Study On The SUMO E3 Ligase MdSIZ1 Which Regulates The Stress Rsponses And Anthocyanin Accumulation In Apple

SUMOylation, the conjugation of target proteins with SUMO(small ubiquitin-related modifier), is a post-translational modification process in eukaryotes and involves the sequential action of activation(E1), conjugation(E2) and ligation(E3) enzymes. In plant,SIZ1 protein is a SUMO E3 ligase and promotes the conjugation of SUMO proteins to target substrates. SIZ1 is mainly involved to regulate the SUMOylation of target proteins associated with stress responses. Recently, SIZ1 has been isolated and functionally characterized in Arabidopsis, rice and Dendrobium. In this study, MdSIZ1 was isolated and functionally characterized in appleSUMOylation analysis showed that MdSIZ1 has SUMO E3 ligase activity in vitro and in vivo. SUMO conjugation increased in response to high temperature, low temperature, and abscisic acid(ABA). The ectopic expression of MdSIZ1 gene in Arabidopsis siz1-2 mutant partially complemented the morphological mutant phenotype and enhanced the levels of SUMO conjugation. Taken together, these results suggested that MdSIZ1-mediated SUMO conjugation of target proteins is an important process that regulates the adaptation of plants to various environmental stresses in apple.Apple MdSIZ1 protein has conservative domains similar to Arabidopsis AtSIZ1.Real-time quantitative RT-PCR analysis showed that MdSIZ1 gene expression was induced by phosphate deficiency. Meanwhile, SUMOylation level was remarkably enhanced. As a phosphorus starvation-dependent protein, MYB transcription factor MdPHR1 is modified by SUMO1. Subsequently, MdSIZ1 expression vector was constructed and genetically transformed into Arabidopsis mutant siz1-2 and apple callus, respectively. The ectopic expression of MdSIZ1 gene partially complemented the mutated phenotype in siz1-2 mutant under phosphate deficient conditions. The survival rate was almost same between transgenic lines and wild type. In addition, the MdSIZ1 transgenic callus exhibited higher SUMO conjugate level and fresh weight than the WT control. Therefore, MdSIZ1 positively regulated the tolerance of apple callus to phosphate deficiency in apple.In this study, it was demonstrated that R2R3 MYB transcription factor MdMYB1 physically interacted with and was SUMOylated by MdSIZ1 in apple. The SUMOylation of MdMYB1 is induced by adverse temperature conditions and stabilizes the MdMYB1 protein by inhibiting its 26 S proteasome-mediated proteolysis. It was also found that differentexpression patterns of MdSIZ1 gene exerted great influence on fruit coloration by MdMYB1,indicating that MdSIZ1 plays an important role in the regulation of apple fruit coloration.Taken together, MdSIZ1 plays an important role in the adaptation to environmental stresses and the regulation of secondary metabolism in apple. Therefore, MdSIZ1 is useful for the improvement of stress tolerance and fruit quality in apple.