Molecular Mechanism On Protein Phosphatase TOPP4in Regulating Gibberellin Signaling In Arabidopsis

Gibberellins (GAs) are a class of phytohormones. They are important regulators of plant growth and development, including seed germination, leaf formation, stem and root elongation, floral initiation, and flowering time. One of the major events during GA-regulated growth is the degradation of DELLA proteins, key negative regulators of the GA signaling pathway. The stability of DELLA proteins is believed to be mediated by protein phosphorylation and dephosphorylation, although the phosphatases involved and its regulatory mechanism have not been identified.By screening an ethyl methane sulfonate (EMS)-mutagenized Arabidopsis thaliana population, we have identified and characterized a dwarfed Arabidopsis mutant, topp4-1. The mutant exhibits lack of apical dominance, aberrant leaf phyllotaxy, tiny, curled, and dark-green rosette leaves, delayed flowering, smaller flowers with loosely arranged sepals, partially twisted petals and siliques, reduced mature pollen grains in anthers, and fewer seeds in mature siliques. TOPP4encodes a catalytic subunit of PP1protein phosphatase in Arabidopsis. TOPP4is ubiquitously expressed in various organs throughout development. TOPP4protein is mainly localized in the nucleus and at the plasma membrane. Mutated topp4-1protein has a dominant-negative effect on plant development, because overexpressing of mutated topp4-1in wild-type plants resulted in topp4-1-mimic phenotypes. Reducing expression of TOPP4using artificial microRNA method caused dwarfed phenotype in wild type, while overexpressing of TOPP4in wild-type plants caused enlarged organs.The transcription of GA biosynthetic enzyme genes GA20oxl and GA3oxl were decreased, while the expression of GA catabolic enzyme gene GA2ox2was increased in topp4-1, suggesting that GA content may be decreased in this mutant. High performance liquid chromatography (HPLC) analysis verified that the content of total GAs were decreased in topp4-1mutant. However, topp4-1is insensitive to exogenously applied GA3. Thus, TOPP4is likely involved in GA signal transduction. GA promoted the accumulation of TOPP4in wild type, while this process was blocked in gidla/b/c.The dwarfed phenotypes of topp4-1were partially rescued by the DELLA deficient mutants rga-t2and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for biological function of TOPP4. Both RGA and GAI accumulated in the topp4-1mutant compared to the wild-type plant but were present in lower concentration in transgenic plants overexpressing TOPP4than in the wild-type plants. In vitro degradation assays showed that the dgradation of RGA was delayed in topp4-1, and adding TOPP4could rescue this defect, therefore, TOPP4may promote the degradation of DELLA proteins in GA signaling. BiFC analysis demonstrated that TOPP4could directly bind RGA and GAI. In vitro dephosphorylation assays confirmed that TOPP4could dephosphorylate RGA and GAI. In conclusion, TOPP4promotes DELLA proteins degradation by directly dephosphorylating them in GA signal transduction. These studies provide strong evidence for a crucial role of protein dephosphorylation of DELLA in GA signaling pathway, and reveal the important function of PP1in plant growth and development.