铝胁迫下小麦MAPKs介导的生长素信号转导系统与苹果酸分泌关系研究

The secretion of organic acid is prominent way of plant resist with Al toxicity. Auxin signal transduction system extensively participates in plant resistance to adverse environment stress. Our previous study showed that IAA participated in malic acid secretion under Al stress, but the regulation mechanisms were still unknown. MAPKs cascades are involved in auxin signal transduction pathway. It had been proofed that MAPK could be activated by Al, then regulated the secretion of organic acid. But this Al-induced protein kinase whether was regulated by auxin signal was still unknown. So further studies were conducted based on the results of our previous studies. First, to further confirm that auxin signal was activated by Al, expression of auxin metabolism key enzyme genes and auxin response genes were investigated. Then, homology comparison of the two MAPKs induced by Al or IAA were studied,by which way if Al-induced MAPK was regulated by IAA was estimated. At last, characteristic of malic secretion and anion electrophysiological under IAA or protein kinase activators (inhibitors) treatments were investigated to further illustrates the relationship between MAPKs-mediated auxin signal transduction and malic acid secretion under aluminum stress. Results of this project will extend the regulation mechanisms of Al induced plant malic acid secretion,and providing scientific basis for the breeding of Al tolerance crops.

小麦通过分泌苹果酸螯合根际毒性铝是其重要抗铝胁迫方式，生长素信号转导系统广泛参与植物抗逆胁迫。申请人前期工作表明IAA参与铝胁迫下小麦苹果酸分泌，但其调控苹果酸分泌机理仍需进一步研究。丝裂原活化蛋白激酶（MAPKs）是生长素信号转导系统重要组成部分，铝胁迫通过活化该蛋白激酶实现有机酸分泌调控，但该蛋白激酶是否受生长素信号调控仍缺乏研究。因此本项目拟在前期研究基础上，通过探索铝胁迫对生长素代谢关键酶及生长素响应基因转录表达影响，明确铝胁迫对生长素信号系统的激活作用；比较研究铝及外源IAA活化的MAPK的生物学特征，解析铝胁迫下生长素信号对MAPK的调控作用；观察IAA及蛋白激酶抑制剂（激活剂）处理下苹果酸分泌及阴离子通道电生理特征，进一步阐明铝胁迫下小麦MAPKs介导的生长素信号转导系统与苹果酸分泌的关系。该项目研究成果将丰富铝胁迫诱导植物有机酸分泌调控机理，并为培育耐铝毒作物提供科学依据。

本研究主要以耐铝型小麦品种ET8和铝敏感品种ES8为试验材料，阐明了铝诱导的小麦苹果酸分泌与MAPKs信号途径的关系；揭示了铝胁迫与小麦体内生长素信号转导系统的关系；探讨了MAPKs信号途径与铝胁迫和生长素的关系。主要结果如下：.1. 铝胁迫下蛋白抑制剂和外源IAA对不同耐铝型小麦品种苹果酸分泌影响.单独IAA处理对小麦苹果酸的分泌无显著作用，但在有铝条件下，则可显著促进小麦苹果酸的分泌。蛋白抑制剂PAO或和CHX单独处理对小麦根尖苹果酸分泌无显著影响。蛋白抑制剂与铝共处理可显著抑制ET8和ES8苹果酸的分泌。IAA与PAO(CHX)共处理可显著促进铝胁迫下ET8和ES8分泌苹果酸。说明，IAA参与了小麦根尖苹果酸的分泌。.2. 铝胁迫下蛋白抑制剂和外源IAA对不同耐铝型小麦品种根尖细胞中ARF2和IAA Protein基因表达影响.1) 铝处理可提高ARF2在ET8根尖细胞中的表达，降低其在ES8中的表达量；降低IAA Protein在ET8和ES8根尖细胞中的表达。2)IAA处理可显著降低ET8和ES8中ARF2和IAA Protein的表达量。3) Al与PAO共处理， ET8和ES8中ARF2和IAA Protein表达量均显著低于Al处理，表明PAO可抑制ARF2和IAA Protein表达。4)IAA、Al和PAO共处理下，ET8和ES8中ARF2和IAA Protein的表达量显著降低，再次表明IAA可抑制ARF2和IAA Protein的表达。.3. 铝胁迫下不同耐铝小麦品种根尖细胞中MAPK2C 和MAPK1A对外源IAA和蛋白抑制剂响应.1)不同耐铝型小麦根尖细胞中MAPK2C的表达量均随铝浓度的升高而降低， MAPK1A的表达量则随铝浓度的升高而升高。随Al处理时间的延长，ET8和ES8中MAPK2C的表达量逐渐下降，MAPK1A的表达量升高。MAPK2C的表达表现为剂量、时间依赖性下调，MAPK1A表现为剂量、时间依赖性上调。2)无铝条件下，IAA可显著抑制ET8和ES8中MAPK2C的表达，促进MAPK1A的表达。3)蛋白抑制剂PAO可显著促进ET8和ES8根尖细胞中MAPK2C的表达。PAO与Al共处理可显著抑制MAPK1A在ET8和ES8中的表达。4) IAA、Al和PAO共处理时MAPK2C和MAPK1A的表达量显著低于无IAA处理。