Acetylation And Mechanism Of Arabidopsis Blue Light Receptor CRY2

Blue light signals play a crucial role in regulating plant growth and development,and cryptochromes are one of the blue light receptors that mediate plant photoreactions.Previous studies have shown that protein-protein interactions of photoreceptors are the main mechanism for cryptochrome signal transduction.In recent years,researchers have identified a series of proteins that interact with CRY2,which have helped to further understand the pathway of blue light signal transduction.However,there has been little research on the characteristics of CRY2 itself,such as post-translational modifications.So far,only ubiquitination and phosphorylation have been identified,but there may be other important post-translational modification mechanisms for CRY2.The discovery and completion of the post-translational modification mechanisms of CRY2 would have significant implications for the entire field of light signal transduction research.In this study,we discovered a new and important posttranslational modification of CRY2-acetylation,which is regulated by blue light.This suggests that acetylation modification of CRY2 may be an important regulatory mechanism,which is also related to the plant’s response to blue light signals in the environment.Using Arabidopsis as the experimental material,we explored the specific mechanism of acetylation modification and regulation of the blue light receptor CRY2.This study provides important references for further understanding the plant blue light signal transduction research.The specific results of this study are as follows:1.By mass spectrometry,five acetylation sites of CRY2 protein were identified as K375,K156,K238,K268,and K422,and it was found that the acetylation of CRY2 is regulated by blue light.The protein HDA19,which interacts with CRY2,was also identified,but its interaction strength remained consistent in both darkness and blue light.A PCI-neo 6X flag HDA19 vector was successfully constructed and verified using HEK293 T cells,demonstrating the existence of a CRY2-HDA19 interaction that is independent of blue light.2.By western blot,the acetylation changes of Arabidopsis thaliana seedlings growing under different light conditions were identified.It was found that there was almost no acetylation under blue light and white light,the acetylation level was the highest under red light,and the acetylation was very weak in the dark.The acetylation of CRY2 was regulated by blue light in both HEK293 T cells and plants,and the acetylation level of CRY2 decreased gradually with the extension of blue light irradiation time.By analyzing the gray values of the protein bands under red and blue light,it is concluded that deacetylation of CRY2 is dependent on blue light.After treatment with TSA,a HDACs-specific deacetylase inhibitor,Arabidopsis thaliana seedlings were identified,and the acetylation level of CRY2 increased at TSA concentration of 10μM,indicating that the blue-dependent deacetylation of CRY2 is dependent on the deacetylases of the HDACs family.3.Five single point mutation vectors of CRY2 acetylation sites were successfully constructed and transferred into HEK293 T cells.Confocal microscopy and Image J software were used to observe and analyze the phase separation phenomenon.It was found that CRY2 with single point mutation of five acetylation sites had phase separation,and the number of phase separation was higher than that of the wild type,indicating that CRY2 with low acetylation,can promote its phase separation.CRY2 and HDA19 were transferred into HEK293 T cells together.Confocal microscopy showed phase separation of CRY2,but no phase separation of HDA19,and no cophase of CRY2.Inverted fluorescence microscopy was used to observe that CRY2-GFP expressing HEK293 T cells and CRY2-GFP protoplasts were inhibited by the addition of TSA at a concentration of 10μM,indicating that CRY2 phase separation could be inhibited by high acetylation.4.Western blot analysis showed that CRY2 could regulate the acetylation of CIB1 under blue light.Further using Argobest experiment,it was found that factors with high acetylation state could regulate the transcription of FT,so it was speculated that the expression of FT might depend on the high acetylation of CIB1.