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

植物含有大量具有氧化还原活性的含巯基蛋白质，即硫氧还蛋白（Trx）、谷氧还蛋白（Grx）、过氧化物氧还蛋白（Prx）和其他氧化还原蛋白。我们专注于一个独特的成员的Grx家族，单硫醇-GrxS 17从拟南芥，由一个单一的Trx和三个Grx结构域。胞质和核定位的蛋白质发生作为二聚体，并含有三个[2Fe-2S]簇。纯合T-DNA插入系（SALK_021301.56.00.X）表现出延迟的开花诱导，在茎尖完全缺乏器官分化，类似于具有扰乱的生长素分布的PIN 1表型。GrxS 17与转录因子和蛋白激酶的相互作用在初步工作中发现，将研究，在体内和体外，使用各种截断和半胱氨酸突变的形式。这些分子特征的功能重要性将在基因敲除背景下的转基因品系中进行测试。GrxS 17及其与转录因子NF-YC 11和PKC样蛋白激酶的相互作用将是拟议工作的重点。为了推断功能上重要的特征，将在瞬时转化的原生质体和稳定转化的植物中测试各种构建体。KO背景将与表达GSH-氧化还原状态（roGFP 2）和生长素分布（DR 5启动子：：YFP）的指示物的细胞系交叉，以监测活细胞中诱导的变化。在长日方案下GrxS 17-KO中的PIN样表型将分别用作受损分化程序和拯救的读出。氧化还原和金属依赖的功能，这个主调节芽分生组织分化的环境调整的发展计划的一部分。以下问题将得到解决：1。 GrxS 17如何反映细胞质/细胞核的氧化还原状态？它是否作为一个金属开关与[2Fe-2S]集群，这取决于氧化还原状态的协调半胱氨酸残基？2. 哪些结构特征和哪些结构域是其与转录因子NF-YC 11和PCK样蛋白激酶相互作用所必需的？3. 什么诱导GrxS 17及其相互作用伴侣的核转移和DNA结合？4. 哪些信号通路通过GrxS 17作为中心枢纽相互连接？5. 光环境、光质和光量、植物激素和温度如何影响A. thaliana？

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?