Analysis Of Anther Proteomics And Ubiquitinome And Functional Analysis Of E3 Ligase TaHUB1/2 Genes In P-type Cytoplasmic Male Sterility In Wheat

The utilization of wheat heterosis plays pivotal roles in increasing yield and improving quality;although researchers have devoted themselves for many years,due to the lack of good male sterile lines,the commercial application of hybrid wheat still lags behind.Therefore,analyze the mechanism of wheat male sterility is important to provide theoretical and technical support for commercial application of hybrid wheat.Studies on this mechanism by combining proteomics and modification omics have not yet been reported.Our study analysed ms(P)-ys(defined as P)and maintainer line(Yanshi 9 hao;defined as Y)anthers morphology,proteomics,ubiquitinome,the correlation genes TaHUB1/2 were revealed,and the function was explored through molecular biological experiments.The results provided scientific basis and data support for further elucidating the sterility mechanism of wheat.The main research results are as follows:(1)Anthers of P were wizened,albescent and indehiscent at the mature pollen grains stage;I₂-KI staining indicated little accumulate of starch.Microspores observations of P showed that nucleus were diffuse at the binucleate stage(defined as stage 4),and almost never develop to the trinuclear stage.The results indicated that stage 4 of P was the key period for abortion.Tapetum appeared vacuolization in the tetrad stage,the early uninucleate stage(defined as stage 2)degradation was early and fast,and degradation was complete in the stage 4.During the tapetum degradation process,abnormal Ubisch bodies were produced,which blocked synthesis of pollen wall.TUNEL experiment again proved the tapetum of P degraded earlier.In summary,the early degradation of the tapetum lead to the lack of energy and material,which inhibits the development of microspores and the synthesis of pollen wall,and finally leads to abortion.So,early tapetum degradation is the key factor of abortion,and stage 2 to stage 4 is the key stage of abortion.(2)In the analysis of proteome,a total of 4,519 proteins were identified,of which2,576 had quantitative information.178 DEPs were identified in stage 2,of which 103 were up-regulated and 75 were down-regulated.717 DEPs were identified in stage 4,of which389 were up-regulated and 328 were down-regulated.Through subcellular localization,GO,KOG,KEGG and other analysis,found that“TCA cycle”,“oxidative phosphorylation”,“glycolysis”,“starch and sucrose metabolism”pathways were main enriched in stage 4,and tapetum related proteins were also down-regulated.These results were consistent with the conclusion that stage 4 was the key stage of abortion,indicated that the lack of energy supply is closely related to anther abortion.(3)q RT-PCR detected the corresponding genes of 15 randomly selected DEPs,The expression patterns of 9 genes on the two materials of P and Y were completely consistent with the gene expression level,and the rest were different.which well verified the accuracy and reliability of protein data.At the same time,indicated the expression pattern of gene transcription level is not strictly consistent with the actual protein level.(4)In the analysis of ubiquitinome,a total of 6015 Kub sites were identified,which distributed in 2495 proteins.Among them,4728 Kub sites were quantified in 2056 proteins.13 conserved motifs were identified,seven of them were also reported in other species.The overlapping motifs indicated that ubiquitination was a highly conserved PTM among different species.(5)527 Kub-DMSs were identified on 387 proteins in stage 2,of which 102 were up-regulated and 425 down-regulated.700 Kub-DMSs were identified on 507 proteins in stage 4,of which 484 were up-regulated and 216 down-regulated.Through subcellular localization,GO,KOG,KEGG,PPI and other analysis,we found that the key pathways(“autophagy”,“pyruvate metabolism”,“oxidative phosphorylation”and“starch and sucrose metabolism”)associated with abortion in stage 4,and large numbers of Kub sites were observed on metabolism-related enzymes.We also observed that Kub-DMSs involves some transcription factors and some RNA binding proteins which play regulatory role in transcription process.Therefore,we believe that ubiquitination plays an important role in regulating the activity of transcription-related proteins during the development of anthers.(6)Ubiquitination has an important effect on the fate of histones,plays an important role in stabilizing DNA and ultimately affects plant fertility.Two comparison groups have15 histone proteins exists significant differences;Combined analysis of anther proteins and their ubiquitination data indicated that there was no clear correlation between protein abundance and ubiquitination levels.(8)The CDS length of TaHUB1/2 genes were 2613bp and 2535bp,respectively.TaHUB1/2 genes were homologue of rice Os HUB1/2 and Arabidopsis thaliana HUB1/2genes,with typical RING zinc finger domain,belong to C3HC4 type E3 ubiquitin ligases.Both localize in the nucleus and can form homodimers and heterodimers in both yeast and tobacco.The expression levels of male sterile lines were significantly lower than that of maintainer lines in stage2 and stage4.When TaHUB1/2 genes were transferred into wild-type Arabidopsis thaliana,the transgenic plants were characterized by promoting root development and inhibiting lateral root development,increasing the number of rosette leaves and earlier flowering time,short fruit pods,and some of them were empty pods without fruiting.Those results indicated that the overexpression of TaHUB1/2 genes in Arabidopsis thaliana affects its growth and development,and severe partial abortion may occur.