Differential Proteomics Study Of Anthers Of K Cytoplasmic Male Sterile In Wheat

K cytoplasmic male sterile line(K-CMS)in wheat is a type of male sterile with Ae.Kotschyi cytoplasm.In this study,the male sterile line KJi5418 A and its isotype maintainer Line Ji5418 B were used as materials.During the early development of anthers,transmission electron microscopy was used to observe the development of anthers;isobaric tags for relative and absolute quantification(iTRAQ)were used to obtain differential protein of anther at uninucleate and binucleate stage from the sterile and its maintainerand bioinformatics analysis was performed on the differential proteins.1.The abnormal development of microspores and tapetums may affect the pollen fertility of the sterile line.Microscopic observation showed that in the late tetrads and early uninucleate,some cell nucleus was lost and the cytoplasm was autolyzed in the anthers of the male sterile line KJi5418A;most of the microspores were empty and deformation in the later uninucleate and binucleate stage.no accumulation of cytoplasm,organelles,plastids,etc.The observation of tapetum showed that during the period of tetrads,there was no significant difference between the tapetums of CMS lines and maintainer lines;in the uninucleate stage,the tapetums were degraded seriously;during the binucleate stage,the tapetums were completely degraded.The wheat K cytoplasmic male sterile line may have abortion in the late tetrad or early mononuclear stage and reach the peak in the uninucleate stage and the binucleate stage.Microspore distortion and abnormal degradation of the tapetum may lead to pollen abortion.2.Proteomic analysis showed that 523 and 420 differential expressed proteins were respectively obtained from the male sterile line and the maintainer line in the uninucleate and binucleate stages.Among the 523 differential proteins in the uninucleate stage(differential multiples>1.2,p<0.05),there were 315 up-regulated proteins,208 down-regulated proteins.In the binucleate stages,420 differential proteins(differential multiples>1.2,p<0.05),there were 211 up-regulated proteins and 209 down-regulated proteins.The number of differential proteins in the uninucleate stage was significantly greater than that in the binucleate stages,which may be related to the dramatic changes in the microspores and tapetum of the sterile lines during this period.3.The differential proteins were respectively classified into 22 and 23 GO types and 25 KOG types.GO analysis showed that the major biological processes involved in stimulatory responses,biological regulation,cell composition processes,metabolic processes,etc..Molecular functions involved in protein association,metal ion binding,catalytic reaction,etc..Cellular componentsincluded organelles,cytoplasmic matrix,cell nucleus and other parts.The functional classification of KOG indicates that differential proteins are mainly classified as Posttranslational modification,protein turnover,chaperones(O),RNA processing and modification(A),Carbohydrate transport and metabolism(G),Energy production and conversion(C)and so on.4.Metabolic pathway analysis revealed that 737 proteins were involved in 98 metabolic pathways,including phenylalanine metabolic pathways,endoplasmic reticulum-related metabolic pathways,and sucrose and starch metabolic pathways.Abnormal metabolism of phenylalanine in male sterile lines may be one of the causes of male infertility.In the male sterile line,the expression of phenylalanine dehydrogenase and 4-coumaryl-CoA ligase was up-regulated,and the expression of chalcone synthase was down-regulated.the expression of catalase and aspartate aminotransferase is closely related to the degradation of styrene.The anomaly of chalcone synthesis in the anther of K-Ji5418 A may affect the pollen fertility.5.The abnormality of endoplasmic reticulum-associated metabolic pathways in CMS male sterile lines may affect pollen fertility of male sterile lines.Endoplasmic reticulumassociated metabolic pathways involve the process of N-glycosylation of secreted proteins and ubiquitin proteasome-mediated protein degradation.In the process of N-glycosylation of the secreted protein in the sterile line,the expression of glucosidase,calreticulin,calmodulin and disulfide isomerase was significantly downregulated.the expression of the four proteasome regulatory and recognition subunits such as Rpn12 was upregulated,and the expression of four catalytic center subunits such as a1 was downregulated;ubiquitin,nucleic acid protein localization proteins and ubiquitin thioesterase were up-regulated in the CMS line.Abnormal endoplasmic reticulum-related metabolic pathways may produce abnormal metabolites,misfolded and transported proteins,and the accumulation of these abnormal substances may lead to male sterility.6.Glycometabolism may significantly affect wheat pollen fertility.The expression of sucrose and starch anabolism-related enzymes in CMS lines decreased significantly,including phosphoglucomutase,sucrose phosphate synthase,and starch synthase,etc.The expression of sucrose and starch catabolism enzymes was also significantly reduced,including furan Fructosidases(transferases)and amylases.Abnormal metabolism of sucrose and starch may lead to insufficient supply of anther development energy and affect pollen fertility.In summary,the wheat K-CMS showed anther abortive happen in the late tetrad or early mononuclear stage and peaked in the uninucleate stage and the binucleate stage.The pollen abortion of the MS line may be related to microspore distortion and tapetums degradation.Differential proteins are mainly involved in phenylalanine metabolism,endoplasmic reticulum-related metabolic pathways,and sugar metabolism processes.These metabolic processes may affect the pollen fertility of wheat K-CMS.