MOLECULAR FRAMEWORK FOR NITRIC OXIDE (NO) SENSING IN SEEDS
Department of Botany and Plant Physiology, Spanish-Portuguese Agricultural Research Institute (CIALE), School of Biology, University of Salamanca, Salamanca, Spain
Contact: Oscar Lorenzo, email@example.com
Seed dormancy and germination are complex traits regulated by the interaction of different signalling molecules such as the phytohormone abscisic acid (ABA) and the gasotransmitter nitric oxide (NO). In order to elucidate their crosstalk, a genetic screening in presence of (+)-S-ABA coupled to NO scavenger (cPTIO) let the identification of two gap (germination in ABA and cPTIO) mutants, that show ABA and cPTIO-insensitive phenotypes in the transition from dormancy to germination. After characterization and positional cloning of both of them, we found GAP1 encodes ANAC089 transcription factor and GAP2 the well-known ABI5 seed master regulator. gap1 mutants lack the critical transmembrane domain of ANAC089 protein that confers the mutated proteins constitutive nuclear localization. Interestingly, mutants exhibited higher endogenous NO levels avoiding the effect of NO-depletion during seed germination. Furthermore, whole-genome transcriptional profiling indicated the existance of different groups of ABA- and redox-related genes differentially regulated by ANAC089. This transcription factor can specifically bind to the core cis-regulatory element GCGTCAGC harbour in the promoters of ANAC089 regulated genes. Consistently, translocation of ANAC089 protein to the nucleus was directed by changes in cell redox status after NO- and redox-related compound treatments. Thus, ANAC089 transcription factor integrates ABA signalling with NO levels to modulate redox homeostasis as a novel master regulator during seed germination in Arabidopsis. Using pharmacological and genetic approaches, we found that ABI5 protein levels are high in NO-deficient mutant backgrounds and low in NO-overaccumulating seeds. S-nitrosylation of ABI5 at a specific cysteine residue facilitates its degradation by CUL4-based and the RING-type KEEP ON GOING (KEG) E3 ligases and promotes seed germination. Thus, ABI5 is regulated through the antagonistic action of ABA and NO, as evidenced by the synergistic effect of GSNO on ABI5 destabilization, suggesting an inverse molecular link between NO and ABA hormonal signalling in gene regulation of early seedling development. Currently, the identification of redoxins able to denitrosylate ABI5 at this developmental checkpoint solves one of the biggest challenges in the understanding of the reversible NO role in plant signal transduction networks and establish a molecular framework for NO function during seed dormancy and germination.