A DEMETER-like DNA demethylase governs tomato fruit ripening

Abstract

In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.

Keywords: DNA glycosylase lyase; active DNA demethylation; epigenetic; fruit ripening; tomato.

Fig. 1.

Differential expression of SlDML genes in tomato organs. Absolute quantification of SlDML1, SlDML2, SlDML3, and SlDML4 mRNA; SlDML4 gene expression is presented in a separate diagram because of its very low expression level. Fruit pericarp is at 5, 10, 20 dpa and at Breaker (BR, 39 dpa), orange (O), and red ripe (RR). Asterisks indicate significant difference [Student’s t test (n = 3)] between SlDML2 and all other SlDML genes: *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate means ± SD. Ap, stem apex; CF, closed flowers; L, leaves at positions 3, 4, 5, 8, 10, 16, and 20 from apex; OP, open flowers 5, 10, and 20; R, roots; S, stem from whole seedlings.

Fig. 2.

Phenotypes of tomato DML RNAi fruits. (A) Fruits (70 dpa) (upper lane) or fruit sections (lower lane) from eight independent representative T0 RNAi plants. (B) Fruits (85 dpa) from T2 plants (left to right); WT plants, line 2 plants (DML2A and DML2B), line 8 plants (DML8A and DML8B), and an azygous plant (AZ). (C) Ripening kinetics of WT (Top), DML8A (Middle), and DML2A (Bottom). (D) WT bicarpel (Upper) DML2B multicarpel fruits (Lower). (E) VIGS experiment on 47-dpa (Br + 5) fruits injected with PVX/SlDML2 [fruits (1) and (3)] or PVX [fruits (2) and (4)] at 12 dpa [fruits (3) and (4)] inside of fruits (1) and (2), respectively. (Scale bars: 1 cm.)

Fig. 3.

Residual expression of SlDML genes in fruits of transgenic DML RNAi plants. Normalized expression of the SlDML genes (A) in 20-dpa transgenic fruits of plants from line 2 (DML2A and -2B), line 8 (DML8A and -8B), an azygous plant (AZ), and the respective WT1 and WT2 controls (B) in WT2 and DML8A fruits at seven developmental stages. Expression of the SlDML genes was normalized to EF1α and to the corresponding WT fruits at 20 dpa. For each SlDML gene, asterisks indicate significant difference [Student’s t test (n = 3)] between transgenic plants and WT controls, respectively, at 20 dpa (A) or at the same age during fruit development (B). *P < 0.05; **P < 0.01; ***P < 0.001). Error bars indicate mean ± SD.

Fig. 4.

Metabolic profiling of carotenoids and primary metabolites in transgenic DML RNAi fruits. (A) Chlorophylls (Top), total carotenoids (Middle), and lycopene (Bottom) content. Asterisks indicate significant difference [Student’s t test (n = 3)] between DML2A and -2B, DML8A and -8B, and WT1 and WT2, respectively, at the same age: *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate means ± SD. (B) PCA using primary metabolites in WT2 (△) and DML8A (○) fruits at seven developmental stages. Color indicates the fruit developmental stages: white is 20 dpa and from light gray to black are 35, 39 (Br), 55, 70, 85, and 110 dpa.

Fig. 5.

Expression and demethylation at key genes controlling ripening are inhibited in DML RNAi plants. (A) Expression of the RIN, NOR, CNR, and PSY1 genes in transgenic DML8A and WT fruits normalized to EF1α and to WT fruits at 20 dpa. Asterisks indicate significant difference [Student’s t test (n = 3)] between WT and DML8A samples at a given stage: *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate means ± SD. (B) McrBC-PCR analysis of selected promoter fragments in fruits of WT, azygous (Azy), and DML8A plants; 1 µg of genomic DNA was digested with McrBC (NEB) during 5h (+); (–) indicates negative control for the digestion reaction that was performed without GTP. In the WT and azygous plants, the part of NOR, RIN, and PSY1 promoter regions analyzed are methylated at 35 dpa (no amplification) but are demethylated at 55 dpa (amplification). In DML8A plants, the three promoter regions behave similarly to WT at 35 dpa but remained methylated at 55 dpa (no amplification in both cases). The pectin-methyl esterase (PME) promoter is used as an unmethylated control, and the CNR promoter fragment used here was found to be sufficiently methylated at all stages for complete digestion by McrBC.

Fig. 6.

Bisulfite-sequencing analysis at the NOR, CNR, and PSY1 promoter fragments in WT and transgenic DML RNAi plants. (A) Heat-map representation of DNA methylation at selected NOR, CNR, and PSY1 promoter regions (SI Appendix, Fig. S8) in fruits of control (WT1 and WT2) and transgenic (DML2A, -2B, -8A, and -8B) plants at five (WT and line 8) or four (line 2) developmental stages. For each promoter, two fragments have been analyzed (fragment 1, gray box; fragment 2, black box), the positions of which are shown in SI Appendix, Fig. S8 and Fig. S9. The position of the Cs within each promoter fragment is also shown (number in the columns on the right side), as defined in SI Appendix, Fig. S8. For each promoter, Cs have been clustered considering the two PCR fragments analyzed together. (B) Changes in expression of SlDML genes in fruits of Ailsa Craig (WT) and near-isogenic mutant lines rin, Cnr, and nor, as determined by microarrays analysis. For fruit development, days postanthesis are shown. Mature green is 40 dpa in Ailsa Craig and then Br is 49 dpa. For nonripening mutants, Br onward are 49 dpa + 1–7 d. Asterisks indicate significant difference (variance ratio, F tests) between WT and mutant lines for the SlDML2 gene only to avoid overloading the figure: *P < 0.05; **P < 0.01; ***P < 0.001). Details of expression results and statistical analyses for all four genes are provided in Dataset S1. Error bars indicate means ± SD. (C) Proposed function of DNA demethylation in the control of fruit ripening; SlDML2 is necessary for the active demethylation of the NOR, CNR, RIN, and PSY1 promoter region, thereby allowing these gene expressions. SlDML2 gene expression is reduced in the rin, nor, and Cnr background, suggesting a regulatory loop. There is at this time no evidence of direct regulation of the SlDML2 gene by the RIN, NOR, or CNR protein. SlDML2 may control the expression of additional ripening induced gene, as shown in this study for the PSY1 gene and suggested by the demethylation of several promoters during fruit ripening (20). Arrows indicate activation. Lines indicate repression: black, direct effects; gray, direct or indirect effects.