Two transcription factors play critical roles in mediating epigenetic regulation of fruit ripening in tomato

Abstract

DNA methylation regulates fruit ripening in tomato, and disruption of the DNA demethylase DEMETER-LIKE 2 (DML2) results in genome-wide DNA hypermethylation and impaired ripening. We report here that the transcription factors Ripening Inhibitor (RIN) and FRUITFULL 1 (FUL1) play critical roles in mediating the effect of DNA methylation on tomato fruit ripening. RIN and FUL1 are silenced in dml2 mutant plants, and the defective ripening phenotype of dml2 is mimicked by the rin/ful1 double mutant. Restoration of RIN expression in dml2 partially rescues its ripening defects. DNA methylation controls ripening not only by regulating the expression of RIN and FUL1 but also by interfering with the genomic binding of RIN. In dml2 mutant plants, RIN cannot bind to some of its targets in vivo even though DNA methylation does not interfere with RIN binding in vitro; this inhibited binding in vivo is correlated with increased DNA methylation and histone H3 enrichment within 100 bp of the binding site. Our work uncovers the molecular mechanisms underlying DNA methylation control of fruit ripening in tomato.

Keywords: DML2; DNA methylation; FUL1; RIN; demethylase.

Fig. 1.

RIN and FUL1 function redundantly downstream of DML2 in the control of tomato fruit ripening. (A) Phenotypes of tomato fruits of AC (the WT), and of rin, ful1, rin/ful1, nor, and dml2 mutants at 17, 25, 34, 38, 45, and 52 dpa. (Scale bar: 2 cm.) (B) Heatmap showing transcript profiles of RIN and FUL1 in AC and dml2-3 fruits. Transcriptome data of AC and dml2-3 fruits at 25 dpa and 34 dpa were used. (C) RT-qPCR analysis of RIN and FUL1 transcript levels in AC and dml2-3 fruits at 25 dpa and 34 dpa. Values are means ± SD. One-tailed Student’s t test was used to determine significance, ***P < 0.001. (D) Methylation levels of promoters of RIN and FUL1 in AC and dml2-3 fruits at 25 dpa and 34 dpa. Bisulfite sequencing data (Bs-seq) were displayed using Integrative Genome Browser software (IGB), where each vertical bar represents an mC, and the height of the bar indicates the methylation level.

Fig. 2.

Comparative analysis of transcript accumulation during fruit ripening of dml2-3 and rin/ ful1-1. (A) Volcan plots displaying DEGs in dml2-3 and rin/ ful1-1 mutants compared with AC fruits at 34 dpa. (B) Venn diagram showing overlaps among the up- and down-regulated DEGs of dml2-3 and rin/ ful1-1 mutants compared with AC fruits at 34 dpa. (C) Heatmaps showing the transcript levels of overlapped up- and down-regulated DEGs in fruits of AC 25 dpa, AC 34 dpa, dml2-3 34 dpa, and rin/ ful1-1 34 dpa. The overlapped 4,390 up- and 4,389 down-regulated DEGs in (B) were used. (D) Clustering of transcriptome data of fruits of AC, dml2-3, rin-cr1, ful1-1, nor-cr1, and rin/ ful1-1 at 34 dpa, according to the mean expression level in three replicates.

Fig. 3.

Restored RIN expression partially rescues the ripening phenotype of dml2-3. (A) Pictures of fruits of AC, dml2-3, pCBC::RIN-3xFLAG/dml2-3 line 1, and pCBC::RIN-3xFLAG/dml2-3 line 2. Fruits at 25, 34, 38, and 45 dpa are shown. (B) β-carotene and lycopene accumulation in the fruits of AC, ful1-1, rin-cr1, rin/ ful1-1, dml2-3, dml2-4, and pCBC::RIN-3xFLAG/dml2-3 line 1 at 45 dpa. (C) Ethylene production in the fruits of AC, ful1-1, rin-cr1, rin/ ful1-1, dml2-3, dml2-4, and pCBC::RIN-3xFLAG/dml2-3 line 1 at 25, 34, 38, and 45 dpa. (D) ACC content in AC, ful1-1, rin-cr1, rin/ ful1-1, dml2-3, and pCBC::RIN-3xFLAG/dml2-3 line 1 fruits at 38 dpa. (E) qRT-PCR analysis showing the relative expression of DML2 in different mutant fruits at 38 dpa. Values are means ± SD (n = 3). For (B–E), one-tailed Student’s t test was used to determine significance, ***P < 0.001.

Fig. 4.

