Moleculargenetical analysis of phytochrome siggnal transduction

光敏色素是一种色素蛋白，存在于所有绿色植物中。光敏色素被认为是介导不同植物对光反应的主要感光器。虽然人们对光敏色素进行了深入的研究，但光敏色素转导光的分子机制仍不清楚。我们借助分子遗传学技术解决了这个问题。本研究探讨了两个主题。首先，建立了激活标记的拟南芥品系，并分离和分析了在光反应方面存在缺陷的突变体。其次，以绿色荧光蛋白(GFP)为荧光标记，对光敏色素B(PhyB)的核定位进行了研究，从激活标记的品系中筛选出在暗光下部分变绿的突变体。其中有一种突变体，类似于油菜素内酯含量较低的突变体。分子分析表明，该突变体过表达了一个新的细胞色素P450基因。我们通过产生过表达该基因的转基因拟南芥品系，进一步证实了该基因确实是导致表型的原因。不幸的是，这种表型似乎不仅与光敏色素有关，而且还与其他光受体有关。然而，该基因将是阐明光和类固醇信号之间关系的有力工具。我们通过在拟南芥PhyB缺失突变体中表达PhyB-GEP融合蛋白来研究PhyB在细胞内的定位。结果表明，该基因具有生物活性。细胞的显微镜观察显示，在光照条件下，PhyB-GFP定位于细胞核。相反，PhyB-GFP在黑暗中分布在整个细胞内。因此，PhyB似乎以一种光依赖的方式移位到细胞核。虽然光敏色素一直被认为在细胞质中起作用，但我们的结果表明，细胞核是光敏色素作用发生的细胞室之一。

Phytochrome is a chromoprotein, which is found in all green plants. Phytochrome is know to be act as a major photoreceptor mediating divergent plant responses to light. Although phytochrome has been studied intensively, molecular mechanism by which phytochrome transduce the light remains unknown. We addressed this problem with the aid of molecular genetical techniques. Two topics were pursued in this study. Firstly, activation-tagged Arabidopsis lines were produced and mutants that show deficiencies in light responses are isolated and analyzed. Secondly, nuclear localization of phytochrome B (phyB) was studied by using GFP as a fluorescent tag.Mutants that exhibit partial greening under dim light were isolated from the activation-tagged lines. Among them is a mutant, which resembles brassinolide-less mutants. The molecular analysis revealed that a novel cytochrome P450 gene is over-expressed in this mutant. We further confirmed that the gene is indeed responsible for the phenotypes by producing transgenic Arabidopsis lines over-expressing this gene. Unfortunately, the phenotype appeared to be related not only to phytochrome but also to other photoreceptors. Nevertheless, the gene will be a powerful tool to elucidate relationships between light and brassionsteroid signals.We examined intracellular localization of phyB by expressing a phyB-GEP fusion protein in the phyB deficient mutant of Arabidopsis. The results indicated that the introduced phyB-GFP is biologically active. Microscopic observation of the cells revealed that phyB-GFP localizes to the nucleue under light conditions. In contrast, phyB-GFP is distributed throughout the cell in darkness. Hence, phyB appears to translocate to the nucleus in a light-dependent manner. Although phytochrome has been believed to function in the cytoplasm, oure results suggest that the nucleus is one of the cellular compartments where the phytochrome action takes place