Phytochrome A: Structure/Function and Signaling Pathways

光敏色素（phyA至phyE）是一个独特的感觉光感受器家族，其响应于来自环境的信息光信号而调节发育重要基因的表达。 这项研究计划的长期目标是确定这些感觉分子感知，解释和识别光信号的分子和细胞机制，以光响应核基因。最近的证据，从这个和其他实验室已经导致了一个范式转变的概念，关于潜在的细胞内途径和机制，在这个信号传导过程中使用。 数据表明，一种途径涉及phy分子从细胞质到细胞核的光激活易位，随后与启动子结合的碱性螺旋-环-螺旋（bHLH）类转录因子（PIF 3）特异性相互作用，以及随后靶基因的转录激活。 此外，基于寡核苷酸微阵列的表达谱表明，这些靶基因可能包括一个主集的转录调节基因，编排在一个phyA调节的转录网络的各个分支的表达。 尽管取得了这一进展，明确的证据，假设直接参与phy分子的转录调控和可能的机制参与缺乏，和组件和电路组成的初级phy调控的转录网络仍然是完全确定的。 我们建议使用phyA，最好的特点和实验最听话的家庭成员来解决这些不足之处。 该提案的具体目标是：（a）鉴定和表征参与phyA信号转导的分子组分，特别关注那些特异于phyA途径的分子组分;（B）探索phyA转录调节的分子基础，鉴于其与bHLH因子PIF 3的已建立的相互作用;（c）探索phyA信号转移至PIF 3的生化机制;和（d）绘制介导phyA调节的幼苗去黄化的初级转录网络。 实验方法将包括：（a）在phyA特异性信号传导中间体的遗传筛选中鉴定的组分的克隆和分子表征;（B）使用新的酵母双杂交筛选的另外的phyA相互作用蛋白的分子克隆和表征;（c）在用phyA-或PIF 3-融合蛋白转化的植物细胞中的转录激活测定，所述phyA-或PIF 3-融合蛋白通过融合的DNA人工靶向转录因子基因启动子，结合结构域;（d）用重组野生型和信号转导受损的突变体phyA蛋白进行酶测定，以确定在phyA制剂中生化检测到的针对PIF 3的蛋白激酶活性是否与体内phyA信号转导活性相关;和（e）野生型和phyA信号转导缺陷型拟南芥突变体的基于寡核苷酸微阵列的综合表达谱。 了解真核细胞感知和处理细胞外信息信号的分子和细胞机制的全谱仍然是生物医学研究的中心目标。 这里提出的实验系统和策略有可能为实现这一目标做出重大贡献。

The phytochromes (phyA to phyE) are a family of unique sensory photoreceptors that regulate expression of developmentally important genes in response to informational light signals from the environment. The long-term goal of this research program is to define the molecular and cellular mechanisms by which these sensory molecules perceive, interpret and transduce light signals to photoresponsive nuclear genes. Recent evidence from this and other laboratories has resulted in a paradigm shift in concepts regarding the potential intracellular pathways and mechanisms utilized in this signaling process. The data suggest that one pathway involves light- activated translocation of phy molecules from the cytoplasm to the nucleus, followed by specific interaction with a promoter- bound basic helix-loop-helix (bHLH)-class transcription factor (PIF3), and consequent transcriptional activation of target genes. In addition, oligonucleotide microarray-based expression profiling suggests that these target genes may include a master set of transcriptional-regulator genes that orchestrate expression in various branches of a phyA-regulated transcriptional network. Despite this progress, definitive evidence of the postulated direct involvement of phy molecules in transcriptional regulation and the possible mechanisms involved are lacking, and the components and circuitry comprising primary phy-regulated transcriptional networks remain to be fully defined. We propose to address these deficiencies using phyA, the best characterized and experimentally most tractable member of the family. The specific objectives of this proposal are: (a) to identify and characterize molecular components involved in phyA signal transduction, with particular focus on those specific to the phyA pathway; (b) to explore the molecular basis of transcriptional regulation by phyA, given its established interaction with the bHLH factor PIF3; (c) to explore the biochemical mechanism of phyA signal transfer to PIF3; and (d) to map the primary transcriptional network that mediates phyA- regulated seedling deetiolation. The experimental approaches will include: (a) cloning and molecular characterization of components identified in genetic screens for phyA-specific signaling intermediates; (b) molecular cloning and characterization of additional phyA-interacting proteins using a novel yeast two-hybrid screen; (c) transcriptional activation assays in plant cells transformed with phyA-or PIF3-fusion proteins artificially targeted to reporter-gene promoters via fused DNA-binding domains; (d) enzymatic assays with recombinant wild-type and signaling-compromised mutant phyA proteins to determine whether protein kinase activity toward PIF3 detected biochemically in phyA preparations is correlated with phyA signaling activity in vivo; and (e) comprehensive oligonucleotide microarray-based expression profiling of wild-type and phyA- signaling-defective Arabidopsis mutants. Understanding the full spectrum of molecular and cellular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals remains a central goal of biomedical research. The experimental system and strategies proposed here have the potential to contribute significantly to this goal.