Research Of TPS And CCD Function Analysis And Their Influence On Petal Color And Scent In Osmanthus Fragrans Lour.

Osmanthus fragrans Lour.,belonging to the genera Osmanthus of family Oleaceae,is one of the top ten traditional famous flowers in China.It is widely used both in ancient and modern gardens as landscape plant.Because of its unique aroma,it is also one of the important natural health plant used for plants aromatherapy and perfume and food process industries.According to the flower different colors and flowering seasons,O.fragrans cultivars are divided into four groups,Albus Group,Luteus Group,Aurantiacus Group and Asiaticus Group.Generally,it is considered that the flower color from Aurantiacus Group is gorgeous,but the floral scent is weaker than that from Albus Group and Luteus Group.Moreover,there are some differences in floral volatile profiles of the different cultivars that are belonged to the same group.Although the major constituents for floral scent and color in O.fragrans flowers have been analyzed in several studies,but the molecular mechanism on the formation of main compounds has not been further reported.Terpenoid is not only the dominant components and important aroma-active compounds for floral scent,but also an important part contributed to floral color in O.fragrans.So far,the research on the metabolism mechanism of their biosynthesis is very few.To study the molecular mechanism for floral scent and color formation in O.fragrans flowers,we isolated and cloned the genes related to the biosynthesis of floral scent and color,analyzed the transcript levels of gene involving in their metabolism pathway,and found the key genes controlling floral scent and color formation.Our study will provide a molecular basis for regulating floral scent and color,and molecular breeding of O.fragrans.The main findings were as follows:1.cDNA-AFLP analysis for different petals and young leaves in 'Liuye Jingui'According to cDNA-AFLP,we analyzed the differential expression profiles in different petals and young leaves in 'Liuye Jingui' and got 283 different differentially expressed gene fragments.Of which,150 gene fragmens had corresponding functional annotations that was divided into 10 categories by the biological processes,including metabolism,development,aging,resistance,transcriptional regulation and so on.The three kinds of biological process such as secondary metabolism,transcription regulation and floral development is likely to be associated with scent and color formation in O.fragrans.2.Comparision of floral scent emission and accumulation in 'Gecheng Dangui'and 'Liuye Jingui'We compared the floral volatiles profiles of 'Gecheng Dangui' and 'Liuye Jingui',and analyzed the emission and accumulation patterns of the major monoterpenes.The results showed that 'Gecheng Dangui' and 'Liuye Jingui' had distinct floral volatiles profiles.The flowers of 'Gecheng Dangui' emitted a large amount of monoterpenes and little carotenoids derived aroma volatiles;while the vast amount of carotenoids derived aroma volatiles and a small portion of monoterpenes were released from flowers of 'Liuye Jingui'.Furthermore,the emission and accumulation of monoterpenes were under the control of floral development and circadian rhythm,and the glycosylation of monoterpenes had an important role in regulating the floral volatiles release during these processes.3.Transcript levels of genes involving in the monoterpene biosynthesis.Comparing the gene expression level in 'Gecheng Dangui' and 'Liuye Jingui' at different flowering stages,the results showed that the expression of genes in the MEP pathway showed small differences between two cultivars or incongruous with monoterpenes production.However,the gene expression of terpene synthase(TPS)genes,especially OfTPSl and OfTPS3,which is the final enzyme converting the precursor GPP to kinds of monoterpenes,showed more than 100-fold higher in'Gecheng' compared to 'Liuye' throughout flowering stages and was consistent with the monoterpene production.4.Cloning and function analysis for terpene synthase(TPS)genesThe full lengths of TPS genes were cloned from O.fragrans flowers by the RACE-PCR method.Total four TPS genes with the complete open reading frames had been got and their encoded proteins had highly conservative DDxxD motif and(N,D)D(L,I,V)x(S,T)xxxE motif.In the four TPS proteins,only OfTPS4 had RR(x)8W motif in the N end.Phylogenetic analysis showed that OfTPS1 and OfTPS2 belonged to the TPS-g subfamily,while OfTPS3 and OfTPS4 clustered into the TPS-b subfamily.The results for gene function analysis showed that OfTPS1 and OfTPS2 were linalool synthase,while OfTPS3 and OfTPS4 were(E)-?-ocimene synthase and a-farnesene synthase,respectively.5.Transcript levels of genes involving in the carotenoid matobalism.Comparing the gene expression level in 'Gecheng Dangui' and 'Liuye Jingui' at tight bud stage and full flowering stage,the results showed that,in carotenoids synthetic metabolism,only PDS1?CRTISO?LCYB1 showed higher expression at full flowering stage than tight bud stage,meanwhile their expressions were higher in'Gecheng Dangui' accumulating more carotenoids in flowers.In carotenoid transformation metabolism,CHYE gene in 'Liuye Jingui' showed higher expression at tight bud stage compared to at fill flowering stage,but there was no obvious difference in its expression between two stages in 'Gecheng Dangui'.ZEP gene showed higher expression at full flower stage compared to at tight bud stage,meanwhile its expression were higher in 'Gecheng Dangui'.The expression of VDE was just opposite with ZEP,showed higher expression at tight bud stage in 'Liuye Jingui'.In carotenoid cleavage metabolism,the expression of NCED1 and CCD4a is higher at full flowering stage than tight bud stage,showed higher expression in 'Liuye Jingui' at the same stage.There were differences in NCED2 expression between'Gecheng Dangui' and 'Liuye Jingui' with different change trends,but its trends in two cultivars were different.There was no obvious difference in CCD1 and CCD4 expression between two cultivars.6.Cloning and function analysis for carotenoid cleavage dioxygenase(CCD)genesTotal three CCD genes with the complete open reading frames had been got by RACE-PCR.In which,OfCCD4b was a new gene that had not been reported,OfCCDl and OfCCD4b were separately highly homology with the reported CCD1 and CCD4 in O.fragrans.The three OfCCD genes had different expression patterns in different tissues and cultivars.The expression of OfCCDl and OfCCD4a was highest in petals and closely related to carotenoid accumulation in different cultivars.OfCCD4b expression was highest in peduncles,receptacles and pistils,and its gene expression was not associated with the accumulation of carotenoid in flowers of different cultivars.Function analysis in vivo showed that OfCCD1 possessed the ability for degradation of lycopene into C8 5-hepten-2-one,6-methyl-and C13(E,E)-pseudoionone,?-carotene and zeaxanthin into P-ionone.OfCCD4a could not degrade these carotenoids produce aroma compounds,but expressing this gene in zeaxanthin-accumulated E.coli produced new carotene compositions.OfCCD4b expressing in carotenoid-accumulated E.coli could not produce new carotene or carotenoid derived arom compounds.Function analysis in vitro showed that OfCCD1 could degrade a-and ?-carotene into C13 ?-and ?-ionone,lutein into C13 dehydro-?-ionone,?-cryptoxanthin into four kinds of C13 products including dihydro-a-ionone,a-ionone,dihydro-?-ionone and ?-ionone.Unexpectedly,OfCCD1 incubating with zeaxanthin in vitro could not produce ?-ionone.There was no carotenoids derived products detected from OfCCD4a reacting with these carotenoids.