| Prunus mume is a famous and traditional flower in China,with typical flower fragrant.Cultivars in P.mume contain abundant germplasm resources and is divided into eight cultivars groups.Floral scent is an important characteristics of P.mume.At present,benzyl acetate and eugenol dominate its floral scent in most of cultivars in ‘Yudie’ mei or apricot mei.However,the diversity of its floral scents remains to be elucidated.To fully understand the fragrance composition in P.mume and metabolism synthesis regulation mechanism,in this work,eight cultivars from four cultivars groups were selected for analyzing floral volatiles and endogenous extraction.Based on this,the aroma composition representative cultivars were chosen.Bioinformatics,plant physiology,biochemistry and molecular biology were used to illustrate the biosynthesis molecular mechanism of different floral compounds,from the transcription level and enzymes activity analysis as well as gene function analysis.The main results were as follows:1.There were obvious differences in the floral composition of different cultivars in P.mume.The exogenous and endogenous floral volatile compounds of eight cultivars were compared and analyzed.It was found that,in addition to benzyl acetate and eugenol,benzyl benzoate,cinnamyl acetate,cinnamyl alcohol and benzyl alcohol were also important floral volatile components in P.mume.Benzyl acetate and cinnamyl acetate were the main floral constituents of ‘Fenpi Gongfen’ and ‘Fenhong Zhusha’.Cinnamyl alcohol and benzyl alcohol were the main floral components of ‘Jiangsha Gongfen’.Besides benzyl acetate,benzyl benzoate also played an important role in the aroma of ‘Zaohua lv’e’,‘Fuban lv’e’ and ‘Subai Taige’.The content of eugenol in ‘Wuyuyu’ had no significant difference with other varieties,only a small amount of benzyl alcohol was detected and no benzyl acetate and cinnamyl acetate were detected in its floral scent.2.The catalytic ability of the enzymes in each step of the synthesis pathway might be the limiting factor for the differences in the synthesis of different cultivars in P.mume.Comparing the enzyme activities of six cultivars with typical fragrance,it was found that the catalytic capacity of benzyl alcohol and cinnamyl alcohol to benzyl acetate and cinnamyl acetate in ‘Jiangsha Gongfen’ and‘Wuyuyu’ was weak,limiting the two esters synthesis.Only ‘Zaohua lv’e’ had the ability to synthesize benzyl benzoate.The ability catalyzing the synthesis of benzaldehyde and cinnamaldehyde into benzyl alcohol and cinnamyl alcohol,respectively,in each variety was obviously different.3.The gene transcriptional level of the corresponding catalytic step in the biosynthesis might regulate the level of enzyme activity.Based on the transcriptome sequencing analysis of flowers different cultivars at the bloom stage,68 of the differentially expressed genes involved in benzenoids/phenylpropanoids pathway were selected.Furthermore,combining with the enzyme activity analysis,we screened three Pm CAD genes related to cinnamic alcohol synthesis,three Pm CFATs genes associated with benzyl acetate,and two Pm BAHDs genes correlation with cinnamyl acetate synthesis.4.Pm CAD1 acted on the synthesis of cinnamyl alcohol in P.mume,and Pm CFAT1 played in the synthesis of benzyl acetate and cinnamyl acetate.With ‘Fenhong Zhusha’ as the plant material,the volatiles at different flowering stages were measured.The release of cinnamyl alcohol and cinnamyl acetate showed dynamic changes at different flowering stages.The release of cinnamyl alcohol gradually increased from bud stage to wilting stage,and the cinnamyl acetate emission amounts showed a trend of increasing from bud stage to flowers starting to open and decreasing until wilting stage.After Pm CADs were further screened through gene expression and phylogenetic tree,Pm CAD1 and Pm CAD2 were cloned.Bioinformatics analysis showed that both sequences contained three conserved domains in the amino acid sequences of CAD.The relative expression analysis showed that the expression trend of Pm CAD1 was consistent with the release of cinnamyl alcohol.Subcellular localization analysis showed that Pm CAD1 was localized in the cytoplasm,while Pm CAD2 was localized in the cytoplasm and nucleus.Enzyme activity analysis in vitro indicated that Pm CAD1 was mainly responsible for catalyzing the synthesis of cinnamyl alcohol from cinnamaldehyde.Pm CFAT1 enabled to catalyze the synthesis of benzyl acetate and cinnamyl acetate from benzyl alcohol and cinnamyl alcohol,respectively.5.Transcription factors regulate the expression of Pm CAD1 to function the floral volatile synthesis.On the basis of transcriptome analysis,combined with the data of different tissues and different flowering stages of P.mume,the flower-special expression transcription factors were screened.With Pm CAD1 and Pm CFAT1 as target genes,the correlation analysis showed that 2 transcription factors(Pm NAC56 and Pm IAA26)that might positively regulate the expression of Pm CAD1;and Pmb ZIP53 might negatively regulate its expression.This study provides more references for the exploitation of related derivative products of mei’s aroma.At the same time,clarifying the floral volatile metabolism mechanism in mei,analyzing the reasons for the formation of different fragrance types of P.mume,and exploring the special floral scent metabolism pathway which might exist in P.mume,so as to provide a theoretical basis for breeding the new cultivars in P.mume even the whole Prunus. |
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