| Protein ubiquitination can mediate the degradation of target proteins through the ubiquitin-proteasome pathway,which is the main mode of protein degradation in eukaryotic cells and also the main form of regulating the fate of proteins in cells.It is very important to understand the different changes of protein ubiquitination in plants to analyze the regulation and influence of protein ubiquitination on plant growth and development.The da3-1 Arabidopsis thaliana mutant is a kind of deubiquitinating enzyme mutant,which has the obvious characteristics of large and curly leaves,large pods and seeds.The mutant mac3 a mac3b is an E3 ubiquitin ligase mutant.Its plant morphological characteristics are opposite to that of the mutant da3-1,with smaller leaves and smaller pods and seeds.It is helpful to understand the difference of ubiquitination between the two mutants compared with wild type in order to understand the possible regulatory mechanism of ubiquitination on plant growth and development.In this study,quantitative proteomics method was used to analyze the ubiquitination of Arabidopsis thaliana da3-1 and mac3 a mac3b mutant compared with wild type.We used K-?-GG ubiquitin specific antibody to enrich the ubiquitin modified peptides and identified 2970 peptides of 1475 ubiquitin modified proteins by highresolution mass spectrometry.Among 1037 ubiquitination modified proteins,1752 peptides showed different ubiquitination levels compared with Arabidopsis wild type.The low level of ubiquitination in mutant da3-1 was more than that in mac3 a mac3b.These proteins are mainly involved in signal transduction,gene expression and transcription,protein synthesis,folding,degradation and modification,protein ubiquitination,stress and defense and metabolism.We also used TMT and highresolution mass spectrometry to analyze the protein abundance of leaves.It was found that there was no direct relationship between the protein abundance of ubiquitination differential protein and the ubiquitination level.Only 464 proteins ubiquitination levels and protein abundances have changed at the same time.The change pattern of these protein ubiquitination levels and abundances has preliminarily resolved the regulation of plant growth and development process or stress response process,mainly involving:(1)Ubiquitination plays an important role in the regulation of transcription factors and RNA polymerase II in DNA damage repair;(2)Ubiquitination modification participates in protein homeostasis through the regulation of protein synthesis,folding,transport and other related proteins;(3)The components in the ubiquitin proteasome system regulate their own activities through self ubiquitination modification,and then regulate the fate of some proteins;(4)In the mutant da3-1,calmodulin regulates the deubiquitinating enzyme UBP6 activity It can regulate the ubiquitination level of substrate proteins;(5)In the mutant da3-1,deubiquitinating enzyme UBP12,UBP14,OTU redundancy play a role in regulating the deubiquitinating enzyme level;(6)The regulation of COI1 E3 ligase on the ubiquitination modification of JAZ family proteins is conducive to weakening the inhibition of JA signal response gene expression and participating in the normal growth and development process of plants;(7)E3 ligase in the ubiquitin-proteasome system can regulate the activity and stability of key proteins in the ABA signaling pathway,though effectively controlling the expression of ABAresponsive genes,thereby regulating the ABA signaling pathway,and participating in plant seed germination.The above results provide important information for the analysis of the possible phenotypic regulation mechanism in the two mutants,and are of great significance for the further analysis of the growth and development regulation mechanism of the two mutants. |
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