Studies On Aging-Related Proteins In Maize Embryo Based On ITRAQ Quantitative Proteomics Technique

Seed vigor was irreparably reduced or even ultimately lost during aging.Clarifying the mechanism of seed aging would lay a good foundation for genetic improvement and effective storage technology of seed.In this study,maize seeds（zhengdan 958）were selected as experimental material.Seeds were respectively treated by artificial aging treatment（temperature: 45℃,RH: 100%）for 1 to 5 days and natural aging treatment for 3 years.We forced on the changes of seed germination ability,the content of MDA and H₂O₂,O₂^·—production rate in seed embryo treated by different aging treatment.We also respectively detected the changes of proteins after artificial aging treatment for 3 days and natural aging treatment for 3 years by the iTRAQ quantitative proteomics technique.All differentially expressed proteins（DEPs）were analysised by bioinformatics and searched for the proteins related to seed aging.Quantitative real-time PCR was performed to analyze the expression patterns of aging-related genes in the different artificial and natural aging times.The results were as follows.Seed germination ability showed a decreasing trend after aging trentment.The contant of MDA in seed embryo increased all the time,which indicated that oxidative stress strengthen and membrane system damage increase during seed aging.The H₂O₂ content and O₂^·—production rate of embryo increased,peaked at the 3 days of artificial aging treatment,and then decreased.The result indicted ROS production and accumulation occur in the initial stage of seed aging.The iTRAQ identified 209 DEPs in maize embryo after artificial aging treatment and 319 DEPs after natural aging treatment.In these DEPs,there were 73 proteins were in common.DEPs cover a wide range of molecular functions,including energy,protein turnover,stress and defence,metabolism,signal transduction,membrane and transport,transcription and cell growth/division.According to KEGG enrichment analysis,we found that DEPs that participated in 138 KEGG pathways in which 14 pathways were significantly enriched after artificial aging treatment.Among these pathways,metabolic,biosynthesis of secondary metabolites,protein processing in endoplasmic reticulum and starch and sucrose metabolism were detected more proteins.DEGs participated in 140 KEGG pathways in which 21 pathways were significantly enriched after natural aging treatment.The pathways analysis show that DEGs are mainly clustered together on the pathways of metabolic,biosynthesis of secondary metabolites,pyruvate metabolism,arginine and proline metabolis and glycolysis/gluconeogenesis.The above results showed that aging trentment affected metabolic and physiological regulation,impaired cell function,and ultimately resulted in seed deterioration.There is a big difference of DEPs between artificial aging treatment and natural aging treatment.Our results suggest that decreased abundance of glyceraldehyde-3-phosphate,pyruvate kinase,dehydrogenase,fructokinase,isocitrate dehydrogenase and malate dehydrogenase,revealed that the glycolysis and tricarboxylic acid cycle pathway were disturbed by aging treatment,which might lead to impaired energy supply.Aging also broke down antioxidant proteins and defence related proteins,such as peroxiredoxin,glutathione transferase and antimicrobial peptide,reduced protection against aging.Proteins involved in protein turnover were downregulated,with regard to protein synthesis,modification and degradation,which might induce a loss of ability of seed protein synthesis and/or increase the accumulation of misfolded proteins.Furthermore,proteins involved in metabolites were altered in expression,including aspartate aminotransferase,glycine cleavage complex P-protein,methylthioribose kinase and farnesyl diphosphate synthase,strongly affected the synthesis of amino acids,nucleotides and secondary metabolites.We analyzed the gene expressions of aging related proteins by quantitative real-time PCR,the mRNA and proteins expression tendencies of 12 proteins,which accounted for 9 and 8 in artificial aging and natural aging treatment respectively,were in common.These proteins were relevant to energy,protein translation and reactive oxygen removal processes,might play a critical role in seed deterioration and vigor loss.Taken together,the inhibition of energy metabolism resulted in decreased energy supply in seed,which would affect seed radical cellular activities.The downregulation of ribosomal proteins and translation initiation factors may induce a loss of ability of seed protein synthesis.The metabolism of amino acids,carbohydrates and lipids were strongly affected by aging.The break-down of the antioxidant proteins and defence related proteins,which reduce the antioxidant capacity and might lead to disturbance of defence system against aging.These results made a result of the destruction of cell integrity and homeostasis,and ultimately lead to seed deterioration and vigor loss.