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Mechanisms of response to environmental stresses with active oxygen species-detoxification in higer plants

高等植物活性氧解毒响应环境胁迫的机制

基本信息

批准号：
12440229
负责人：
KOBAYASHI Hirokazu
金额：
$ 9.73万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Salt tolerance in plants has been attributed to many mechanisms, including accumulation of osmoregulatory solutes inside cells, enhanced ability of cells to take up water or K^+ from surrounding solutions of higher salt concentration, elimination of excess NaCl, and the ability to maintain photosynthetic activity under salt stress. To further investigate the mechanisms of salt tolerance, we have begun to screen and characterize mutants of Arabidopsis thaliana tolerant to high NaCl concentrations during photoautotrophic growth. Several mutants of A. thaliana with altered responses to salt have been reported, including mutants tolerant of salt during germination and mutants that are salt-hypersensitive. However, there have been no reports of mutants whose photoautotrophic growth is salt tolerant.We selected pst (photoautotrohic salt tolerance) mutant lines of Arabidopsis, and characterized pst1 plants in which activities of active oxygen species-detoxifying enzymes were enhanced (Plant Cell, 11, 1195-1206, 1999). However, it was difficult to specify the event crucial for conferring salt tolerance on pst2 plants. The cDNA macroarray and oligo-microarray were performed for pst2 and wild-type lines grown under a non-stress condition. Higher expression of a transcription factor with bHLH motif in pst2 plants was detected, and further confirmed by real-time RT-PCR. There was another specie of mRNA produced by alternative splicing under the salt stress. The RACE has clarified that transcription initiation site is single. The salt-induced mRNA retained an intron on C-terminal side among two introns, and the newly generated stop codon of first ORF and its subsequent initiation codon of new second ORF may encode two separate peptide molecules which looked like cleaved forms of the original protein. Protein species derived from the bHLH gene are being analyzed by immuno-blotting, and the function of bHLH transcription factor is also examined with its transgenic plants.

植物的耐盐性与许多机制有关，包括细胞内盐调节溶质的积累、细胞从高盐溶液中吸收水分或K^+的能力增强、清除过量的NaCl以及在盐胁迫下维持光合活性的能力。为了进一步研究耐盐机制，我们已经开始筛选和表征拟南芥耐高浓度NaCl的突变体在光合自养生长。A.已经报道了对盐具有改变的响应的拟南芥，包括在萌发期间耐盐的突变体和盐超敏感的突变体。然而，还没有报道其光合自养生长是耐盐的突变体，我们选择了拟南芥属的pst（光合自养耐盐）突变株系，并表征了其中活性氧类解毒酶活性增强的pst 1植物（Plant Cell，11，1195-1206，1999）。然而，很难确定赋予pst 2植物耐盐性的关键事件。对生长在非胁迫条件下的pst 2和野生型株系进行cDNA宏阵列和寡核苷酸微阵列。荧光定量RT-PCR检测到一个具有bHLH基序的转录因子在pst 2中有较高的表达。在盐胁迫下，还存在另一种由选择性剪接产生的mRNA。RACE已经阐明了转录起始位点是单一的。盐诱导的mRNA在两个内含子中保留了一个位于C端的内含子，第一个ORF的新终止密码子和第二个ORF的新起始密码子可能编码两个独立的肽分子，看起来像原始蛋白的切割形式。通过免疫印迹分析了bHLH基因的蛋白种类，并通过其转基因植物检测了bHLH转录因子的功能。