Discrete And Essential Roles Of The Multiple Domains Of Arabidopsis FHY3 In Mediating Phytochrome A Signal Transduction

As sessile organisms, higher plants have evolved a network of photoreceptors tosense changes in the ambient light environment and undergo adaptive growth anddevelopment. Among these photoreceptors, phytochromes are the best characterizedand exist in two distinct forms, Pr and Pfr. In Arabidopsis (Arabidopsis thaliana),phytochromes are encoded by a five-member gene family, PHYTOCHROME A(PHYA) to PHYE. Phytochrome A is the primary photoreceptor for mediating variousfar-red light-induced responses in higher plants. Intensive molecular genetic studieswith Arabidopsis have helped to establish a framework of the molecular eventslinking phyA activation to regulated nuclear gene expression to induce atranscriptional cascade necessary to implement FR light-mediated photomorphogenicresponses.FAR-RED ELONGATED HYPOCOTYLS 3 (FHY3) and (FAR-RED-IMPAIREDRESPONSE 1 (FAR1), a pair of homologous proteins identified as two essentialpositive regulators of phyA signaling, act as novel transcription factors essential foractivating the expression of FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) andFHY1-like (FHL), whose products are required for light-induced phytochrome Anuclear accumulation and subsequent light responses. Arabidopsis FHY3 and FAR1share significant sequence homology to Mutator-like transposases. FHY3, FAR1, andMutator-like transposases also share a similar domain structure, including anN-terminal C2H2 zinc finger domain, a central putative core transposase domain, anda C-terminal SWIM motif (named after SWI2/SNF and MuDR transposases). To gaina better understanding of the function of FHY3 in phyA signaling, in this study, weperformed a promoter-swapping analysis of FHY3 and FAR1 to explore themolecular basis of partially overlapping function of FHY3 and FAR1, and usedsite-directed mutagenesis and transgenic approaches to understand the molecularmode of FHY3 function.To explore the molecular basis of partially overlapping function of FHY3 andFAR1, we generated a series of transgenic plants, includeing FHY3 endogenous promoter driving the full-length FHY3 gene in fhy3-4 mutant background, FAR1endogenous promoter driving the full-length FAR1 gene in far1-2 mutant background,FHY3 endogenous promoter driving the full-length FAR1 gene in fhy3-4 and far1-2mutant background, and FAR1 endogenous promoter driving the full-length FHY3gene in fhy3-4 and far1-2 mutant background. The result of promoter-swappinganalysis suggests that the partially overlapping functions of FHY3 and FAR1 entaildivergence of their promoter activities and protein subfunctionalization.To further define the structure-function relationship of FHY3, we performedmutagenesis studies of a group of highly conserved amino acids in the predictedN-terminal C2H2 zinc finger domain, the central core transposase domain, and theC-terminal SWIM motif. The results reveal that the multiple domains of ArabidopsisFHY3 are of discrete and essential roles in mediating phyA Signal Transduction. As aconclusion, (1) the results of a yeast one-hybrid assay, an electrophoresis mobilityshift assay (EMSA) and transgenic plants analysis showed that the conservedN-terminal C2H2 zinc finger domain is essential for direct DNA binding andbiological function of FHY3 in mediating light signaling by; (2) the results of a yeastone-hybrid assay, an Arabidopsis protoplasts transformation and transgenic plantsanalysis showed that the central core transposase domain and C-terminal SWIMdomain are essential for the transcriptional regulatory activity of FHY3 by; (3) theresult of a yeast two-hybrid assay showed that the central core transposase domainand C-terminal SWIM domain are essential for its homodimerization orheterodimerization with FAR1 by, and the ability to form homodimers orheterodimers largely correlates with the transcriptional regulatory activity of FHY3 inplant cells; (4) the observation of yellow fluorescence protein (YFP) signal in aconfocal microscope showed that FHY3 and FAR1 proteins are localized in thenucleus in plant cells and the D288A, E323A, and H591A amino acid substitutions donot affect nuclear localization of the FHY3 protein. Together, our results providefunctional support for the proposition that FHY3 and FAR1 represent transcriptionfactors derived from a Mutator-like transposase(s).