| Chinese olive [Canarium album(Lour.)Raeusch.] is a famous and characteristic fruit tree in Fujian Province.Its fruit is rich in polyphenols,flavonoids and other physiologically active ingredients,which is a wellknown medicinal and edible fruit.Phenolic compounds are important indicators that affect the flavor quality and pharmacological activity of fruit,and they are also a hot spot of research.Chinese olive has rich varieties resources,and there are differences in the metabolism of phenolic among different varieties(lines).The shikimic acid pathway is the only way for phenolic metabolism playing an important role in the synthesis of phenolic substances.Therefore,the study of shikimic acid metabolism in fruits of different varieties(lines)can further reveal the mechanism of phenolic metabolism in Chinese olive fruit,which is of great significance to the study the flavor formation of fruit.In this study,the fruits of ‘Tanxiang’,‘Changying’ and ‘Lingfeng’ were used as test materials.The contents of total phenols,total flavonoids and lignin in mature fruits were measured and combined with the widely targeted metabolomics data of mature fruits,the results were analyzed.The contents of phenolic compounds,quinic acid and shikimic acid were determined in three varieties(lines)of Chinese olive fruit during growth and development.Shikimic acid metabolism related genes were screened from transcriptome data,and their complete ORF sequences were cloned and analyzed by bioinformatics.The subcellular localization of some genes was analyzed by transient expression system.The expression of shikimic acid metabolism related genes and the activity of key enzyme SDH were measured during fruit growth and development.The relationship between phenolic substances and genes related to shikimic acid metabolism was analyzed,and the key genes of shikimic acid metabolism were screened.Thereby,exploring the differences of shikimic acid metabolism in fruits of different varieties(lines)can provide a theoretical basis for the study of shikimic acid metabolism in Chinese olive fruits and the formation of differences in phenolic metabolism among different varieties(lines),which is of great significance to the study of Chinese olive fruit quality.The main findings are as follows:1.There were differences in the contents of total phenols,total flavonoids and lignin in the fruits of the three varieties(lines)at the mature stage.The contents of total phenols and total flavonoids in ‘Tanxiang’ were significantly higher than that of the other two varieties(lines).The contents of total phenols,total flavonoids and lignin in ‘Lingfeng’ were the lowest among the three varieties(lines).The lignin content of ‘Changying’ was very high,2.34 times that of ‘Tanxiang’ and 4.18 times that of ‘Lingfeng’.Metabolome data showed that flavonoid metabolites were the most important differential metabolites among the fruits of the three varieties(lines).The main types of flavonoids metabolites were quercetin derivatives,kaempferol derivatives and luteolin derivatives.The contents of most flavonoids metabolites of ‘Tanxiang’ were significantly higher than that of the other two varieties(lines).The content of phenolic substances,quinic acid and shikimic acid in the fruit during the growth and development of the three varieties(lines)were determined.The results show that gallic acid,ellagic acid,quercetin,kaempferol and luteolin in Chinese olive fruit mostly existed in the form of bound state.The changes of(iso)corilagin,bound gallic acid,bound quercetin,bound kaempferol and shikimic acid in fruits of different varieties(lines)showed the same trend.Among the three varieties(lines),the contents of(iso)corilagin,ellagic acid,bound quercetin,bound kaempferol and other phenolic substances in ‘Tanxiang’ were relatively high.The contents of ellagic acid,bound quercetin,bound kaempferol and other flavonoids of ‘Lingfeng’were relatively low.During fruit development,the content of quinic acid was relatively high,while the content of shikimic acid was relatively low.The difference of these two shikimic acid intermediate metabolites between varieties(lines)was relatively small.The significant differences in phenolic substances and lignin contents of the fruits of the three varieties(lines)were consistent with their flavor and texture performance,and they were excellent materials for studying the metabolism of shikimic acid in Chinese olive fruits.2.DAHPS-1,DAHPS-2,DAHPS-3,DHQS,DHD/SDH-1,DHD/SDH-2,DHD/SDH-3,DHD/SDH-4,DHD/SDH-5,CS,CM-1 and CM-2 were screened out based on transcriptome data,and the complete ORF sequences were cloned.The results of bioinformatics analysis and subcellular localization of DAHPSs and CMs indicated that the number of amino acid residues of these 12 genes ranged from 771 to 1608,and the isoelectric point