Genetic characterization of phytochrome nuclear bodies in plant light signaling

描述（由申请人提供）：核体（NB）是存在于动物和植物细胞中的不同亚核结构域。虽然许多NB，如Cajal小体，PML（早幼粒细胞白血病）NB，和IGC（染色质间颗粒簇）已被广泛研究在哺乳动物系统中，这些核体的形态和组成的变化与人类疾病，NB的精确功能和调节仍然知之甚少。一个可能的原因是缺乏研究NB功能的遗传模型。我们建议使用光敏色素NB在植物光信号作为遗传模型系统，调查NB功能背后的一般原则。光敏色素是一类红色和远红色光感受器，通过转录调控调控植物的生长发育。光敏色素在黑暗中定位于细胞质中。在光激活时，它们重新定位到细胞核并形成光敏色素NB。我们证明了光稳定的光敏色素B（PHYB）的形成含有NBs直接受光调控，并与光敏色素反应密切相关。基于这些观察，我们假设光敏色素NB直接参与光敏色素信号事件。为了验证这一假设，我们提出：（1）通过基于共焦的遗传筛选鉴定和表征PHYB-GFP NB形成所需的新组分。我们已经发现了29个这样的突变体。一个位点，HMR，已被克隆，和其他两个粗略定位。引人注目的是，我们的初步研究表明，从这个屏幕上确定的第一个基因（HMR）是所需的PHYB-GFP NB的形成和早期光敏色素信号事件，包括光依赖性蛋白水解的PHYA，光不稳定的光敏色素。有趣的是，HMR在结构上类似于酵母蛋白质RAD 23的哺乳动物直系同源物，其是参与蛋白质降解的多泛素结合蛋白。这些结果首次提供了光敏色素核体与蛋白质降解相关的遗传学证据，并表明该遗传筛选将有可能发现光敏色素核体功能与早期光敏色素信号事件相关的新组分;（2）研究光敏色素核体在早期光敏色素信号事件中的功能。我们提出了许多实验来直接测试光敏色素NB是否是PHYA降解和/或转录调节的位点;（3）定义HMR在PHYA降解中的功能。我们将测试HMR是否通过将PHYA递送到蛋白酶体进行降解而充当RAD 23。总的来说，拟议的实验应有助于更好地了解植物中的光信号。更重要的是，他们还将开始解开NB在细胞信号传导中的一般原理。 公共卫生相关性：核体是存在于动物和植物系统中的不同亚核域。许多哺乳动物核体的形态和组成的变化与人类疾病有关。我们将利用植物光敏色素核体系统，并将其作为一个遗传模型来研究核体在细胞信号传导中的功能和调控。我们的研究应该对我们了解人类疾病的细胞和分子基础做出重大贡献。

DESCRIPTION (provided by applicant): Nuclear bodies (NBs) are distinct subnuclear domains present in both animal and plant cells. Although numerous NBs, such as Cajal bodies, PML (promyelocytic leukemia) NBs , and IGC (interchromatin granule cluster) have been extensively studied in mammalian systems, and changes in morphology and constitution of these nuclear bodies are associated with human diseases, the precise function and regulation of NBs are still poorly understood. One possible reason for this is the lack of genetic models to study NB functions. We propose to use phytochrome NBs in plant light signaling as a genetic model system to investigate general principles behind NB functions. Phytochromes are red and far-red photoreceptors regulating plant development and growth through transcription regulation. Phytochromes localize in the cytoplasm in the dark. Upon light activation, they relocate to the nucleus and form phytochrome NBs. We demonstrated that the formation of a photo-stable phytochrome B (PHYB) containing NBs is directly regulated by light and is tightly correlated to phytochrome responses. Based on these observations, we hypothesize that phytochrome NBs are directly involved in phytochrome signaling events. To test this hypothesis, we propose to: (1) identify and characterize new components required for PHYB-GFP NB formation by a confocal-based genetic screen. We have already identified twenty-nine such mutants. One locus, HMR, has been cloned, and two others rough-mapped. Strikingly, our preliminary studies show that the first gene (HMR) identified from this screen is required for both PHYB-GFP NB formation and early phytochrome signaling events including the light-dependent proteolysis of PHYA, a photo-labile phytochrome. Interestingly, HMR is structurally similar to the mammalian ortholog of a yeast protein RAD23, which is a multiubiquitin binding protein involved in protein degradation. These results provide the first genetic evidence linking phytochrome nuclear bodies with protein degradation, and demonstrate that this genetic screen will likely identify novel components linking phytochrome NB function and early phytochrome signaling events; (2) investigate the function of phytochrome NBs in early phytochrome signaling events. We propose a number of experiments to directly test whether phytochrome NBs are sites for PHYA degradation and/or transcription regulation; (3) define the function of HMR in PHYA degradation. We will test whether HMR acts as RAD23 by delivering PHYA to the proteasome for degradation. Collectively, the proposed experiments should contribute significantly to a better understanding of light signaling in plants. More importantly, they will also start to unravel general principles of NBs in cell signaling. PUBLIC HEALTH RELEVANCE: Nuclear bodies are distinct subnuclear domains present in both animal and plant systems. Changes in morphology and constitution of numerous mammalian nuclear bodies are associated with human diseases. We will take advantage of the plant phytochrome nuclear body system and use it as a genetic model to investigate the function and regulation of nuclear bodies in the context of cell signaling. Our studies should contribute significantly to our understanding of the cellular and molecular basis for human diseases.