Functional analysis of PAT1

光敏色素是一种红色/远红色的光感受器，在植物的整个生命过程中起着关键作用。光敏色素A （Phytochrome A, phyA）是唯一的远红光受体，在富含远红光的环境中萌发和去黄化是必需的。phyA在黑暗中定位于细胞质，但在光线激活后转运到细胞核中。我们之前已经证明phyA的核转运对于远红光感知至关重要，并且它依赖于两个同源蛋白FHY1（远红细长下胚轴1）和FHL （FHY1样）。FHY1和FHL都含有一个NLS（核定位序列），并以轻调控的方式与phyA相互作用。FHY1/FHL的c端phya结合位点高度保守，是FHY1/FHL功能不可缺少的。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表型，这是由c端截断的PAT1的表达引起的。我们的数据表明，这种截断的PAT1与FHY1/FHL的c端phyA结合位点相互作用，并且几乎完全阻断phyA核运输。将NLS直接融合到phyA和通过过表达增加FHY1的量都可以挽救pat1-1表型，从而将pat1-1突变体确定为phyA核转运突变体。根据我们的工作模型，PAT1调节可用于phyA核运输的FHY1/FHL的数量，因此，对远红光的敏感性。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，这种调节可能受到干扰。通过结合分子、细胞生物学和生理学的方法，我想确定与PAT1调控域相互作用的因素，以及利用PAT1与远红光感知进行串扰的信号/信号传导途径。

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.