| Gibberellic acids (GAs) are a class of growth-promoting hormones and play important roles in seed germination, hypocotyl and stem elongation, leaf expansion,juvenile-to-adult phase transition, pollen maturation, and flowering. In the gibberellin signaling pathway, DELLA proteins are the central transcription repressor that repress almost all known GA-dependent processes. Although DELLA proteins are known to exhibit phosphorylation and de-phosphorylation states at the molecular level, and protein serine (Ser)/threonine (Thr) de-phosphorylation activity is suggested to be necessary for the degradation of DELLAs, neither a protein kinase nor Ser/Thr phosphatase that can modify DELLA proteins in Arabidopsis has been discovered until now. In addition, little is known regarding the effects of different modifications of DELLA proteins on the regulation of their bioactivity and stability at the genetic level. To investigate further whether DELLA proteins can be phosphorylated/de-phosphorylated modification, and whether the conserved Ser/Thr site-directed substitutions are important for DELLA functions and stability, we deiermined the de-phosphorylated modification of DELLA proteins in molecular biological methods and genetic level by yeast two hybrid, pull-down,co-immunoprecipitation, site-directed substitution and transgenic technology.1. TOPP4 interacts with DELLA protein. Protein phosphatase TOPP4 interacts with DELLA proteins in vitro and in vivo. Furthermore, TOPP4 interacts with the C-terminal, GRAS domain, of RGA and GAI.2. TOPP4 de-phosphorylates DELLA proteins, RGA and GAI. Both RGA and GAI exhibited the same band shift by treating with calf intestinal alkaline phosphatase(CIP) and recombinant TOPP4, suggesting that protein phosphatase TOPP4 can de-phosphorylate DELLA proteins.3. TOPP4 mediate the degradation of DELLA proteins, RGA and GAI. Both RGA and GAI were accumulated in topp4-1, which exhibited dwarf phenotypes; and both proteins decreased in 35S:TOPP4, whose phenotypes of whole plant height, leaf area, and influence were higher and greater.4. Mimicking constitutive de-phosphorylated state of RGA activates GA signaling and is unstable in the presence of GA. The overexpression of de-phosphomimic RGA in Arabidopsis Col-0 plants caused GA-overdose phenotypes,which were similar to DELLA-deficient mutant dellaP, in leaf size, plant height,flowering time, flower number, pistil and silique lengths, fertility, and physiological sensitivity to GA3 and paclobutrazol (PAC) . These phenotypes were probably caused because de-phosphomimic RGA retained its transcription activator activity that induces GA biosynthesis genes, but lost the transcription repressor function that inhibits GA-responsive genes. Further, the de-phosphomimic RGA protein was unstable and easily degradable unlike the wild-type RGA protein, suggesting that the de-phosphorylated form is necessary for its degradation.5. Mimicking constitutive phosphorylated state of RGA represses GA signaling and is stable in the presence of GA. The overexpression of phosphomimic RGA caused phenotypes that were deficient in GA and had non-degradable RGA, such as dwarf, dark green, and sterility. These phenotypes were probably caused because phosphomimic RGA is unable to induce GA biosynthesis genes, but can repress the expression of GA-responsive genes. In addition, the phosphomimic RGA was stable and hardly degradable, which aggravated the RGA-inhibiting function in GA signaling. Furthermore,non-degraded RGA affected plant fertility more severe in the Col-0 ecotype than in the Ler ecotype.Our biochemical and genetic analyses revealed that DELLA proteins might be de-phosphated by the protein phosphatase TOPP4, and they performed various biological functions and regulated their stability by the phosphorylated/de-phosphorylated modification of the six conserved Ser/Thr sites. |
PDF Full Text Request