Studies On The Functions Of U-box Protein PUB13in Arabidopsis

The ubiquitination pathway is widely involved in regulating plant growth, development and defense. Few studies, however, were reported about the function of this pathway in correlating the signaling between plant development and defense. Rice Spl11gene encodes a U-box/ARM protein endowed with E3ubiquitin ligase activity. PUB13negatively regulates rice disease resistance against some bacterial and fungal pathogens, and also regulates rice flowering time via monoubiquitinating its target SPIN1. In this study, we mainly aim at the biological and biochemical functions of the U-box/ARM protein PUB13which is ortholog of SPL11in Arabidopsis. The results of ubiquitination reaction assay show that PUB13possesses E3ligase activity, and the conserved amino acid Val273in U-box domain is required for the activity of PUB13. During the planting of pub13, a T-DNA insertional mutant of PUB13, we found this mutant spontaneously displays lesion mimic phenotype, especially under long day growth conditions. The pub13leaves, correspondingly, show abundant H2O2accumulation and cell death based on DAB and trypan blue staining. Moreover, the expression of pathogenesis related gene PR1and content of salicylic acid (SA) are increased in pub13, which lead us to analyze the ability of pub13in disease resistance. Under long day conditions, pub13shows enhanced resistance against biotrophic pathogens but not necrotrophic pathogens. To test the affection of SA signaling in pub13phenotypes, SID2and PAD4which are components of SA signaling pathway are knocked out in pub13respectively, and got pub3sid2-2and publ3pad4double mutants. The knock out of SID2and PAD suppressed phenotypes of H2O2accumulation, cell death and disease resistance obviously in pub13, which indicates that SA signaling is required for the cell death and defense regulated by PUB13. In addition, the humidity was also found to affect the phenotypes of pub13:cell death and resistance to biotrophic pathogens are further enhanced in pub13after high humidity treatment while the resistance against necrotrophic pathogens is suppressed.Another important function of PUB13was found in this study, which is regulating flowering time of Arabidopsis. pub13flowers earlier than wild-type under long day and middle day conditions but not short day conditions. The flowering time regulated by PUB13is also required SA signaling pathway. Because of the highly similar amino acids sequence between PUB13and SPL11, rice Spl11was transformed into pub13mutant, and found that Spl11complemented the phenotypes of cell death and flowering time in pub13. The above results demonstrate that the E3ligase PUB13comprehensively regulates cell death, disease resistance and flowering time, indicating the cross-talk between plant development and innate immunity signaling, and also provide new clues in study the functions of ubiquitination in comprehensively regulating plant signaling transduction.