A comparative transcriptomics and eQTL approach identifies SlWD40 as a tomato fruit ripening regulator

Abstract

Although multiple vital genes with strong effects on the tomato (Solanum lycopersicum) ripening process have been identified via the positional cloning of ripening mutants and cloning of ripening-related transcription factors (TFs), recent studies suggest that it is unlikely that we have fully characterized the gene regulatory networks underpinning this process. Here, combining comparative transcriptomics and expression QTLs, we identified 16 candidate genes involved in tomato fruit ripening and validated them through virus-induced gene silencing analysis. To further confirm the accuracy of the approach, one potential ripening regulator, SlWD40 (WD-40 repeats), was chosen for in-depth analysis. Co-expression network analysis indicated that master regulators such as RIN (ripening inhibitor) and NOR (nonripening) as well as vital TFs including FUL1 (FRUITFUL1), SlNAC4 (NAM, ATAF1,2, and CUC2 4), and AP2a (Activating enhancer binding Protein 2 alpha) strongly co-expressed with SlWD40. Furthermore, SlWD40 overexpression and RNAi lines exhibited substantially accelerated and delayed ripening phenotypes compared with the wild type, respectively. Moreover, transcriptome analysis of these transgenics revealed that expression patterns of ethylene biosynthesis genes, phytoene synthase, pectate lyase, and branched chain amino transferase 2, in SlWD40-RNAi lines were similar to those of rin and nor fruits, which further demonstrated that SlWD40 may act as an important ripening regulator in conjunction with RIN and NOR. These results are discussed in the context of current models of ripening and in terms of the use of comparative genomics and transcriptomics as an effective route for isolating causal genes underlying differences in genotypes.

Figure 1

Candidate gene filtration by integrating comparative transcriptomics and eQTL mapping. A, Pipelines for candidate gene filtration of eQTL approach. Filters are shown in bullet points. B, Heat map of relative expression level of filtered candidates. Lyco/Penn, Genes were sorted by ratio of expression value for S. lycopersicum and S. pennellii. ILs are arranged as per the number of chromosome (X-axis). Genes are arranged according to their Gene IDs (Y-axis). Regions of red or blue indicate that the gene expression is increased or decreased, respectively, over that of M82. Chr, chromosome. C, Heat map of relative expression level of filtered candidates. Penn/Lyco, Genes were sorted by the ratio of expression value for S. pennellii and S. lycopersicum. ILs are arranged as per the number of chromosome (X-axis). Genes are arranged according to their Gene IDs (Y-axis). Regions of red or blue indicate that the gene expression is increased or decreased, respectively, over that of M82. D, Pipelines for candidate gene filtration of TFs approach. Filters are shown in bullet points. Br, Break.

Figure 2

VIGS of empty vector (silencing of Del/Ros1) and SlWD40 in Microtom Del/Ros1 fruit.

Figure 3

Co-expression network of SlWD40 with tomato ripening pathway-specific genes. Well-characterized key regulators such as RIN (ripening inhibitor), NOR (nonripening), and FUL1 (FRUITFUL1) (labeled in blue) strongly co-expressed with SlWD40. ELIP, early light induced protein; NF-Y, nuclear factor Y; MADS, MADS domain protein.

Figure 4

Photographs and pigments content of WT and T1 generation RNAi (lines 1 and 2) and OE (lines 1 and 2) lines at 28, 34 (MG), 37 (Br), and 45 (Pink) DPA fruits. A, Photographs of WT and transgenic SlWD40 fruits. Images were digitally extracted for comparison. B, Chlorophylls and carotenoids of WT and transgenic SlWD40 fruits. The values in each column are the mean of at least three biological replicates. Error bars indicate sd. The asterisks indicate statistically significant differences determined by the Student’s t test (two-tail): *P < 0.05; **P < 0.01. ND, not detected.

Figure 5

The effect of SlWD40 on fruit transcriptome. A, PCA of transcriptome of different genotypes in the three fruit stages. PC, principal component. B, Heat map of transcriptome profiles of pericarp tissues of T1 SlWD40 RNAi, OE lines, and WT. X-axis indicates different transgenic lines along with different fruit ripening stages. The hierarchical clustering based on transcriptome indicated that the development of RNAi samples is delayed especially on Br stage which are cluster with MG stage samples. C, Venn diagram showing the overlap DEGs between SlWD40 transgenic fruits, rin and nor mutant fruits.

Figure 6

The different expression of SlWD40 and other 17 ripening-related genes of T1 transformants of RNAi and OE lines compared with WT fruits. The values in each column are the mean of three biological replicates. Error bars indicate sd.

Figure 7

The effect of SlWD40 on fruit metabolism. A, PCA of metabolite levels of different genotypes in the three fruit stages. PC, principal component. B, Heat map of metabolite profiles of pericarp tissues of T1 SlWD40 RNAi, OE lines, and WT. X-axis indicates different transgenic lines along with different fruit ripening stages. Y-axis indicates metabolites. The hierarchical clustering based on all metabolites indicated that the development of RNAi samples is delayed especially in the Pink stage which are cluster with Br stage samples of OE and WT. C, Venn diagram showing the overlap different primary metabolite between SlWD40 RNAi fruits, rin and nor mutant fruits at the Pink stage.

Figure 8

The scheme of major metabolic changes of the transgenic lines. The difference of sugar and amino acid-related metabolites between transgenic lines and WT fruit at MG (A), Br (B), and Pink (C) stage. Blue and red color depicts a decrease and increase in metabolic levels compared with the WT fruit samples, respectively.

Figure 9

The difference of representative lipids and secondary metabolites of T1 transformants of RNAi and OE lines compared with WT fruits. The values in each column are the mean of at least three biological replicates. Error bars indicate sd. The asterisks indicate statistically significant differences determined by the Student’s t test (two-tail): *P < 0.05; **P < 0.01.

Figure 10

Proposed schematic overview of network of regulatory factors controlling tomato fruit ripening.