Sakamoto K, Nagatani A
Laboratory for Photoperception and Signal Transduction, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Plant J. 1996 Nov;10(5):859-68. doi: 10.1046/j.1365-313x.1996.10050859.x.
Phytochromes are soluble red/far-red-light photoreceptor proteins which mediate various photomorphogenic responses of plants. Despite much effort, the signal transduction mechanism of phytochrome has remained obscure. Phytochromes are encoded by a small multigene family in Arabidopsis. Among the members of the family, phytochrome A (phyA) and B (phyB) are the best characterized. PhyB contains putative nuclear localization signals within its C-terminal region. Transgenic Arabidopsis plants were produced with expressed a fusion protein consisting of GUS and C-terminal fragments of phyB. GUS staining from the fusion protein in these transgenic plants was observed in the nucleus, which suggests that the nuclear localization signal of the fragment is functional. Next, it was examined whether the endogenous phyB was detected in the nucleus. Nuclei were isolated from the light-grown wild-type Arabidopsis leaves and subjected to the immunoblot analysis. The result indicated that a substantial fraction of total phyB was recovered in the isolated nuclei. This result was further confirmed by the immunocytochemical analysis of the protoplasts. Finally, the effects of light treatments on the levels of phyB in the isolated nuclei were examined. Dark adaptation of the plants before the nuclear isolation reduced the levels of phyB. The reduction was accelerated by irradiation of plants with far-red light before the transfer to darkness. Thus, nuclear localization of phyB was suggested to be light-dependent.
光敏色素是可溶的红光/远红光光受体蛋白，介导植物的各种光形态建成反应。尽管付出了很多努力，光敏色素的信号转导机制仍然不清楚。光敏色素由拟南芥中的一个小多基因家族编码。在该家族成员中，光敏色素A（phyA）和B（phyB）的特征最为明显。PhyB在其C末端区域含有假定的核定位信号。构建了表达由GUS和phyB的C末端片段组成的融合蛋白的转基因拟南芥植株。在这些转基因植株中观察到融合蛋白的GUS染色出现在细胞核中，这表明该片段的核定位信号是有功能的。接下来，检测了细胞核中是否能检测到内源性phyB。从光照生长的野生型拟南芥叶片中分离细胞核，并进行免疫印迹分析。结果表明，分离的细胞核中回收了相当一部分总phyB。原生质体的免疫细胞化学分析进一步证实了这一结果。最后，检测了光照处理对分离细胞核中phyB水平的影响。在细胞核分离前对植株进行暗适应降低了phyB的水平。在转移到黑暗之前用远红光照射植株加速了这种降低。因此，phyB的核定位被认为是光依赖的。
