Spatio-temporal Distribution Of Volatiles And Identification Of Synthesis Related TPS Genes During Pomelo Fruit Ripening

As one of three basic species of citrus,pomelo(Citrus grandis)is of economic importance in citrus industry.Pomelo is native in China whose planting area and production yield are the highest in the world.Regional characteristic cultivars were developed due to different geographic conditions and long cultivation history,i.e.Yuhuan(C.grandis osbeck cv.Yuhuan)in Zhejiang province.Aroma related volatiles are an important factor determining fruit quality,essential oils from pomelo has been widely applicated in industry.Basically,there are four different parts of pomelo fruit,cosisted of flavedo,albedo,segment membrane and juice sacs.Spatial and temporal distribution of volatile compounds in during pomelo fruit ripening is still not clear.Genes associated to volatile formation has not been identified.In present study,Yuhuan(C.grandis osbeck cv.Yuhuan)pomelo fruits were used as material.The main results are as follows:(1)A total of 64 volatile compounds were identified throughout pomelo fruit ripening stages.Major volatile classes of pomelo fruit consisted of monoterpenes,oxygen monoterpenes,sesquiterpenes,oxygen sesquiterpenes,aldehydes,alcohols,ester and phenypropanoid.Compared to previous studies,15 volatiles were reported in pomelo fruit in the first time,including 1 monoterpene,6 oxygen monoterpenes,4 sesquiterpenes,2 aldehydes,2 alcohols.Venn diagram analysis showed that 26 volatiles could be detected in flavedo,albedo,segment membrane(SM)and juice sacs(JS).A total of 12(18.8%)volatiles were only detected in flavedo,including 6 oxygen monoterpenes,3 sesquiterpenes,3 aldehydes.(E)-Pinocarveol and Perilla alcohol were unique produced by albedo.Volatile(E)-2-Octenal was only detected in JS.In present study,no unique volatiles were detected in SM.Thirty-nine volatiles produced by SM could also detected in other three fruit parts.(2)Pomelo fruit flavedo produced the highest content of volatiles,where monoterpene was the most abundant volatile class and the highest content was detected for limonene.Among volatiles detected in pomelo fruit,51 chemcials had the highest content in the flavedo,7 in albedo,2 in SM,and 4 in JS,respectively.For ripe fruit at 200DAB,volatile content was approximately 80009.87 ?g/g FW in flavedo,being 1000-fold higher than that summary in albedo,SM and JS.Monoterpene accounted for 94.61%of total volatiles in flavedo,being approximately 75695.44 ?g/g FW.As the most abundant volatile compound,content of limonene was 45981.77 ?g/g FW,accounting for approximately 57.47%of total volatiles.Relative content of monoterpene was 77.12%,79.24%and 67.43%in albedo,SM and JS,respectively.Fruit tissues of flavedo,albedo,SM and JS could be separated in a PC A plot using volatiles as variables,where flavedo samples were mainly clustered on the positive PC1 axis.(3)Increased content of volatiles was observed for whole fruit during ripening,while different volatile profiles were detected for various fruit parts.As fruit weight increased from approximately 650 g(80DAB)to 1940 g(200DAB),total volatile content increased from 47990.79 ?g/g FW to 80093.61 ?g/g FW.Approximately 1.67-and 5.66-fold accumulation of volatiles during fruit ripening were observed for flavedo and albedo,respectively.The content of volatile compounds in fruit SM did not show significant difference between 200DAB and 80DAB.For fruit JS,the content of total volatile compounds at 200DAB was decreased by 91%in relative to that at 80DAB.For monoterpene in flavedo,content increased from 43237.25 ?g/g FW to 75695.44?g/g F W as pomelo fruit ripening.As the most abundant volatile,content of limonene in flavedo tissue at 80DAB and 200DAB were 25670.2 ?g/g FW and 45981.77 ?g/g FW,respectively.However,opposite trend was observed for limonene in JS,where content decreased from 15.00 ?g/g FW to 2.90 ?g/g FW as fruit ripened from 80DAB to 200DAB.Significant changes were also observed for ocimene and linalool during fruit ripening.For linalool,content decreased throughout fruit ripening process,decling from 701.75 ?g/g FW to 448.07 ?g/g FW.(4)Volatile biosynthesis related TPSs were identified based on correlation analysis and enzyme activity in vitro.Significant high transcript levels were observed for genes derived from MEP and MVA pathways,which are responsible for volatile terpenoid formation in pomelo fruit.Terpene synthase TPS is the last enzyme involved in terpene biosynthesis pathway.A total of 63 TPS genes were characterized based on pomelo genome sequence.Five TPS genes were screened as candidate genes associated with volatile formation in pomelo fruit based on correlation analysis between transcript levels and volatile contents.Finally,two TPS genes were cloned expressed in E.coli for recombinant enzyme activity analysis in vitro.Member belong to TPS-b subfamily Cg8g012040 catalyzed ocimeme formation,Cg2g040850 from TPS-g was associated with linalool synthesis.