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

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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?