活性，但对照叶片细胞已观察不到。这些结果说明，高温锻炼和SA诱导幼苗抗热性的提高与质膜上的H~+-ATP酶和Ca~(2+)-ATP酶活性有关，并且具有相似的调控机制。另外，高温锻炼预处理也可提高质膜Fe(CN)_6~(3-)还原活性，在热激下保持较高的水平。 采用Ca~(2+)和CaM抑制剂预处理来研究Ca~(2+)对SA诱导幼苗耐热性的影响。结果表明，Ca~(2+)可促进SA对幼苗耐热性诱导，而Ca~(2+)抑制剂EGTA、Ca~(2+)通道抑制剂La~(3+)以及CaM拮抗剂W7对SA诱导的耐热性产生抑制作用。高温热激后，SA或SA加Ca~(2+)处理可诱导叶片内CaM的积累，EGTA、La~(3+)或W7则抑制CaM的积累。高温下，SA通过维持高水平的SOD和CAT的活性，降低MDA含量来抵抗高温造成的氧化胁迫，外源Ca~(2+)可促进SA对SOD和CAT的诱导，而EGTA、La~(3+)或W7则产生相反的作用。在高温前后，各处理叶片内的POD和APX的活性并没有明显的变化。另外，SA或SA加Ca~(2+)处理可增加叶片中的脯氨酸含量，并在高温下维持较高的水平。这些试验结果表明，Ca~(2+)可调控SA诱导的耐热性，而且在此过程中，要求细胞外的Ca~(2+)穿过质膜进入胞内，并有抗氧化酶的参与。 对幼苗植株中部叶片饲喂~(14)C-SA，而其对侧上方叶和对侧下方叶进行40±0.5℃高温处理。结果发现，高温处理增加了饲喂叶~(14)C-SA的向外运转量，改变了~(14)C-SA在各器官的分配比例，处理叶片中的~(14)C_SA积累量至少为对照的3倍以上。采用提取试材的SA，然后再测定~(14)C比放射强度的方法，证明在试验中，直接用仪器测得的~(14)C比放射强度至少有70％以上是~(14)C-SA。 在幼苗根部饲喂~(14)C-SA，用40±0.5℃的高温处理植株的地上部分。结果发现，在处理6h之内，处理植株根部吸收的~(14)C-SA向地上部的分配率明显高于对照；处理植株地上部各器官(叶、韧皮部和木质部)的~(14)C-SA含量也显著高于对照，而根部的~(14)C-SA含量与对照没有明显差异，这是首次有关植物根部SA对高温胁迫应答反应的报告。另外，正常葡萄植株根部的结合态SA含量为游离态的3.59倍，当用40±0.5℃的高温处理离体根2h时，根内游离态SA的含量急剧升高，为对照的6.02倍，随后又迅速下降。上述结果表明，在葡萄幼苗对高温胁迫的响应中，根部的SA具有极其重要的作用。
The young grape plants (Vitis viniferaXV.labrussa L. cv. Jingxiu)were used as materials to study on the mechanisms of thermotolerance induced by heat acclimation or exogenous salicylic acid(SA), and probe the way of SA signal transduction. The results obtained were as following:Ultrastructural changes of the mesophyll cells under heat stress were observed via transmission electron microscope. Under heat stress at 45±0.5℃ for 3h or 6h, the ultrastructure of normal mesophyll cells was damaged remarkably. There was a little change of ultrastructure after the leaves pretreated with heat acclimation, but heat acclimation pretreatment delayed the ultrastructural damage induced by heat stress, principally showing in plasmolemma, vacuole membrane, nucleus and chloroplasts. No ultrastructural change showed after SA pretreatment, but the degree of ultrastructional damage after SA pretreatment was less than that of H2O pretreatment while the leaves under heat stress.The activity of H+-ATPase or Ca2+-ATPase of plasmolemma was studied with biochemistry assay in which the method of two-phase partition was used to purify plasmolemma and electromicroscopic-cytochemical location in which cerium trichloride precipitation method was adopted. At last, the results were almost same. After the young plants were treated with heat acclimation or SA, H+-ATPase or Ca2+-ATPase activity of plasmolemma was higher than that of control or H2O pretreatment, and enhanced the stability of H+-ATPase or Ca2+-ATPase activity under the following heat stress. If the young plants were stressed at 45 ± 0.5℃ for 6h, the H+-ATPase activity of plasmolemma of heat acclimation or SA pretreatment was 7.1 or 3.7 times than that of control or H2O pretreatment, however, the Ca2+-ATPase activitiy of plasmolemma of heat acclimationor SA pretreatment was 4.4 or 4.6 times than that of control or H2O pretreatment. Through observation of electromicroscope, the H+-ATPase or Ca2+-ATPase activity of plasmolemma of heat acclimation or SA pretreatment remained active after heat stress at 45±0.5℃ for 6h, but the activity of control or H2O pretreatment was completely inactive. These results showed that the young plantings thermotolerance induced by heat acclimation or SA pretreatment was related with the H+-ATPase or Ca2+-ATPase activity of plasmolemma, heat acclimation and SA almost had the same regulating mechanism. In addition, the Fe (CN) 63- reduction of plasmolemma in leaf cells of heat-acclimated young plants was enhanced and kept higher level under the following heat stress, but SA pretreatment had no effect on the Fe (CN)63- reduction of plasmolemma.Effect of Ca2+ on the young plants thermotolerance induced by SA was studied with pretreatment of Ca2+ and antagonists of Ca2+ or CaM. The results showed that Ca2+ enhanced the SA-induced thermotolerance. In contrast, pretreatment with the Ca2+ chelator EGTA, the plasmolemma Ca2+ channel blockers La3+, which was expected to inhibit the influx of extracellular Ca2+ into cells, and the CaM antagonist W7 weakened the SA-induced thermotolerance. Pretreatmentwith SA or Ca2+( SA+Ca2+) increased CaM content of leaves, but EGTA(SA+ EGTA ), La3+(SA+ La3+) or W7(SA+ W7) decreased the CaM content of leaves under heat stress at 45±0.5# for 3h. SA pretreatment enabled young plants to maintain higher activity of SOD or CAT and a lower level of MDA to weaken oxidative stress induced by heat stress. However, Ca2+ pretreatment enhanced the activity of SOD or CAT SA-induced, but EGTA, La3+ and W7 had a contrary effect. POD or APX activity of each treatment had little change before and after heat stress. In addition, pretreatment of SA or Ca2+ (SA+Ca2+) enhanced the content of proline and kept higher level under heat stress. These results showed that SA-induced thermotolerance was medicated by Ca2+ and required the entry of extracellular Ca2+ into cells through plasmolemma, and was related with antioxidant enzymes and proline.The midst leaf of young planting was fed with 14C-SA, and the other side leaf above
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