Yamaguchi R, Nakamura M, Mochizuki N, Kay S A, Nagatani A
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
J Cell Biol. 1999 May 3;145(3):437-45. doi: 10.1083/jcb.145.3.437.
Phytochrome is a ubiquitous photoreceptor of plants and is encoded by a small multigene family. We have shown recently that a functional nuclear localization signal may reside within the COOH-terminal region of a major member of the family, phytochrome B (phyB) (Sakamoto, K., and A. Nagatani. 1996. Plant J. 10:859-868). In the present study, a fusion protein consisting of full-length phyB and the green fluorescent protein (GFP) was overexpressed in the phyB mutant of Arabidopsis to examine subcellular localization of phyB in intact tissues. The resulting transgenic lines exhibited pleiotropic phenotypes reported previously for phyB overexpressing plants, suggesting that the fusion protein is biologically active. Immunoblot analysis with anti-phyB and anti-GFP monoclonal antibodies confirmed that the fusion protein accumulated to high levels in these lines. Fluorescence microscopy of the seedlings revealed that the phyB-GFP fusion protein was localized to the nucleus in light grown tissues. Interestingly, the fusion protein formed speckles in the nucleus. Analysis of confocal optical sections confirmed that the speckles were distributed within the nucleus. In contrast, phyB-GFP fluorescence was observed throughout the cell in dark-grown seedlings. Therefore, phyB translocates to specific sites within the nucleus upon photoreceptor activation.
光敏色素是植物中普遍存在的光感受器，由一个小的多基因家族编码。我们最近发现，一个功能性的核定位信号可能存在于该家族主要成员光敏色素B（phyB）的COOH末端区域内（坂本，K.，和A. 永谷。1996年。植物杂志。10:859 - 868）。在本研究中，由全长phyB和绿色荧光蛋白（GFP）组成的融合蛋白在拟南芥的phyB突变体中过表达，以研究phyB在完整组织中的亚细胞定位。所得的转基因株系表现出先前报道的phyB过表达植物的多效性表型，这表明融合蛋白具有生物活性。用抗phyB和抗GFP单克隆抗体进行的免疫印迹分析证实，融合蛋白在这些株系中高水平积累。对幼苗的荧光显微镜观察显示，phyB - GFP融合蛋白定位于光生长组织的细胞核中。有趣的是，融合蛋白在细胞核中形成斑点。共聚焦光学切片分析证实，斑点分布在细胞核内。相比之下，在黑暗生长的幼苗中，整个细胞都观察到phyB - GFP荧光。因此，光敏色素B在光感受器激活后会转移到细胞核内的特定部位。
