| Euonymus japonicus Thunb is an evergreen broad-leaved shrub in Euonymus family,which has strong adaptability,wide distribution and cultivation range,and is one of the important tree species for landscaping.E.japonicus can endure the low temperature of-28.5℃ in winter in temperate zone,but the molecular mechanism of its resistance to freezing stress in winter is not clear.In this study,the leaves of E.japonicus in autumn and winter were used as materials,and the relative physiological and biochemical indexes such as electrolyte leakage and antioxidant enzymes were measured,the comparative analysis of plastid proteome and metabolome provides important data for revealing the metabolic regulation strategy and related molecular regulation mechanism of E.japonicus under winter freeze stress.The results can be summarized as follows:The physiological indexes such as cell membrane damage,osmotic adjustment substance content,antioxidant enzyme activity and chlorophyll content of leaves in autumn and winter were measured.The results showed that the contents of relative electrolyte leakage and MDA in the leaves of E.japonicus in winter were significantly higher than those in autumn,which indicated that freezing stress caused damage to the cell membrane,and the contents of soluble sugar and proline were significantly higher than those in autumn,the results showed that osmotic adjustment substances participated in the process of frost stress tolerance of E.japonicus in winter,and the activities of SOD,POD,CAT and APX increased,which indicated that Ros scavenging system was involved in the process of frost stress tolerance of E.japonicus in winter.The decrease of chlorophyll content,net photosynthetic rate and stomatal conductance indicated that the leaves of E.japonicus could adapt to winter freezing stress by regulating photosynthesis.In order to reveal the regulation of gene expression mechanism of winter freeze stress on E.japonicus leaves,transcriptome sequencing technique was used to analyze the differential expression of genes between autumn and winter leaves.A database of 138731 unigene transcriptions of the spear of wintergreen has been established for the first time,with 87353 unigene transcriptions possessing functional annotations.Compared with autum,the expression of 21031 genes in winter leaves changed significantly,including 8358 up-regulated genes and 12673 down-regulated genes,differentially expressed genes are involved in signal transduction,transcriptional regulation,antioxidant activity and other processes.The enriched part include the apoplast of E.japonicus.KEGG pathway enrichment analysis shows that,the metabolic pathways of secondary metabolites are biosynthesis,transport and catabolism,membrane transport,protein folding,protein sorting and degradation,hormone signal transduction,carbohydrate metabolism,lipid metabolism and amino acid metabolism.KEGG data also showed that ABA signal transduction was involved in the response of E.japonicus to winter freezing stress.Some winter-overexpressed genes,such as Golsl,may play an important role in freezing tolerance of E.japonicus。The changes of proteins and metabolites in the apoplast may be involved in the winter freezing stress tolerance of E.japonicus.In order to establish a liquid extraction method for the apoplast of E.japonicus,the extraction method was optimized from the aspects of centrifugation speed and buffer type.The results show that the centrifugal force is 2500 g,and the buffer is ascorbic acid plus anhydrous calcium chloride aposome liquid extraction effect is better.The differences in the apoplast proteome of leaves of E.japonicus in autumn and winter were analyzed by label-free LC/MS.A total of 838 peptides were obtained and 194 proteins were identified.Compared with autumn,the abundance of 43 proteins in apoplasts changed significantly in winter,among which 26 differentially accumulated proteins were up-regulated and 17 were down-regulated under winter freezing stress.The differentially accumulated proteins were mainly related to cell wall modification,disease resistance,free radical scavenging and other functions.A large number of proteins accumulated in winter,including cysteine protease and aspartic protease,may be involved in the adaptation of E.japonicus to freezing stress.Metabolomic analysis based on liquid chromatography-mass spectrometry(LC-MS)technique,a total of 1542 metabolites were obtained in positive mode and 736 metabolites were obtained in anion mode in E.japonicus vulgaris leaves.The total number of significantly different metabolites was 721(cation mode),349(anion mode).The total number of metabolites significantly up-regulated in positive mode was 487,and the number of metabolites significantly down-regulated was 234;the total number of metabolites significantly up-regulated in anionic mode was 192,and the number of significantly down-regulated metabolites was 157.KEGG enrichment analysis showed that the significantly enriched metabolic pathways were flavonoid biosynthesis,glycerophospholipid metabolism,secondary metabolite biosynthesis,plant hormone signal transduction,monoterpene biosynthesis,anthocyanin biosynthesis,citric acid cycle,etc.The results of multi-group analysis show that E.japonicus has many ways to improve its adaptability to freezing stress in winter,the activation of flavonoid synthesis pathway and the accumulation of flavonoid in ectoplasm are very important to improve the frost resistance of E.japonicus.The physiological and biochemical indexes of E.japonicus under freezing stress in winter were determined,and the differentially expressed genes,proteins and metabolites were identified,which provided valuable clues for understanding the tolerance of E.japonicus to freezing stress. |
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