Analysis of the function, localization and regulation of RGA, a negative regulator of gibberellin signaling in Arabidopsis thaliana

The plant hormone gibberellin (GA) regulates plant growth and development. RGA and GAI are negative regulators of GA signaling in Arabidopsis thaliana. Functional redundancy between these two homologous genes was investigated through the use of the null alleles rga-24 and gai-t6. rga-24 but not gai-t6 alone partially restored several of the mutant phenotypes of the GA-deficient ga1-3 mutant. Phenotypes rescued include abaxial trichome initiation, rosette radius, flowering time, stem elongation and apical dominance. The trigenic rga-24/gai-t6 /ga1-3 mutant is restored to wild-type or GA-overdose morphology for all of the phenotypes partially rescued by rga-24, indicating that RGA and GAI have a synergistic effect on plant growth. These results showed that these genes are partially but not completely redundant and that RGA is a more important repressor of GA signaling than GAI. Histochemical localization of a GUS-RGA fusion protein showed staining in cotyledons, hypocotyls, root tip, true leaves, and inflorescence stem but not in germinating seedlings or anthers. GUS-RGA staining and immunoblotting with anti-RGA antibodies showed that RGA levels were higher in younger organs and decreased as tissue aged, suggesting that RGA is fine-tuning growth, not stopping it. A functional analysis was done on RGA protein to identify regions important for RGA function. The GA-unresponsive semi-dwarf gai-1 mutant contains gai protein with a deletion in the N-terminal DELLA domain. This region is identical between RGA and GAI, so the role of the DELLA domain in GA signaling was investigated using a rga allele (rga-Δ17) containing an equivalent mutation. rga-Δ17 confers a GA-unresponsive dwarf phenotype on transgenic Arabidopsis and this phenotype can be partially suppressed by gar2 and spy-8, suppressors of gai-1. These results suggest that RGA and GAI are regulated, at least partially, by the same mechanism. RGA protein is degraded in response to GA but rga-Δ17 protein does not, indicating that the DELLA domain plays a regulatory role in GA-induced RGA degradation and that deletion of this motif stabilizes the protein. Examination of RGA protein levels in other rga mutant alleles indicated that the C-terminal region of RGA is also a regulatory region for GA-induced degradation.