The six conserved serine/threonine sites of REPRESSOR OF ga1-3 protein are important for its functionality and stability in gibberellin signaling in Arabidopsis.

MAIN CONCLUSION: 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.

Arabidopsis DELLA protein degradation is controlled by a type-one protein phosphatase, TOPP4.

Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway.

Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp(y11) mutant embryos or cardia bifida phenotypes in fau(tm236a) mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.