CONSTITUTIVE PHOTOMORPHOGENIC 1 is involved in gibberellic acid-mediated germination by repressing RGA-LIKE 2 in Arabidopsis thaliana

Abstract

In flowering plants, germination is a sophisticated process, which is regulated by the cross-talk between endogenous signals and environmental cues such as hormones, light, water and temperature. GA hormone is a key regulator of seed germination. Many genes are involved in GA-mediated seed germination pathway. Among them, RGL2 has been considered to be the major negative regulator by repressing germination associated genes. Here, we showed that COP1 is closely involved in regulating GA-mediated seed germination. We found that the germination rate of cop1 mutants were strongly decreased by paclobutrazol (PAC) treatments, that is inhibitor of GA biosynthesis, compared with Wild-type. However, germination of COP1 overexpressed-transgenic plants is insensitive to PAC. Analysis of western blot using antibody against COP1 recombinant proteins demonstrated that GA affected on COP1 protein stability. While imbibed wild-type seeds under GA present condition significantly increased COP1 protein than that in mock (distilled water) condition, COP1 protein was decreased by PAC treatment. The genetic study of cop1-4 rgl2 double mutants provided strong evidence that COP1 act as upstream negative regulator of RGL2. Further analysis by BiFC and in vitro pull-down assay indicated that COP1 physically interacts with RGL2. RGL2 protein was degraded in COP1 overexpressed plants. These results suggested that COP1 is partially involved negatively regulates in RGL2 protein stability. COP1 regulates the transcript levels of the genes associated with germination such as GASA6, EXPA1, EXPA2, EXPA8, and XTH33. Taken together, our results suggested that COP1 regulates GA-mediated seed germination through degradation of RGL2 proteins.