Phytochrome A: Structure/Function and Signaling Pathways

描述(申请人提供)：感觉性光感受器的五成员光敏色素(PHY)家族对信息光信号的感知启动了一个细胞内转导过程，最终导致核基因表达的改变，该核基因指导生长和发育反应，称为光形态发生，适合于当时的环境。该计划的长期目标是确定这一过程发生的细胞、分子和生化机制。目前的数据表明，转导过程包括光激活的光感受器分子从细胞质快速移位到核，在那里它与bHLH转录因子家族的一部分成员物理上相互作用，称为光敏色素相互作用因子(PIF)，在靶基因中诱导转录反应。最近的证据表明，PIF家族的成员共同抑制暗生长幼苗的光形态发生，而光激活PHY通过诱导PIF分子在最初暴露于光下时迅速降解来逆转这种抑制。这个过程包括PHY诱导相互作用的bHLH蛋白的快速磷酸化，然后通过泛素蛋白酶体系统降解。尽管取得了这些进展，但仍有几个核心问题存在，包括负责PIF磷酸化的蛋白激酶的身份以及PHY-PIF信号通路的主要靶基因。我们建议在这里使用拟南芥作为模型系统来解决这些不足。这个建议的具体目标是：(A)确定不同的PIF家族成员在控制早期和萌发后幼苗发育方面的功能；(B)剖析PHY-PIF信号接口上发生的分子和生化交易，包括负责PHY诱导的PIF蛋白磷酸化的蛋白激酶成分(S)的分子鉴定；以及(C)确定PHY相互作用的bHLH因子和它们的目的基因之间的转录接口。实验方法将包括：(A)对PIF-突变组合进行遗传筛选和反向遗传分析，以剖析家族成员的差异和冗余功能，并确定其他因素；(B)对转基因植物中PHY和PIF蛋白的靶向、定点、错义突变进行分子遗传学功能分析，以确定细胞中相互作用、磷酸化和泛素化所需的残基；(C)蛋白质相互作用筛选，包括酵母“三联”筛选和拟南芥亲和纯化复合体的质谱分析，旨在确定与PHY和/或PIF蛋白质在体内光诱导的信号交易之前、期间和之后相关的成分；以及(D)综合全基因组表达谱和染色质免疫沉淀(CHIP)分析，旨在确定PIF-bHLH转录因子在PHY调控的转录网络中的直接主要靶点。

DESCRIPTION (provided by applicant): The perception of informational light signals by the five-membered phytochrome (phy) family of sensory photoreceptors (phyA through phyE) initiates an intracellular transduction process that culminates in the altered expression of nuclear genes that direct growth and developmental responses, termed photomorphogenesis, appropriate to the prevailing environment. The long-term goal of this program is to define the cellular, molecular and biochemical mechanisms by which this process occurs. Current data indicate that the transduction process involves rapid translocation of the light-activated photoreceptor molecule from the cytoplasm to the nucleus, where it interacts physically with a subset of members of the bHLH transcription- factor family, termed phytochrome-interacting factors (PIFs), inducing transcriptional responses in target genes. Recent evidence shows that members of the PIF family collectively repress photomorphogenesis in young dark-grown seedlings, and that photoactivated phy reverses this repression by inducing rapid degradation of the PIF molecules upon initial exposure to light. This process involves rapid, phy-induced phosphorylation of the interacting bHLH protein, followed by degradation via the ubiquitin proteasome system. Despite these advances, several central questions remain, including the identity of the protein kinase responsible for PIF phosphorylation and the primary target genes of the phy-PIF signaling pathway. We propose to address these deficiencies here, using Arabidopsis as a model system. The specific objectives of this proposal are: (a) To define the functions of the different PIF-family members in controlling early, post- germinative seedling development; (b) To dissect the molecular and biochemical transactions occurring at the phy-PIF signaling interface, including molecular identification of the protein kinase component(s) responsible for phy-induced phosphorylation of the PIF proteins; and (c) To define the transcriptional interface between the phy-interacting bHLH factors and their target genes. The experimental approaches will include: (a) Genetic screens and reverse-genetic analyses of pif-mutant combinations to dissect out the differential and redundant functions of the family members and to identify additional factors; (b) Molecular-genetic functional analyses of targeted, site-specific, missense mutants of the phy and PIF proteins in transgenic plants to define residues necessary for interaction, phosphorylation and ubiquitylation in the cell; (c) Protein-interaction screens, including a yeast "tribrid" screen and mass-spectrometry of affinity-purified complexes from Arabidopsis, directed at identifying components that associate with either phy and/or PIF proteins before, during and after the light- induced signaling transaction between the two molecules in vivo; and (d) Integrated genome-wide expression profiling and chromatin-immunoprecipitation (ChIP) analyses aimed at identifying direct, primary targets of the PIF-bHLH transcription factors in the phy-regulated transcriptional network.