Genetic characterization of phytochrome nuclear bodies in plant light signaling

描述（由申请人提供）：核体（NBs）是存在于动物和植物细胞中的不同亚核结构域。尽管许多NBs，如Cajal小体、PML（早幼粒细胞白血病）NBs和IGC（染色质间颗粒簇）已经在哺乳动物系统中得到了广泛的研究，并且这些核小体的形态和构成的变化与人类疾病有关，但NBs的确切功能和调控仍然知之甚少。一个可能的原因是缺乏研究NB功能的遗传模型。我们建议利用植物光信号中的光敏色素NBs作为遗传模型系统来研究NBs功能背后的一般原理。光敏色素是通过转录调控植物发育和生长的红色和远红色光感受器。光敏色素在黑暗中定位于细胞质中。光激活后，它们迁移到细胞核并形成光敏色素NBs。我们证明了含有NBs的光稳定光敏色素B （PHYB）的形成是由光直接调节的，并且与光敏色素的反应密切相关。基于这些观察，我们假设光敏色素NBs直接参与光敏色素信号转导事件。为了验证这一假设，我们建议：(1)通过基于共聚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。其中一个基因座HMR已被克隆，另外两个基因座已被粗略绘制。引人注目的是，我们的初步研究表明，从该筛选中鉴定出的第一个基因（HMR）是PHYB-GFP NB形成和早期光敏色素信号事件（包括光不稳定光敏色素PHYA的光依赖性蛋白水解）所必需的。有趣的是，HMR在结构上类似于酵母蛋白RAD23的哺乳动物同源物，RAD23是一种参与蛋白质降解的多泛素结合蛋白。这些结果提供了将光敏色素核体与蛋白质降解联系起来的第一个遗传证据，并表明这种遗传筛选可能会发现将光敏色素NB功能与早期光敏色素信号事件联系起来的新成分；(2)探讨光敏色素NBs在光敏色素早期信号事件中的作用。我们提出了一些实验来直接测试光敏色素NBs是否是PHYA降解和/或转录调控的位点；(3)明确HMR在PHYA降解中的作用。我们将通过将PHYA传递给蛋白酶体进行降解来测试HMR是否作为RAD23。总的来说，提出的实验应该有助于更好地理解植物的光信号。更重要的是，他们也将开始揭示NBs在细胞信号传导中的一般原理。

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.