Functional analysis of PAT1

光敏色素是一种红色/远红光感受器，在植物的整个生命过程中发挥着关键作用。光敏色素A(PhyA)是唯一的远红光受体，在远红光丰富的环境中萌发和去黄化是必需的。PhyA在黑暗中定位于胞浆，但在光激活后移位到细胞核中。我们以前已经证明，PhyA的核运输是远红光感知所必需的，并且它依赖于两个同源蛋白FHY1(远红细长下胚轴1)和FHL(FHY1-like)。FHY1和FHL都含有一个NLS(核定位序列)，并以光调控的方式与PhyA相互作用。FHY1/FHL基因C末端的PhyA结合位点高度保守，是FHY1/FHL功能所必需的。值得注意的是，在远红光下，pat1-1突变体表现出强烈的显性负性表型，这是由C端截短的PAT1表达引起的。我们的数据表明，这个截短版本的PAT1与FHY1/FHL的C端PhyA结合位点相互作用，几乎完全阻止了PhyA的核运输。将NLS直接与PhyA融合，并通过过表达增加FHY1的数量，都可以挽救pat1-1的表型，从而将pat1-1突变体鉴定为PhyA核运输突变体。根据我们的工作模型，PAT1调节可用于PhyA核运输的FHY1/FHL的量，因此对远红光敏感。因此，任何调节PAT1的信号都会调制远红光信号。在pat1-1突变体中，这一调节可能被扰乱。使用分子、细胞生物学和生理学相结合的方法，我想确定与PAT1的调控结构域相互作用的因素，以及使用PAT1进行远红光感知串扰的信号/信号通路。

Phytochromes are red/far-red photoreceptors, which play a key role throughout the life of plants. Phytochrome A (phyA) is the only far-red light receptor and required for germination and de-etiolation in far-red light enriched environments. phyA localises to the cytosol in the dark but translocates into the nucleus after activation by light. We have previously shown that nuclear transport of phyA is essential for far-red light perception and that it depends on the two homologous proteins FHY1 (far-red elongated hypocotyl 1) and FHL (FHY1-like). Both FHY1 and FHL contain an NLS (nuclear localisation sequence) and interact with phyA in a light regulated manner. The phyA-binding site at the C-terminus of FHY1/FHL is highly conserved and indispensable for FHY1/FHL function. Remarkably, in far-red light the pat1-1 mutant exhibits a strong dominant negative phenotype, which is caused by the expression of a C-terminally truncated PAT1. Our data show that this truncated version of PAT1 interacts with the C-terminal phyA-binding site of FHY1/FHL and that it almost completely blocks phyA nuclear transport. Both fusing an NLS directly to phyA and increasing the amount of FHY1 by overexpression rescues the pat1-1 phenotype, which identifies the pat1-1 mutant as a phyA nuclear transport mutant. According to our working model PAT1 regulates the amount of FHY1/FHL available for phyA nuclear transport and, as a consequence, sensitivity to far-red light. Thus, any signal regulating PAT1 would modulate far-red light signalling. In the pat1-1 mutant this regulation may be disturbed. Using a combined molecular, cell biological and physiological approach I want to identify factors interacting with the regulatory domain of PAT1 as well as the signal/signalling pathway that employs PAT1 for cross talk with far-red light perception.