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

植物的耐盐性有多种机制，包括细胞内渗透调节溶质的积累、细胞从周围高盐浓度溶液中吸收水分或K^+的能力增强、过量NaCl的消除以及在盐胁迫下维持光合活性的能力。为了进一步研究拟南芥耐盐机制，我们开始筛选和鉴定光自养生长过程中耐高浓度NaCl的突变体。据报道，拟南芥的几个突变体对盐的反应发生了改变，包括发芽时耐盐的突变体和盐过敏的突变体。然而，目前还没有关于光自养生长耐盐突变体的报道。我们选择了拟南芥（拟南芥）的pst（光自养耐盐性）突变系，并对其活性氧解毒酶活性增强的pst1植株进行了鉴定（Plant Cell, 11, 1195-1206, 1999）。然而，很难确定使pst2植株具有耐盐性的关键事件。对非胁迫条件下生长的pst2和野生型细胞系进行了cDNA大阵列和寡核苷酸微阵列分析。在pst2植物中检测到bHLH基序转录因子的高表达，并通过实时RT-PCR进一步证实。盐胁迫下还产生了另一种选择性剪接的mRNA。RACE已经明确了转录起始位点是单一的。盐诱导的mRNA在两个内含子之间的c端保留了一个内含子，新生成的第一个ORF的停止密码子和随后新的第二个ORF的起始密码子可能编码两个独立的肽分子，它们看起来像原始蛋白质的切割形式。利用免疫印迹法分析了bHLH基因衍生的蛋白种类，并利用bHLH转录因子的转基因植株对其功能进行了研究。

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.