Responses of rin/ ful1-1 fruits to exogenous ethylene are similar to those of dml2-3 fruits. The effect of exogenous ethylene treatment on the ripening of AC, rin-cr1, ful1-1, rin/ ful1-1, and dml2-3 fruits was examined. All fruits were picked at the MG stage (30 dpa) and treated with external ethephon (2 mM) for 5 min, or with 1-MCP (5 ppm) continually for up to 8 d. Photos of representative fruits are shown. (Scale bar: 2 cm.)

Fig. 5.

DML2 is required for RIN binding at a subset RIN targets. (A) Heatmaps showing RIN ChIP signals in pCBC::RIN-3xFLAG and pCBC::RIN-3xFLAG/dml2-3 fruits at 34 dpa. Antibody against the FLAG tag was used in this assay. ChIP signals within ±2.0 kb of the summit of RIN-binding peaks are shown. (B and C) IGB display of RIN enrichment at the promoters of downstream genes (AP2a, PG2a, NAC-NOR, and Z-ISO) of RIN. Compared with ChIP signals in pCBC::RIN-3xFLAG, the RIN enrichment was maintained at the promoters of AP2a and PG2a (B), but was lost at the promoters of NAC-NOR and Z-ISO (C) in pCBC::RIN-3xFLAG/dml2-3 fruits. ChIP signals in AC served as a negative control. (D) ChIP-qPCR analysis of RIN enrichment in the promoters of AP2a, PG2a, NAC-NOR, and Z-ISO. The AC, pCBC::RIN-3xFLAG, and pCBC::RIN-3xFLAG/dml2-3 fruits at 34 dpa were used. SlACTIN2 was used as a nonspecific control for the target genes. ChIP signal is displayed as the percentage of total input DNA. Values are means ± SD (n = 3). One-tailed Student’s t test was used to determine significance, ***P < 0.001.

Fig. 6.

The loss of RIN binding in dml2-3 is highly correlated with the increase in DNA methylation. (A) Boxplots showing DNA methylation levels of the 68,230 dml2-3 hyper DMRs in AC, dml2-3, and pCBC::RIN-3xFLAG/dml2-3. Methylation levels in C, CG, CHG, and CHH contexts are shown. (B) Profile plots showing the average RIN signal for AC and dml2-3 in the three groups of RIN-binding regions, where the DNA methylation was not increased (difference < 0.05), moderately increased (difference > 0.05 and < 0.2), or greatly increased (difference > 0.2) in dml2-3 relative to the WT. (C) Smoothed Scatter plots showing the correlation between the gain of DNA methylation and RIN enrichment in dml2-3. RIN binding peaks in pCBC::RIN-3xFLAG fruits at 34 dpa were used in this analysis. Randomly selected regions were used as control. Correlation coefficients are shown. (D) IGB display of RIN ChIP signal and BS-seq data showing the effect of cytosine methylation on RIN enrichment at the promoters of Solyc01g094320 and Solyc03g079850 in dml2-3.

Fig. 7.

DNA hypermethylation within 100 bp of the RIN binding site blocks RIN binding. (A) Heatmaps showing RIN ChIP signals of hyper DMR-associated RIN-binding regions in pCBC::RIN-3xFLAG and pCBC::RIN-3xFLAG/dml2-3 fruits at 34 dpa. The RIN enrichment was maintained at 7,836 regions but was blocked at 1,144 regions in dml2-3 relative to the WT. (B) Boxplots showing the change in DNA methylation in dml2-3 relative to the AC in the two groups of RIN-binding regions. The DNA methylation level of bins at the center [±100 bp] is greatly increased in the 1,144 blocked RIN-binding regions but not in the 7,836 maintained RIN-binding regions. (C) The change of RIN ChIP signal is correlated with the changes in DNA methylation and H3 enrichment in dml2-3 relative to AC. The correlations were observed for bins located within 100 bp from the binding site [±100 bp], but not for bins located 100 bp away from the binding site. (D) EMSA experiments using a GST-RIN (1-157aa) truncated protein and GST protein. CArG-box was predicted as the RIN-binding site. mCArG-box, methylated CArG-box. The CArG-box Cy5-Probe and mCArG-box Cy5-Probe were labeled with Cy5. Unlabeled probes named CArG-box cold Probe and mCArG-box cold Probe were used as competitors. The concentration of CArG-box Cy5-Probe and mCArG-box Cy5-Probe was 250 nM. The increasing concentrations of CArG-box cold Probe and mCArG-box cold Probe were 2.5 μM (+), 25 μM (++), and 125 μM (+++).