Transcriptomic Analysis And Regulation Mechanism Of Anthocyanin Accumulation In Peels Of Ficus Carica

Fig(Ficus carica L.)belongs to Ficus of the Moraceae,and its fruit contains a special structure,syconium.The flowers are hidden inside the fruit,so it is named "fig".Fig fruit contains various nutrients that are beneficial to humans and it provides amounts of anthocyanins,flavonoids and other antioxidant substances.Fruit color is an important appearance quality to attract consumers.In Arabidopsis,apple and other vascular plants,anthocyanin biosynthesis pathway and transcriptional factors involved in regulating anthocyanin biosynthesis have been reported widely,but there are few reports in fig fruits.As far as we know,only FcANS1 was cloned with sequence information available.In this study,a red fig variety‘Bojihong'was used as the material,and the peels of the yellow stage and commercial maturity stages were selected for transcriptomic analysis to identify the key structural and regulatory genes of anthocyanin biosynthesis.It laid a foundation for us to clarify the biological mechanism of anthocyanin biosynthesis of fig.The main results are as follows:1.In this study,uncolored(yellow stage,the beginning of turning stage)and colored(red stage,the fruits just developed to the marketable mature stage)fig peels were used as materials for high-throughput RNA-sequencing and obtained a large number of sequences and annotation information.In total,we obtained 186,048 unigenes,with an average length of 1,498 bp,of which 138,194 were annotated in the Nr database.With foldchange ?2 and Padj<0.05 as the thresholds,6,224 differentially expressed genes(DEGs)were identified,of which 3,619 genes were up-regulated and 2,605 genes were down-regulated.The analysis of KEGG pathway showed that phenylpropanoid biosynthesis(ko00940),flavonoid biosynthesis(ko00941)and flavone and flavonol biosynthesis were enriched.At the same time,33 anthocyanin related structural genes and 85 transcription factors that may be involved in the regulation of anthocyanin biosynthesis were identified.The analysis of hormone signal transduction showed that the number of ethylene related genes was largest among the nine groups.In comparison of colored and uncolored stages,the expression levels of positive regulators in ethylene signal were mostly up-regulated,and negative regulators were downregulated.The results indicate that ethylene may play a positive role in the regulation of anthocyanin accumulation in fig.2.According to the sequence data of transcriptomic analysis on anthocyanin biosynthesis,FcCHSl,FcCHI1,FcDFRl and FcFLS1 were cloned from fig peels,and their full length open reading frames were 1170,774,1023 and 1092 bp,respectively.Multiple alignment showed that FcCHS1,FcCHI1,FcDFRl and FcFLSl all contained conservative active sites and substrate specific sites of their own family.The phylogenetic analysis showed that the proteins encoded by the four cloned genes showed close relationships with the homologous proteins in Morus notabilis,a member of Moraceae.FcCHS1,FcCHI1 and FcDFR1 all grouped in the same branch with proteins from the Rosaceae,while FcFLS1 was in a separate group,which was far from other plants.The expression levels of FcCHS1,FcCHI1 and FcDFR1 were consistent with anthocyanin contents in fig peels and their expression levels in fig peels were significantly higher than other tissues.While,the expression level of FcFLS1 decreased rapidly during anthocyanin accumulation in fig peels and it was highly expressed in mature leaf than other tissues.3.In DEGs,19 members of the MYB family were filtrated.Prediction of the conserved motif by SMART showed that seven of the 19 MYB members belonged to R2R3-MYB family.According to the phylogenetic tree which was constructed using R2R3-MYB proteins from Arabidopsis and fig,two of them were identified as regulator in anthocyanin accumulation of fig,which were named FcMYB21 and FcMYB123,respectively.Transformation of FcMYB21 or FcMYB123 could promote anthocyanin accumulation in both apple peels and calli,and increased the expression levels of anthocyanin structural genes.At the same time,the transcription levels of MdMYC2 and MdbHLH3 increased sharply in FcMYB21-and FcMYB123-overexpressed apple peels and calli in comparison with wild type,respectively.In contrast,the expression levels of MdMYB9 and MdMYBll decreased in FcMYB21-and FcMYB123-overexpressed apple peels and calli.These results indicate that the effects of FcMYB21 and FcMYB123 on anthocyanin synthesis may be independent of endogenous MdMYB9 and MdMYB11,but related to bHLH partners.4.Transcriptomic analysis showed that a MADS-box gene may participate in anthocyanin accumulation of fig.It was named FcMADS9 and the amino acid encoded by this gene belongs to MIKCC family and SEP subfamily,containing MADS-box,I-domain,k-domain and C-domain.Overexpression of FcMADS9 promoted the anthocyanin accumulation in apple peels and calli,and increased the expression levels of anthocyanin related structural genes,such as MdDFR,MdANS and MdUFGT.Exogenous 150 mg·L-1 ethephon was significantly promoted the pigmentation of fig.Besides,the expression of FcMADS9 in ethephon-treated fruits was 8.24 times higher than that of control,which implied that FcMADS9 could be induced by ethephon.When FcMADS9 was overexpressed in apple calli,ethephon treatment increased the anthocyanin contents 2 times compared with the untreated calli.The anthocyanin contents of FcMADS9-overexpressed apple calli treated with 1-MCP was 44.22%lower than that of untreated ones.However,the anthocyanin contents of FcMADS9-overexpressed apple calli with 1-MCP treatment were 50.37%higher than that of empty vector untreated with 1-MCP.The results indicate that FcMADS9-promoted anthocyanin accumulation does not totally depend on ethylene.Those findings provide new insight in exploring the mechanism of anthocyanin biosynthesis in Ficus carica peels,and lay a foundation for further understanding of its molecular mechanism.