ranged from 5.67 to 8.84.Except that DHD/SDH-1 was a hydrophobic protein,other proteins were hydrophilic proteins.DAHPS-1,DAHPS-2 and DAHPS-3 were located in chloroplasts.The amino acid sequence of DAHPS-1 was highly similar to AtDAHPS2 of Arabidopsis,which might be regulated by tyrosine and tryptophan.DAHPS-2 and DAHPS-3 were highly similar to VvDAHPS-1 and VvDAHPS-2 of grape,respectively.DAHPSs might play different physiological functions in shikimate metabolism.DHD/SDHs belonged to three different types.Among them,DHD/SDH-1 and DHD/SDH-5 belonged to QDH,and the amino acid sequence of DHD/SDH-5 contained a motif binding to NAD.DHD/SDH-2 and DHD/SDH-3 belonged to the SDH,which were involved in gallic acid synthesis.DHD/SDH-4 belonged to the “typical” DHD/SDH.Two distinct types of CM existed in Chinese olives.Among them,CM-1was located in chloroplasts,which contained sites of allosteric regulation.Its sequence was highly similar to AtCM1,AtCM3 of Arabidopsis and PhCM1 of petunia,which might be allosterically regulated by aromatic amino acids.CM-2 was located in the cytoplasm,and its sequence was highly similar to AtCM2 and PhCM2.CM-2 was involved in phenylalanine synthesis through the phenylpyruvate pathway in the cytoplasm.3.The expression of twelve related genes of shikimate metabolism and the activity of SDH enzymes during growth and development of fruit in the three varieties(lines)were determined.The results showed that DAHPS-1,DHQS,CS,CM-1 were all up-regulated with fruit ripening.DHD/SDH-5 and CM-2 were down-regulated with fruit ripening.The expression levels of DHD/SDH-2 and DHD/SDH-3 were only higher at 50 days after flowering.Other genes all showed fluctuating variation during fruit development,and they were less diverse among cultivars(lines).There were differences in the expression trend of DAHPSs and the expression level in different varieties(lines)during fruit development,which might be related to the regulation of carbon fluxes in different synthetic directions in the shikimic acid pathway.DAHPS-1 might be a key gene that affected the synthesis of phenolic substances in Chinese olive fruit There was little expression of DAHPS-1 in ‘Lingfeng’,and there were genetic differences between it and the other two varieties(lines).The expression level of DAHPS-1 in the DAHPSs of ‘Tanxiang’ was relatively high,which was a significant correlation with the content of hydrolyzed tannins.The expression levels of DAHPS-2 and DAHPS-3 were higher in the DAHPSs of ‘Changying’.The expression level of DAHPS-3 in the DAHPSs of ‘Lingfeng’ was relatively high,which was significantly negatively correlated with the content of flavonoids.And the differences in the expression levels of DAHPS-1 among the three varieties(lines)were consistent with their differences in the content of phenolic substances.There was a strong positive correlation between DHQS expression and shikimic acid content during fruit development,and there was(extremely)significant positive correlation between the expression of CS and DHQS.It showed that DHQS and CS jointly promote the biosynthesis of aromatic amino acids in shikimic acid pathway.There were differences in the expression during fruit development of DHD/SDH-1 and DHD/SDH-5,DHD/SDH-1 was highly expressed in DHD/SDHs,which might be the reason for the higher quinic acid content in the fruits.There was a negative correlation between the expression of DHD/SDH-5 and the content of quinic acid to a certain extent.There was a strong correlation between the expression of DHD/SDH-2,DHD/SDH-3 and the contents of products related to gallic acid metabolism.The expression level of DHD/SDH-2 was higher than DHD/SDH-3.The variation trend of DHD/SDH-4 expression during fruit development of different varieties(lines)were slightly different,and there was a certain degree of positive correlation between DHD/SDH-4 expression and SDH activity.The expression level of CM-2 was significantly higher than that of CM-1.CM-2 might play a major role in decomposition of chorismic acid.The expression levels of CM-1 and CM-2 in ‘Tanxiang’ and ‘Changying’ were relatively higher after 110 days after flowering.The expression of ‘Lingfeng’ CM-1 was relatively low during fruit development,and the expression of its CM-2 was low after 110 days after flowering.The differences in the expression of CMs in the fruits of the three varieties(lines)were consistent with the differences in the contents of flavonoids and lignin in their fruits.CMs might be the key genes affecting the metabolism of phenylalanine in Chinese olive fruits.SDH activity showed an upward trend during fruit development,and there was a significant positive correlation with the expression levels of DHQS,CS and CM-1,indicating that shikimic acid metabolism was continuously enhanced with olive fruit development. |
