Main Metabolites And Transcriptional Regulation Of Carotenoids In 'Hongrou Huyou' And Its Donor Plants

'Hongrou Huyou'(Citrus unshiu+C.changshan-huyou)is a periclinal chimera,which was found at a tree with poncirus trifoliate as the stock,satsuma mandarin(C.unshiu)as the inter stock,then top-grafted 'Changshan Huyou'(C.changshan-huyou).Layer 1 cells of 'Hongrou Huyou' were derived from 'Owari' satsuma mandarin,Layer 2&Layer 3 cells were derived from 'Changshan Huyou'.In order to investigate the metabolites accumulation patterns of 'Hongrou Huyou' in different tissues,we measured the primary metabolites,volatiles and carotenoids in 'Hongrou Huyou' and its donors;transcriptome sequencing was performed on 3 cultivars' fruits at three different development stages(140d-200d after full bloom),and 9 key enzyme genes of carotenoid biosynthesis pathway were screened and analyzed;Th key genes in the sequencing results were verified by qRT-PCR,and the correlation between 'Hongrou Huyou' and its donors in carotenoid gene expressions and the content changes of 3 related development stages were analysed.The main research results are as follows:1.The metabolites in different parts of the fiuits of 'Hongrou Huyou' showed certain parental dominance.The contents of metabolites in the peels were very close to that of the L2&L3 donor'Changshan Huyou',while the metabolites in the juice sacs were closer to the L1 donor‘Owari' satsuma mandarin.For example,in peels,4-aminobutyric acid,shikimic acid,palmitic acid and 2,4-di-tert-butylphenol were only existed in 'Hongrou Huyou' and donor 'Changshan Huyou';in the juice sacs,4-aminobutyric acid,palmitic acid,allose and a-carotene were only found in the 'Hongrou Huyou'and donor 'Owari' satsuma mandarin.However,for the chimera 'Hongrou Huyou' itself,some new metabolic traits have also been observed.The concentrations of 13 metabolites(carbamic acid,fucose,etc.)and 9 metabolites(xylose,mannose,etc.)in 'Hongrou Huyou' were significantly higher or lower than that of the two donor plants;in particular,the content of Germacrene D in the juice sac of the'Hongrou Huyou' was more than 6-17 times that of the two donors;while oxalic acid and rhamnose were only disappeared in the jucie sac of 'Hongrou Huyou';interestingly,a new metabolite a-ylangene only appeared in the jucie sac of 'Hongrou Huyou',which was undetectable in two donors' fruits.2.9 key enzyme genes screened from the transcriptome sequencing showed different expression quantities among 'Hongrou Huyou' and 2 donors.Firstly,in peels,FC(Fold Change)values of PSY,PDS,ZDS,CRTISO and LCYE during 140-200d between 3 cultivars were<1,so there was no obvious difference in expression;during 140d-200d,the expression levels of LCYB1,LCYB2 and ZEP in the peels of 'Hongrou Huyou' and 'Changshan Huyou' were much closer,but the expression levels of these 3 genes in the peel of 'Owari' were decreased.HYB was the gene with the greatest expression difference in the peels from 140-170d.Secondly,in the juice sacs,PSY,PDS,CRTISO,LCYE and ZEP genes showed no obvious difference in 3 cultivars during 140-200d;ZDS(140d,200d),LCYB1(170d,200d)and LCYB2(140-200d)in 'Hongrou Huyou' expressed the closer quantities to 'Owari',and all of them were up-regulated compared with 'Changshan Huyou'.HYB was still the gene with the greatest difference,and its expression level in 'Hongrou Huyou' and donor 'Owari' were significantly increased in 140d and 170d,compared with that in‘Changshan Huyou'.3.Verify 9 genes screened from transcriptome results,and in combination with the changes of carotenoid content during 140-200d.The results indicated that the expression of genes and carotenoids contents in the peel of 'Hongrou Huyou' during 140-200d were much closer to 'Changshan Huyou'.and lower than that of donor 'Owari';but in juice sacs,were much closer to 'Owari'.and generally up-regulated compared with that in peels.Consistent with the sequencing results.HYB was indeed the most differentially expressed gene among the cultivars.In peels,comparing with the other cultivars.HYB were significantly up-regulated in 'Owari' in 170-200d;in juice sacs,'Hongrou Huyou' and'Owari' shared the closer HYB expression quantities,which were significantly higher than 'Changshan Huyou' during 170-200d.In addition.LCYB2 expressed the highest level in 'Owari' during 170-200d.but in juice sacs,it showed an higher expression level in 'Hongrou Huyou' than its donors during 140-200d.Correlation analysis suggested that the carotenoids content changes and the gene expressions of 'Hongrou Huyou' in peels were significantly correlated with 'Changshan Huyou'(L2&L3 donor),while in juice scs.was strongly correlated with 'Owari'(L1 donor).The changes of carotenoid content and the expression of enzyme genes in the fruit of 'Hongrou Huyou' have parental dominance:co-expression and co-regulation effect of heterologous donors are the reasons for the genetic variation of chimera metabolites.