Phosphoproteomic Analysis Of Rice Developing Seed And Functional Analysis Of OsbZIP72

Protein phosphorylation is part of the most important forms of post-translational modification and plays an important role in the rice seed development.This study was mainly about quantifying the dynamic phosphorylation during the process of rice fertilization to seed filling.A comprehensive phosphoproteomic analysis was carried out based on unfertilized pistil(S0),fertilized seed after 3 days(S3),fertilized seed after 7 days(S7).The largest phosphoprotein data about rice seed development were produced in this study.A gene OsbZIP72,selected from the phosphoproteomic,phosphorylated status was validated by experiment and its signal transduction pathway was studied.The main results were shown as following:(1)A total of 3885,4313 and 4135 phosphopeptides and 1236,1511,1585 were identified covering 4120,4611 and 4135 phosphosites in S0,S3 and S7 samples respectively.We got 5412 unique phosphosites after combining all the phosphosites from the three stages and removing the redundant phosphosites for the Motif-X analysis and found ten overrepresented Motifs(sP,sS,Rxxs,Lxxxxs,sF,Rsx S,LxRxxs,sDxE,Tp,Pt).After the quantitative normalization of the phosphorylation intensity of three biological replicates,2487 proteins were found to be differentially phosphorylated(DP)with twofold change or more among the S0,S3 and S7(P<0.05),including 286,329 and 581 phosphoproteins that were specifically phosphorylated at S0,S3 and S7 respectively,and 341,109 and 287 that were specifically not phosphorylated at S0,S3 and S7 respectively.554 proteins showed a constitutive phosphorylation pattern among the three stages.These DP proteins covered 166 transcription factors,89 epigenetic controlling factors and 209 kinases.GO analysis shown DP proteins were preferentially cataloged into‘‘cellular process,’’ ‘‘metabolic process’ ’ and ‘‘response to stimulus.’’ Molecular function was mainly related to catalysis,binding,transcriptional regulation and other functions.Semi-quantified the protein abundance of six DP proteins which are master regulators of rice seed development at S0,S3 and S7 was carried out,the result show that the phosphorylation intensity of DP protein maybe was independent with protein abundance.(2)Funtional analysis of OsbZIP72,selected from the phosphoproteomics data in this study was carried out.OsbZIP72 was constitutive expressed in almost every rice tissue but most highly expressed in rice leaves.After treated with five plant hormones,relative expression analysis showed that OsbZIP72 expression could induce by ABA.Two phosphosites(ser71 and ser116)in Osb ZIP72,found in our phosphoproteomic data.were phosphorylated by SAPK10 confirmed by yeast hybrid,pull down and phostag assay.The EMSA assay showed that phosphorylated OsbZIP72 could directly bind to the promotor of OsAOC,which was a key enzyme of JA synthesis.Our result indicated that ABA maybe cross-talk with JA regulated the rice development.