Key transcription factors regulate fruit ripening and metabolite accumulation in tomato

Abstract

Fruit ripening is a complex process involving dynamic changes to metabolites and is controlled by multiple factors, including transcription factors (TFs). Several TFs are reportedly essential regulators of tomato (Solanum lycopersicum) fruit ripening. To evaluate the effects of specific TFs on metabolite accumulation during fruit ripening, we combined CRISPR/Cas9-mediated mutagenesis with metabolome and transcriptome analyses to explore regulatory mechanisms. Specifically, we generated various genetically engineered tomato lines that differed regarding metabolite contents and fruit colors. The metabolite and transcript profiles indicated that the selected TFs have distinct functions that control fruit metabolite contents, especially carotenoids and sugars. Moreover, a mutation to ELONGATED HYPOCOTYL5 (HY5) increased tomato fruit fructose and glucose contents by approximately 20% (relative to the wild-type levels). Our in vitro assay showed that HY5 can bind directly to the G-box cis-element in the Sugars Will Eventually be Exported Transporter (SWEET12c) promoter to activate expression, thereby modulating sugar transport. Our findings provide insights into the mechanisms regulating tomato fruit ripening and metabolic networks, providing the theoretical basis for breeding horticultural crops that produce fruit with diverse flavors and colors.

Figure 1.

Phenotypes of mutant fruit and differences in the fruit ripening process between the mutants and “Ailsa Craig” (AC). A) Phenotypes of the mutant and AC fruit collected at 46 DPA. Scale bar, 1 cm. B) Carotenoid contents of the mutants and AC. C) Ethylene production in the mutants and AC at 46 DPA. Data are presented as the mean ± Sd (n = 3) in panels B and C. Lowercase letters indicate significantly differences by Duncan's multiple range test with P < 0.05.

Figure 2.

Genome-wide profiling of ripening-associated genes regulated by TFs. A) PCA plot and cluster dendrogram of the transcriptome data for the mutant and “Ailsa Craig” (AC) fruit. B) Heatmap representation of the expression levels of the differentially expressed red ripening-related genes (RRGs) in the mutants and AC. C) Distribution of the downregulated and upregulated genes among the TF-related DEGs and RRGs. RR vs MG refers to DEGs in the RR stage fruit compared with MG stage fruit. Heatmaps were also constructed for the DEGs involved in ethylene biosynthesis and signal transduction (D), ABA signaling (E), auxin (IAA) signaling (F), and cell wall metabolism (G). For (B, D to G), the heatmaps show expression levels as Log₂(FPKM + 1).

Figure 3.

Schematic representation of carotenoid biosynthesis in tomato. The carotenoid contents and relative ABA contents in the ripe mutant and “Ailsa Craig” (AC) tomato fruit are presented in boxes. Data are presented as the mean ± Sd (n = 3). Different letters indicate significant differences (P < 0.05) relative to the AC values by Duncan's multiple range test. Expression patterns of genes involved in carotenoid metabolism in nine mutants and AC.

Figure 4.

Metabolome analysis of the mutants and “Ailsa Craig” (AC) during the fruit ripening stage. A) Pie chart with metabolite categories. B) PCA plot and cluster dendrogram of the metabolome data for the fruit ripening stage of nine mutants and AC. C) Differences in the metabolites identified in the mutants and AC. D) Overview of the DAMs between the mutants and AC. The heatmap shows relative content levels of metabolites.

Figure 5.

Phenolic acid and flavonoid contents in the mutant and WT tomato fruit. A) Phenolic acid and flavonoid synthesis pathways in tomato. The metabolites that were detected in this study are indicated in red. B, C) Heatmaps of the DAMs (B) and DEGs (C) related to the flavonoid, flavone, and flavanol synthesis pathways among the mutants and “Ailsa Craig” (AC). D, E) Heatmaps of the DAMs (D) and DEGs (E) related to the phenolic acid synthesis pathway among the mutants and AC. The gray block indicates the metabolite was undetectable. The metabolite and transcript data in each row were normalized and standardized. For (B) and (D), the heatmaps show relative content levels of metabolites.

Figure 6.

Overview of linoleic acid metabolism and SGA biosynthesis in mutant tomato fruit. A) Schematic representation of linoleic acid metabolism in tomato. B) Relative linolenic acid, α-linolenic acid and γ-linolenic acid contents in five mutants and “Ailsa Craig” (AC) tomato fruit. Data are presented as the mean ± Sd (n = 3). Asterisks indicate significant differences between the mutation and AC by Student's t test with P < 0.05. C) Expression patterns of linoleic acid metabolism-related genes in five mutants and AC. Gene expression data were normalized to −1.5 to 2. D) Schematic representation of glycoalkaloid biosynthesis and regulation in tomato. Heatmap of the DAMs (E) and DEGs (F) related to glycoalkaloid biosynthesis in nine mutants and AC. For (E), the heatmap shows relative content levels of metabolites. The metabolite and transcript data in each row were normalized and standardized.

Figure 7.

HY5 activates SWEET12c expression to regulate sugar accumulation in tomato fruit. Relative fructose and glucose contents in hy5-cr1 and AC tomato fruit. Data are presented as the mean ± Sd (n = 3). Asterisks indicate significant differences (*P < 0.05) were determined by Student's t test. B) Phenotypes of the hy5 mutant and AC tomato fruit at different development stages. The hy5-cr1, hy5-cr2, and WT fruit at the mature green (25 DPA), break (38 DPA), and ripening (46 DPA) stages are presented. C) Fructose and glucose contents of the hy5 mutants and AC at 46 DPA. Data are presented as the mean ± Sd (n = 3). Asterisks indicate significant differences (*P < 0.05) were determined by Student's t test. D) Soluble solids content (SSC) of the hy5 mutants and AC at 46 DPA. Data are presented as the mean ± Sd (n = 3). Asterisks indicate significant differences (***P < 0.001) were determined by Student's t test. E) Relative Susy and SWEET12c transcript levels in fruit at 46 DPA. Values are expressed relative to the wild-type level. Data are presented as the mean ± Sd (n = 3). Asterisks indicate significant differences (**P < 0.01) were determined by Student's t test. F) A Y1H assay indicated that HY5 can bind directly to the SWEET12c promoter containing the G-box motif. The arrow represents the C/G-box motif. G) A dual-luciferase reporter assay verified that HY5 affects the SWEET12c promoter activity. ProSweet12 (L2) represents the fragment L2 of the SWEET12c promoter, and the ProSweet12 (L2ΔG-box3) represents the fragment L2 without G-Box 3 of the SWEET12c promoter. Error bars represent the Sd (n = 6). Asterisks indicate significant differences (two-tailed Student's t-test; ***P < 0.001). H) The EMSA results reflected the ability of HY5-GST to bind directly to the SWEET12c promoter. Lanes 3 and 4 represent the negative control (GST incubated with the labeled probe). Unlabeled probes (4-, 20-, 100-, and 1,000-fold excess) were used as competitors. The probes used for the EMSA were Probe 1 (G-box 1), Probe 2 (G-box 2), and Probe 3 (G-box 3), which were designed and labeled with Cy5. I) Expression patterns of SWEET12c and Susy, which influence sucrose metabolism in tomato.