Genome-Wide Analysis of Anthocyanin Biosynthesis Regulatory WD40 Gene FcTTG1 and Related Family in Ficus carica L

Abstract

WD40 proteins serve as crucial regulators in a broad spectrum of plant developmental and physiological processes, including anthocyanin biosynthesis. However, in fig (Ficus carica L.), neither the WD40 family nor any member involved in anthocyanin biosynthesis has been elucidated. In the present study, 204 WD40 genes were identified from the fig genome and phylogenetically classified into 5 clusters and 12 subfamilies. Bioinformatics analysis prediction localized 109, 69, and 26 FcWD40 proteins to the cytoplasm, nucleus and other cellular compartments, respectively. RNA-seq data mining revealed 127 FcWD40s expressed at FPKM > 10 in fig fruit. Most of these genes demonstrated higher expression in the early stages of fruit development. FcWD40-97 was recruited according to three criteria: high expression in fig fruit, predicted nuclear localization, and closest clustering with TTG1s identified in other plants. FcWD40-97, encoding 339 amino acids including 5 WD-repeat motifs, showed 88.01 and 87.94% amino acid sequence similarity to apple and peach TTG1, respectively. The gene is located on fig chromosome 4, and is composed of 1 intron and 2 exons. Promoter analysis revealed multiple light-responsive elements, one salicylic acid-responsive element, three methyl jasmonate-responsive elements, and one MYB-binding site involved in flavonoid biosynthesis gene regulation. FcWD40-97 was in the FPKM > 100 expression level group in fig fruit, and higher expression was consistently found in the peel compared to the flesh at the same development stages. Expression level did not change significantly under light deprivation, whereas in leaves and roots, its expression was relatively low. Transient expression verified FcWD40-97's localization to the nucleus. Yeast two-hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays revealed that FcWD40-97 interacts with FcMYB114, FcMYB123, and FcbHLH42 proteins in vitro and in vivo, showing that FcWD40-97 functions as a member of the MYB-bHLH-WD40 (MBW) complex in anthocyanin-biosynthesis regulation in fig. We therefore renamed FcWD40-97 as FcTTG1. Our results provide the first systematic analysis of the FcWD40 family and identification of FcTTG1 in fig pigmentation.

Keywords: MBW complex; TTG1; WD40; anthocyanin biosynthesis; expression profile; fig (Ficus carica L.).

FIGURE 1

Chromosomal locations, phylogenetic relationships, and collinearity analysis of the FcWD40 gene family. (A) Phylogenetic tree of 437 WD40 genes identified in Ficus carica and Arabidopsis thaliana genomes. Orange, red, blue, purple, and green highlighted areas show clusters I–V. Red squares represent fig WD40. The numbers on the branch points represent total number of WD40 gene; scale length represents genetic distance. (B) FcWD40 genes are marked on chromosomes; scale bar on the left indicates length of fig chromosome (Mb). (C) Collinearity relationship of WD40 genes among Ficus carica, Ficus hispida, and Ficus microcarpa. Identified collinear genes are linked by red lines.

FIGURE 2

Analysis of domain structure and promoter of FcWD40s. (A) Distribution of primary domains of 66 FcWD40 proteins with nuclear localization and genes expressed in fig fruit. (B) Introns number of 66 FcWD40s. (C) Promoter analysis of FcWD40s by Plant CARE, based on 2000-bp sequence upstream of the genes.

FIGURE 3

Expression profiles of FcWD40 genes in the flesh and peel tissue of developing and ripening fruit. (A) Photographs of fig flesh and peel color at different stages. (B) Members of FcWD40 in group S1 (FPKM ≥ 100). (C) Members of FcWD40 in group S2 (100 > FPKM ≥ 50). (D) Members of FcWD40 in group S3 (50 > FPKM > 10). Color bars represent level of downregulation (blue) and upregulation (red).

FIGURE 4

Expression profiles of FcWD40 genes under light deprivation. PFM and PPM represent the mature-stage flesh and peel of “Purple-Peel,” respectively; BPFM and BPPM represent the bagging mature-stage flesh and peel of ‘Purple-Peel’ fruit, respectively.

FIGURE 5

Phylogenetic analysis of 43 FcWD40 proteins and TTG1s of other species. (A) Phylogenetic relationships of TTG1 proteins. GenBank accession numbers: Arabidopsis thaliana AtTTG1 (NM_122360), Malus domestica MdTTG1 (GU173813), Pyrus pyrifolia PyWD40 (HQ641374.1), Fragaria × ananassa FaTTG1 (JQ989287), Camellia sinensis CsWD40 (MH618664), Punica granatum PgTTG1 (HQ199314), Gossypium hirsutum GhTTG3 (AAM95645), and Medicago truncatula, MtWD40-1 (EU040206.1). FcWD40-97 is indicated with red dot. (B) Alignment of deduced amino acid sequences of FcWD40 proteins and other known TTG1 homologs. The four conserved amino acid residue pairs (WD, FD, LD, and WE) are marked by different-colored straight lines.

FIGURE 6

Relative expression levels of FcTTG1 in different tissues and at different developmental stages in Ficus carica. (A) Fig plant tissues; flesh and peel are from ripe fruit. (B) Fig fruit flesh at developmental stages 1–6 corresponding to Figure 3A. (C) Fig fruit peel at developmental stages 1–6 corresponding to Figure 3A. Relative expression levels are shown as mean ± SD from three replications. Different letters between bars indicate significant difference at p < 0.05.

FIGURE 7

Subcellular localization and interaction network prediction of FcTTG1. (A) Subcellular localization of FcTTG1 in Nicotiana benthamiana. Images under fluorescence (left), bright field (middle) and merged (right). Bars = 50 μm. (B) Predicted interaction network for FcTTG1. Pink lines – experimentally determined physical interactions; yellow lines – interactions by text mining; black lines – co-expression; purple lines – protein homology.

FIGURE 8

Interaction of FcTTG1 with FcMYBs and FcbHLHs in vitro and in vivo. (A) Yeast two-hybrid test to determine the in vitro interaction with different constructs. (B) In vivo verification of interactions between FcTTG1 and FcMYB114, FcMYB123, and FcbHLH42. Bars = 50 μm.