Study On The Mechanism Of Protein Expression And Phosphorylation Modification On Fertility Conversion Of YS-type Thermo-senesitive Male Sterile Lines In Wheat (Triticum Aestivum L.)

Utilization of heterosis is an important way to improve crop yield.However,the shortage of sterile genes and handy sterile lines limits the manufacturing scale and utility of hybrid wheat.The cultivation area of hybrid wheat is much less than 1% of the world’s complete wheat acreage.At present,improving wheat yield relies upon conventional breeding methods and the utilization of heterosis in wheat genetics;however,wheat hybrids has not yet achieved a major breakthrough.Cytoplasmic male sterility(CMS)has enabled the utilization of heterosis in wheat.Thus,applications of thermosensitive sterile genes and CMS in wheat improvement are presently being explored.To study the possible molecular mechanisms regulating protein activity during male sterility induction,we used proteomics and phosphorylated proteomics analysis to analyze the binuclear anthers of thermo-sensitive sterile line YS3038.Moreover,the abortion regulation network of YS3038-A was preliminarily evaluated by cytological physiological and biochemical methods.Through amino acid sequence alignment and molecular biology methods,candidate genes were identified,and their functions were verified.The main conclusions are as follows:The anther of YS3038-A was thinner without cracks,and its microspores were shrunk with aborted pollens.Y S3038-A anther walls and microspores initiated the programmed cell death(PCD)process.Additionally,YS3038-A anther had insufficient carbohydrate deposition,high free fatty acid content,high H2O2 and MDA accumulation,low POD and CAT production rate,and low total ATP content.The energy deficiency and abnormal ROS deposition anthers were important causes of sterility in YS3038-A.1.Specific peptides were 9072,of which 6959 were quantifiable,669 were significantly up-regulated,and 372 were significantly down-regulated.Differential abundance proteins(DAPs)were mainly localized in the chloroplast,nucleus,and cytoplasm.The gene ontology(GO)analysis showed that most down-regulated differentially expressed proteins were mainly enriched in sporan synthesis,pollen wall formation,and pollen development.However,the KEGG pathway was mainly enriched in oxidative phosphorylation,RNA splicing,and lipid metabolism.A total of 126 transcription factors were identified in proteomic data.Amino acid sequence alignment revealed that 28 differential proteins were homologous to known proteins related to male abortion in rice,and most of these were down-regulated.Abnormal energy metabolism is an important cause of abortion in YS3038-A.2.Phosphoproteomics was used to analyze the anthers of YS3038-A and YS3038-B.A total of 11074 peptides were identified by spectral analysis,of which the phosphorylated peptides were 9995.Moreover,5019 phosphoproteins were identified,of which 4169 were quantified,and 975 were found to be significantly up-regulated while 998 were down-regulated.The phosphorylation events of YS3038 anther mainly occurred on serine residues.Differential abundance phosphoproteins(DAPPs)were mainly localized in the nucleus,chloroplasts,and cytoplasm.Furthermore,GO analysis found that several down-regulated phosphoproteins were enriched in ‘microsporocyte differentiation,’ and these differentially expressed phosphorylation sites were significantly enriched in ‘RNA splicing.’ A total of 174 phosphoproteins from 33 transcription factor families were identified in the phosphoproteome data,among which the phosphorylated ones were 95 epigenetically related proteins,369 kinases,and 82 phosphatases.The kinase-substrate network regulation map of the YS3038 sterile line was also p redicted.3.Some homologs of rice proteins related to male sterility were found through amino acid sequence alignment in the identified DAPs and DAPPs.Ta PDCD5 was significantly up-regulated at both protein and transcription levels and was localized to the membrane and nucleus.YS3038 anther development is sensitive to Ta PDCD5 transcript abundance,which initiates male fertility in null wheat by affecting the normal PCD of tapetum and microspores.4.The wheat mitogen-activated protein kinase(MAPKs)gene family is implicated in TCMS fertility regulation.This study identified 46 MAPK gene family members,which were divided into four groups(A,B,C,and D)based on their evolutionary relationship and physicochemical properties.Each group of Ta MAPKs had diverse gene structures,but members of the same group exhibited conserved gene structures.The analysis of gene duplication events showed that fragment duplication contributed the most to the expansion of the wheat MAPK gene family.The expression of Ta MAPKs exhibits specific temporal and spatial characteristics in wheat.This study found that the expression of Ta MAPKs in wheat anthers was significantly induced under high and low-temperature treatments.The Ta MAPK011,which regulates male fertility in wheat by affecting the wheat anther primordium mitosis and differentiation of sporogenous cells.Ta MAPK011 was localized in the nucleus.5.The lipid metabolism,starch and sucrose metabolism,and Ox Phos metabolic pathways in the regulation network of abortion energy metabolism greatly affected the energy supply in wheat anthers of YS3038 wheat thermosensitive cytoplasmic male sterile line.The mitochondrial respiratory chain plays a central role in energy metabolism.Mild and continuous oxidative stress in a sterile environment increases respiratory electron transport,ROS production,and oxidative stress amplification,thus initiating PCD of YS3038-A anther leading to pollen abortion.