Analysis Of Fruit Aroma Profiles During Postharvest And Functional Identification Of Lipoxygenase Gene LOX2a Of Binzi Fruit

Volatile aroma components are important secondary metabolites essential to unique fruit flavor quality.Identification and selection of major aroma compounds and genes related aroma play important roles in analyzing the formation mechanism of fruit aroma quality.As a traditional economic forest in China,Binzi has many varieties and has been widely planted in Beijing,Hebei,and Shanxi.One of the Binzi’s varieties,‘Xiangbinzi’ is known for its attractive and lasting aroma,favored by consumers.However,studies on its aroma components and the mechanism of aroma metabolism have not been reported.In this study,the high-fragrant ‘Xiangbinzi’ fruit and the low-fragrant ‘Hulabin’ fruit were used as experimental materials.The dynamic changes of postharvest fruit quality and aroma compounds were studied by transcriptomics,molecular biology,biochemistry,pharmacology,and other methods.The important genes related to aroma synthesis were explored,and further explore the important reasons for the formation of the ‘Xiangbinzi’ aroma.The main research results are as follows:1.Binzi is a typical respiratory climacteric fruit.Binzi fruit has the characteristics of postharvest ripening.During the postharvest period,the respiration rate and ethylene release increased,accompanied by changes in firmness,soluble solids content(SSC),soluble sugar,linoleic acid,linolenic acid,amino acids,and other quality characteristics.Linolenic acid,amino acids,and branched chain amino acids are important precursors for ester aroma synthesis.Linoleic acid and linolenic acid in ‘Xiangbinzi’ fruit showed a trend of increasing first and then decreasing in the postharvest stage,indicating that these two free fatty acids were involved in the synthesis of aroma substances in the post-ripening stage of Binzi.The content of branched-chain amino acids Val,Ile,and Leu in ‘Xiangbinzi’ fruit was higher than that in ‘Hulabin’,which was speculated to promote the synthesis of aroma substances in the ‘Xiangbinzi’ fruit.2.Binzi is an ester-flavor fruit.Esters,aldehydes,alcohols,ketones,and other volatile aroma substances were detected in ‘Xiangbinzi’ and ‘Hulabin’ fruits,and the esters were the main aroma compounds.The content of volatile aroma components in the ‘Xiangbinzi’ fruit was significantly higher than that in the ‘Hulabin’.Therefore,it was speculated that the reason why ‘Xiangbinzi’ fruit was more fragrant than ‘Hulabin’was the higher content of aroma components in ‘Xiangbinzi’ fruit.Furthermore,the main aroma compounds in ‘Xiangbinzi’ fruit were ethyl 2-methylbutyrate,hexyl 2-methylbutyrate,ethyl butyrate,(E)2-hexenal and 1-hexanol,whose odor activity values(OVAs)were greater than 1.3.Based on transcriptome sequencing,RT-q PCR results,and correlation analysis,LOX2 a,LOX5a,ADH1,and AAT1 may be involved in the metabolism of postharvest aroma of Binzi fruit.A total of 24 samples of ‘Xiangbinzi’ and ‘Hulabin’ fruits(Day 0,4,8,and 12 after harvest)were subjected to parametric transcriptome sequencing(Apple,Malus ′ domestica).A total of 160.83 G Clean Data was obtained.The number of differentially expressed genes in the four groups was 5,401,5,508,3,962,and 4,714,respectively.In this study,seven candidate genes LOX1 a,LOX2a,LOX5 a,LOX7a,ADH1,HPL1,and AAT1 were screened from the X4-vs-H4 group,and the candidate genes were further screened by RT-q PCR and correlation analysis.Based on the above results,LOX2 a,LOX5a,ADH1,and AAT1 may be related to the metabolism of postharvest aroma of ‘Xiangbinzi’ fruit.4.Overexpression of LOX2 a promotes the synthesis of volatile aroma compounds.Due to the significant correlation between LOX2 a gene and aroma components,the property and function of LOX2 a gene were further explored.The open reading frame of the LOX2 a gene was 2,721 bp,encoding 906 amino acids.And it contained a conserved Lipoxygenase domain and located in the cytoplasm.Meanwhile,phylogenetic analysis showed that ‘Xiangbinzi’ LOX2 a had the highest homology with apple LOX2 a.The expression level of LOX2 a was the highest in root and the lowest in seed.The expression level of LOX2 a was the highest in fruit at 30 days after flowering(DAF),then decreased and maintained at a certain level,and increased at maturity(150DAF).Furthermore,Overexpression of LOX2 a in ‘Xiangbinzi’ fruit can promote the synthesis of esters and alcohols,and increase the expression levels of LOX2 a,HPL1,ADH1,and AAT1.Overexpression of LOX2 a in tobacco callus increased the content of aldehydes and alcohols.The ‘Xiangbinzi’ LOX2 a promoter contained cis-acting elements responding to abscisic acid(ABA),methyl jasmonate(Me JA),gibberellin(GA),and salicylic acid(SA),and its expression was induced by these four hormones.The LOX2 a gene expression was also induced by ethylene(ETH).Proteins that may interact with LOX2 a were screened by protein interaction prediction website(STRING).Y2 H,LCI and GST Pull down assays demonstrated that LOX2 a interacts with transcription factor TIFY6b.