SINAT E3 Ligases Control The Light-mediated Stability Of Sinat Itself In Arabidopsis

Drosophila protein SINA?seven in absentia?is an E3 ubiquitin ligase in the ubiquitin-26S proteasome degradation pathway.In plants,monologues of SINA play a key role in development and stress response.In Arabidopsis thaliana,SINATs?SINA of Arabidopsis thaliana?are regulated by light.The protein levels of SINATs decreased when plants were moved from light to dark.However,how light regulates SINATs remains unknown.In this study,we used biochemistry,cell biology,plant physiology,and genetics methods and studied how light regulates SINATs.SINATs are degraded by itself from light to dark.First,we constructed35S::SINAT2^C63S-FLAG and 35S::SINAT4^C67S-FLAG transgenic plants.When the plants were moved from light to dark,the protein levels of SINAT2^C63S63S and SINAT4^C67S67S did not change significantly compared with wild type.Second,we performed qRT-PCR assays and protein quantitative analyses in 35S::SINAT2^C63S-FLAG and 35S::SINAT2-FLAG lines.We found that SINAT2^C63S63S protein in 35S::SINAT2^C63S-FLAG lines was more stable than SINAT2 protein in 35S::SINAT2-FLAG lines.Third,we measured the half-life of SINAT2 protein in 35S::SINAT2-FLAG and 35S::SINAT2^C63S-FLAG lines.We observed that SINAT2 degraded faster in 35S::SINAT2-FLAG over-expression lines than SINAT2^C63S63S in 35S::SINAT2^C63S-FLAG lines.These results indicate that the stability of SINATs depends on its own E3 ubiquitin ligase activity.phyB is a key role in regulating SINATs protein stability.It's known that SINATs regulated by red light and blue light and the degradation of SINATs is independent of COP1.We detected whether SINATs interact with phyB and CRY1,and found that SINATs interacted with phyB and CRY1 in the cytoplasm.We constructed35S::SINAT2-FLAG/phyB-9 lines,and found that SINAT2 protein showed no observable changes from light to dark in 35S::SINAT2-FLAG/phyB-9.We also found SINAT2 protein in 35S::SINAT2-FLAG/phyB-9 line was more stable.These results suggested that phyB promotes SINATs degradation.In summary,we found that SINATs,the regulator of protein stability,can regulate its own protein stability by self-regulatory mechanism,and we found a new mechanism of26S-ubiquitin proteasome pathway participating in light signaling.The understanding of how SINATs response to light is not only beneficial to lay the theoretical foundations of photomorphogenesis in Arabidopsis thaliana,but also help us to understand how other plants response to light.