Central role of the monothiol-glutaredoxin GrxS17 in shoot apex differentiation upon flowering induction

植物体内含有大量具有氧化还原活性的硫醇蛋白，即硫氧还蛋白(TRX)、谷氧还蛋白(GRX)、过氧化还蛋白(PRX)和其他氧化还蛋白。我们重点研究了GRX家族中一个独特的成员--拟南芥中的单硫醇-GrxS17，它由一个TRX结构域和三个GRX结构域组成。胞质和核定位的蛋白质以二聚体的形式存在，包含三个[2Fe-2S]簇。纯合子T-DNA插入系(Salk_021301.56.00.X)开花诱导延迟，顶端器官完全缺乏分化，类似Pin1的表现型，生长素分布紊乱。GrxS17与前期工作中发现的转录因子和蛋白激酶的相互作用将在体内和体外使用各种截断和半胱氨酸突变的形式进行研究。这些分子特征的功能重要性将在基因敲除背景下在转基因品系中进行测试。GrxS17及其与转录因子NF-YC11和PKC样蛋白激酶的相互作用将是拟议工作的重点。为了推断出重要的功能特征，各种结构将在瞬时转化的原生质体和稳定转化的植物中进行测试。KO背景将与表达GSH氧化还原状态指示物(RoGFP2)和生长素分布(DR5启动子：：YFP)的线交叉，以监测活细胞中诱导的变化。GrxS17-KO在长日照条件下的PIN样表型将分别作为受损分化计划和救援的读出。茎分生组织分化的主要调节因子依赖氧化还原和金属的功能是环境调节发育计划的一部分。将解决以下问题：1.GrxS17如何反映胞浆/细胞核的氧化还原状态？它是否起到金属开关的作用，具有依赖于协调Cys残基氧化还原状态的[2Fe-2S]簇？2.它与转录因子NF-YC11和类PCK蛋白激酶相互作用的结构特征和结构域是什么？3.是什么诱导了GrxS17及其相互作用伙伴的核转移和DNA结合？4.GrxS17作为中心枢纽连接了哪些信号通路？5.对光环境、光质量和数量、植物激素和温度的反应如何？

Plants possess a remarkable amount of redox-active thiol-containing proteins, namely thioredoxins (Trx), glutaredoxins (Grx), peroxiredoxins (Prx) and other redoxins. We focus on a unique member of the Grx family, the monothiol-GrxS17 from Arabidopsis thaliana that consists of a single Trx and three Grx domains. The cytosolic and nuclear-localized protein occurs as a dimer and contains three [2Fe-2S] clusters. The homozygous T-DNA insertion line (SALK_021301.56.00.X) exhibits delayed flowering induction with a complete lack of organ differentiation at the shoot apex resembling the PIN1 phenotype with disturbed auxin distribution. The interaction of GrxS17 with transcription factors and protein kinases found in preliminary work will be studied, both in vivo and in vitro, using various truncated and Cys-mutated forms. The functional importance of these molecular characteristics will be tested in transgenic lines on the knockout background. GrxS17 and its interaction with the transcription factor NF-YC11 and the PKC-like protein kinase will be in the focus of the proposed work. In order to deduce functionally important features, various constructs will be tested in transiently transformed protoplasts and in stably transformed plants. The KO background will be crossed with lines expressing indicators of GSH-redox state (roGFP2) and auxin distribution (DR5 promoter::YFP) in order monitor the induced changes in the living cells. The PIN-like phenotype in GrxS17-KO under long-day regime will be used as a read-out for an impaired differentiation programme and rescue, respectively. The redox- and metal-dependent function of this master regulator of shoot meristem differentiation is part of environmentally adjusted developmental programme. The following questions will be addressed: 1. How does GrxS17 reflect the redox-state of the cytosol/nucleus? Does it function as a metal switch with a [2Fe-2S] cluster that depends on the redox-state of the coordinating Cys residues?2. Which structural characteristics and which domains are essential for its interactions with the transcription factors NF-YC11 and the PCK-like protein kinase? 3. What induces the nuclear transfer and DNA binding of GrxS17 and of its interaction partners? 4. Which signaling pathways are interconnected by GrxS17 acting as a central hub? 5. How is the response to light regime, light quality and quantity, phytohormones and temperature affected in mutant and reconstituted lines of A. thaliana?