Molecular mechanism of the acquirement and restriction of differentiation potency in plant cells

1.分离的叶肉细胞转分化多能性获得分析我们分离到12个转录本在百日草叶肉细胞中优先表达的cDNA克隆。这些克隆被分为三组，1)创伤或感染诱导的基因，2)编码蛋白质合成的基因，3)其他基因。所有这些克隆的mRNAs在培养12h内都能被伤害诱导，但随后以激素依赖的方式发生变化，表明早期的过程涉及蛋白质合成的激活。2)分化潜力的限制分析a)TED3启动子的管状元件前体细胞特异性表达分析转TED3启动子和GUS基因的转基因拟南芥的分析表明，该启动子引导了管状元件前体细胞特异性表达。b)TED3功能的分析我们将TED3基因的反义DNA导入百日草叶片，诱导了许多转化的毛状根。在根无性系中，抑制TED3mRNAs积累的无性系表现出对根生长的抑制作用。结合TED3mRNAs在气管前体细胞中的表达，我们提出了一个假设，即TED3产物可能与气管细胞的伸长有关。c)细胞死亡过程的分析我们克隆了半胱氨酸蛋白酶和DNA酶的基因克隆，它们可能参与了气管细胞分化的自溶过程。这些克隆的mRNAs以非常相似的模式表达，只是在液泡破裂之前的一瞬间。这表明了与细胞死亡过程有关的基因表达的共同机制。基于这些结果，我们提出了关于分化潜力变化的假设。

1.Analysis of the acquirement of pluripotency of transdifferentiation of isolated mesophyll cellsWe isolated 12 cDNA clones whose transcripts are expressed preferentially in isolated Zinnia mesophyllcells. These clones were divided into teree groups, 1)wound or infection-induced genes, 2)genes encoding proteins involved in protein synthesis, and 3) others. mRNAs for all of these clones were induced by wounding within 12 h of culture but thereafter chenged in hormone-dependent manners, suggesting that the early process involves the activation of protein synthesis.2.Analysis of the restriction of the potency of differentiationa) Analysis of tracheary element precursor cell-specific expression of TED3 promoterThe analysis of transgenic Arabidopsis with chimeric genes of TED3 promoter and GUS gene indicated that this promoter directed tracheary element precursor cell-specific expression.b) Analysis of TED3 functionWe introduced an antisense DNA of TED3 gene into Zinnia leaves and induced many transformed hairy roots. Among the root clones, clones in which the accumulation of TED3 mRNAs were suppressed showed the inhibition of root growth. Taking together with the expression of TED3 mRNAs is localized in tracheary element precursor cells, we presented a hypothesis that TED3 product may be responsible for the elongation of tracheary elements.c)Analysis of cell death processWe isolated cDNA clones for cysteine protease and DNase which may be involved in autolytic process in differentiating tracheary elements. The mRNAs for these clones expressed in very similar pattern, transiently just before vacuole disruption. This suggests a common mechanism of gene expression in relation to cell death process.Based on these results, we presented a hypothesis on the Change in the potency of differentiation.