Differential color development and response to light deprivation of fig (Ficus carica L.) syconia peel and female flower tissues: transcriptome elucidation

Abstract

Background: Color directly affects fruit quality and consumer preference. In fig syconia, the female flower tissue is contained in a receptacle. Anthocyanin pigmentation of this tissue and the peel differs temporally and spatially. A transcriptome study was carried out to elucidate key genes and transcription factors regulating differences in fig coloring.

Results: Anthocyanins in the female flower tissue were identified mainly as pelargonidin-3-glucoside and cyanidin-3-rutinoside; in the peel, the major anthocyanins were cyanidin 3-O-glucoside and cyanidin-3-rutinoside. Anthocyanin content was significantly higher in the female flower tissue vs. peel before fig ripening, whereas at ripening, the anthocyanin content in the peel was 5.39 times higher than that in the female flower tissue. Light-deprivation treatment strongly inhibited peel, but not female flower tissue, anthocyanin pigmentation. RNA-Seq revealed 522 differentially expressed genes (recruited with criteria log₂ ≥ 2 and P < 0.05) at fig ripening, with 50 upregulated and 472 downregulated genes in the female flower tissue. Light deprivation upregulated 1180 and downregulated 856 genes in the peel, and upregulated 909 and downregulated 817 genes in the female flower tissue. KEGG enrichment revealed significantly changed expression in the phenylpropanoid-biosynthesis and flavonoid-biosynthesis pathways in the peel, but not in the female flower tissue, with significant repression of FcCHS, FcCHI, FcF3H, FcF3'H, FcDFR and FcUFGT transcripts. Light deprivation led to differential expression of 71 and 80 transcription factor genes in the peel and female flower tissue, respectively. Yeast one-hybrid screen revealed that FcHY5 and FcMYB114 bind the promoter regions of FcCHS and FcDFR, respectively in the flavonoid-biosynthesis pathway.

Conclusions: Phenylpropanoid- and flavonoid-biosynthesis pathways were differentially expressed spatially and temporally in the peel and female flower tissue of fig syconia; pathway expression in the peel was strongly regulated by light signal. Differentially expressed transcription factors were recruited as candidates to screen important expression regulators in the light-dependent and light-independent anthocyanin-synthesis pathway. Our study lays the groundwork for further elucidation of crucial players in fig pigmentation.

Keywords: Anthocyanin biosynthesis; Differentially expressed gene; Female flower tissue; Fig (Ficus carica L,); Fruit peel; Light deprivation; RNA-Seq; Transcription factor.

Fig. 1

The phenotype of fig (Ficus carica L.) cv. Zibao at young and mature phases. a Developmental stages T1, T2, T3, T4 and light-deprivation treatment. b Fig fruit quality. c Anthocyanin content in peel and female flower tissue

Fig. 2

Anthocyanins in fig peel and female flower tissue. a Anthocyanins in the peel at stage T4. b Anthocyanins in the peel after light-deprivation treatment. c Anthocyanins in the female flower tissue at stage T4. d Anthocyanins in the female flower tissue after light-deprivation treatment

Fig. 3

Differentially expressed genes (DEGs) between fig peel and female flower tissue and induced by light deprivation. a DEGs of the three comparison groups. DEGs were recruited by P < 0.05 and |log₂FC| ≥ 1. b Venn diagram showing shared and unique DEGs in the peel and female flower tissue after light-deprivation treatment. c Category distribution of shared DEGs in peel and female flower tissue after light-deprivation treatment. T4-P, peel at stage T4; T4-F, female flower tissue at stage T4; LD-P, stage 4 peel following light-deprivation treatment; LD-F, stage 4 female flower tissue following light-deprivation treatment

Fig. 4

Differentially expressed structural genes (DEGs) of anthocyanin-biosynthesis pathway induced by light deprivation. a DEGs were recruited by |log₂FC| ≥ 2 or FRKM ≥50. Blue and red boxes indicate downregulated and upregulated transcripts, respectively. Expression patterns are indicated at the side of each gene. CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3’H: flavanoid 3′-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanidin synthase; UFGT: UDP glucose-flavonoid 3-O-glcosyl-transferase. b Phylogenetic clustering with corresponding genes of other fruit species. T4-P, peel at stage T4; T4-F, female flower tissue at stage T4; LD-P, stage 4 peel following light-deprivation treatment; LD-F, stage 4 female flower tissue following light-deprivation treatment

Fig. 5

Light deprivation induces differential expression of MYB and HY5 genes in fig peel. a Potentially important MYBs recruited by |log₂FC| ≥ 1 and FRKM ≥20 in at least one sample. b Phylogenetic clustering of the recruited MYBs with anthocyanin biosynthesis-related MYBs from other plants. c R2R3 motif sequence alignment. d Expression, domain alignment and phylogenetic clustering of the fig HY5 gene

Fig. 6

qRT-PCR validation. Nineteen unigenes from the flavonoid-biosynthesis pathway were selected to validate the RNA-Seq results. Relative expression of the genes in the peel and female flower tissue in T1, T2, T3, T4 and light-deprived samples is shown. Three biological replicates were used

Fig. 7

Fig CHS and DFR gene promoter clone, sequence analysis and transcription factor binding validation. a FcCHS and FcDFR gene promoter clone. b Nucleotide sequence of the 1791-bp upstream region of FcCHS. Functional elements and all putative cis-elements are underlined. c Nucleotide sequence of the 1122-bp upstream region of FcDFR. Functional elements and all putative cis-elements are underlined. d Yeast one-hybrid test using FcCHS promoter as bait and FcHY5 as prey. e Yeast one-hybrid test using FcDFR promoter as bait and FcMYB114 as prey. Representative growth status of yeast cells is shown on SD/−leucine agar media with or without AbA from triplicate independent trials. Numbers at the top of each photograph indicate relative densities of the cells