The Regulatory Role Of Light Quality On Polymethoxylated Flavones And Anthocyanins In Blood Orange Fruit

Citrus fruits are widely loved by consumers because of their rich nutrition,beautiful color and unique flavor.Flavonoids are one of the important functional components in citrus fruit.Among them,polymethoxylated flavones(PMFs)and anthocyanins are not only the health-promoting components of citrus fruits,but also the main pigments of blood orange varieties.Therefore,blood orange is a good material to study the synthesis mechanism of both PMFs and anthocyanins in citrus fruits.Light is one of the important environmental factors that affects fruit nutrition and color quality.In the present study,the composition and content of PMFs and anthocyanins in ‘Moro’ blood orange during fruit ripening under different light treatments were detected by Ultra Performance Liquid Chromatography(UPLC)and High Performance Liquid Chromatography(HPLC),respectively.Meanwhile,RNA-sequencing was conducted to the treated samples.The GO classification,KEGG enrichment and WGCNA analysis methods were employed to identify candidate genes related to light-induced PMFs and anthocyanins metabolism.Based on the correlation analysis between gene expression and the content of PMFs and anthocyanins,a co-expression network and a regulation model of PMFs and anthocyanin synthesis mechanism under light signal transduction in citrus fruits were preliminary proposed.The main results are as follows:1.During blood orange fruits ripeing,the contents of PMFs and anthocyanins were significantly changed under blue light,red light,white light,dark and CK treatments(P <0.05).The content of PMFs in the pulp gradually decreased,while anthocyanins accumulated significantly.The effect of blue light treatment was significantly higher than that of other light quality treatments at turning stage(DAB210)with the most abundant PMFs,and at the ripening stage(DAB273)with the most abundant anthocyanins(P < 0.05),of which the total content reached 15.95 ± 0.51 mg/kg FW and 942.13 ± 1.67 [(A530-0.25×A657)/kg] FW,respectively.2.Compared with CK group,the number of differentially expressed genes(DEGs)at ripening stage(DAB273)under blue light was the highest,with a total of 873 genes,the number of common DEGs was the highest between DAB210 and DAB273,with a total of 53 genes.Through GO and KEGG enrichment analysis,a total of 1178 DEGs between blue light and CK treatments were enriched into 48 GO classifications and 216 KEGG pathways.Among significant enrichment pathways,there are 6 PMFs and anthocyanins related pathways,including “Biosynthesis of secondary metabolites”,“Metabolic pathways”,“Phenylpropanoid biosynthesis”,“Flavonoid biosynthesis”,“Phenylalanine metabolism”and“Flavone and flavonol biosynthesis”,indicated blue light induced PMFs and anthocyanins related genes differentially expressed.3.Screening biosynthesis structural genes of PMFs and anthocyanins based on annotations,the expression of upstream gene CHS1 and branch genes OMT3 and OMT4 were significantly induced by blue light and positively correlated with the PMFs content(log2(Blue210 / CK210)≥1);the expression of upstream genes PAL1,C4 H,4CL-like,CHS and branch genes F3H-like,F3’5’H,DFR,ANS,3-UFGT were significantly induced by blue light and positively correlated with anthocyanins content(log2(Blue273 / CK273)≥1).4.Screening photoreceptors based on annotations,the expression of CRY3 was significantly positive correlated with the content of anthocyanins and up-regulated under blue light treatment(log2(Blue210 / CK210)=0.28,log2(Blue273 / CK273)=0.65),suggesting CRY3 may be involved in regulation of PMFs and anthocyanins under blue light condition in fruits.5.Weighted gene co-exoression network analysis(WGCNA)showed that there were 39 TF families related to PMFs content,including ERF,BHLH,NAC,BZIP and WRKY etc,of which 5 TFs were differentially expressed under blue light treatment(| log2(Blue210 /CK210)| ≥1),including ERF3,AP2,LBD1,WRKY7 and NAC90;49 TF families related to anthocyanin content,including MYB,MYB-related,NAC,ERF and BHLH,etc,of which21 TFs were differentially expressed under blue light condition(| log2(Blue273 / CK273)|≥1).Combined with sequence analysis result,9 TFs were further screened as candidate TFs,which were MYB114,MYB308,BHLH130,BHLH149,DREB2 a,NAC72,NAC2,DOF2 and TCP.These TFs may play crucial roles in regulation of PMFs and anthocyanins under blue light treatment during fruit ripening.6.The expression of 16 candidate genes were verified by qRT-PCR,and expression trends of all tested genes in qRT-PCR and RNA-seq were consistent.The Pearson correlation coefficient between these expression data was 0.91,indicated the transcriptome sequencing data were reliable.Based on the expression pattern of candidate genes,a blue light regulation model of PMFs and anthocyanins during fruits ripening were preliminary poroposed.