Abstract
Our results provide further insight into the regulation of DELLA proteins in Arabidopsis . We clarified that phosphorylation modification of the six conserved sites is important for RGA functions and stability. The DELLA proteins, important plant growth and development repressors mediate the gibberellin (GA) signaling pathway. Although these proteins exhibit phosphorylation and de-phosphorylation states at the molecular level, little is known regarding the effects of different modifications of DELLA proteins on the regulation of their bioactivity and stability at the genetic level. In this study, six conserved serine (Ser)/threonine (Thr) sites of REPRESSOR OF ga1-3 (RGA) were substituted with alanine (RGA6A) or aspartic acid (RGA6D) to mimic the states of constitutive de-phosphorylation and phosphorylation, respectively. We found that the overexpression of de-phosphomimic RGA in Col-0 plants caused GA-overdose phenotypes, which were similar to DELLA-deficient mutant. These phenotypes were probably attributed to de-phosphomimic RGA, which retained its transcriptional activation activity that induces GA biosynthetic genes, but lost the transcription repressor function that inhibits GA-responsive genes. Further, de-phosphomimic RGA was unstable and easily degradable unlike the wild-type RGA, suggesting that the de-phosphorylated form is necessary for its degradation. In contrast, phosphomimic RGA overexpression caused GA-deficient phenotypes with non-degradable RGA. These phenotypes were probably due to phosphomimic RGA, which represses GA-responsive gene expression instead of inducing GA biosynthetic genes. In addition, phosphomimic RGA was stable and hardly degradable, which aggravated the RGA-inhibiting function in GA signaling. In conclusion, we show that the six conserved Ser/Thr sites are important for the different bioactivities of the RGA protein that regulate the GA response, and also for RGA stability via the mimicking of phosphorylation/de-phosphorylation.
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