DNA methylation is involved in the regulation of pepper fruit ripening and interacts with phytohormones

Abstract

There is growing evidence to suggest that epigenetic tags, especially DNA methylation, are critical regulators of fruit ripening. To examine whether this is the case in sweet pepper (Capsicum annuum) we conducted experiments at the transcriptional, epigenetic, and physiological levels. McrBC PCR, bisulfite sequencing, and real-time PCR demonstrated that DNA hypomethylation occurred in the upstream region of the transcription start site of some genes related to pepper ripening at the turning stage, which may be attributed to up-regulation of CaDML2-like and down-regulation of CaMET1-like1, CaMET1-like2, CaCMT2-like, and CaCMT4-like. Silencing of CaMET1-like1 by virus-induced gene silencing led to DNA hypomethylation, increased content of soluble solids, and accumulation of carotenoids in the fruit, which was accompanied by changes in expression of genes involved in capsanthin/capsorubin biosynthesis, cell wall degradation, and phytohormone metabolism and signaling. Endogenous ABA increased during fruit ripening, whereas endogenous IAA showed an opposite trend. No ethylene signal was detected during ripening. DNA hypomethylation repressed the expression of auxin and gibberellin biosynthesis genes as well as cytokinin degradation genes, but induced the expression of ABA biosynthesis genes. In mature-green pericarp, exogenous ABA induced expression of CaDML2-like but repressed that of CaCMT4-like. IAA treatment promoted the transcription of CaMET1-like1 and CaCMT3-like. Ethephon significantly up-regulated the expression of CaDML2-like. Treatment with GA3 and 6-BA showed indistinct effects on DNA methylation at the transcriptional level. On the basis of the results, a model is proposed that suggests a high likelihood of a role for DNA methylation in the regulation of ripening in the non-climacteric pepper fruit.

Keywords: Carotenoids; DNA methylation; fruit ripening; gene expression; pepper; plant hormone.

Fig. 1.

Dynamic changes of DNA methylation and expression levels of ripening-related genes during ripening of pepper fruit. (A) McrBC-PCR analysis of the upstream region of the transcriptional start site (UROT) of ripening-related genes in the pericarp at the immature-green (IM) and turning (T) stages. + and – indicate the presence or absence, respectively, of McrBC and GTP. (B) Bisulfite sequencing of the UROT of ripening-related genes in the pericarp at the IM and T stages. The scale at the top of each chart represents the position of each cytosine in the tested region. (C) Relative expression of the tested genes during pepper fruit ripening. Data were generated by real-time PCR and are log₂-transformed. For each gene, expression at the MG stage was set as 1. (D) Pepper fruit at five different developmental stages (MG, mature-green; B, breaker; R, red-ripening).

Fig. 2.

Identification of DNA methyltransferase and demethylase genes in pepper. (A) Phylogenetic tree of DNA methyltransferase and demethylase genes in pepper (Ca), tomato (Sl), and Arabidopsis (At). (B) Conserved domains of the DNA methyltransferase and demethylase genes. (C–F) Dynamic expression changes of CaMETs (C), CaCMTs (D), CaDRMs (E), and CaDMLs (F) during pepper fruit development and ripening. Significant differences were determined using ANOVA followed by Tukey’s HSD test (P<0.05).

Fig. 3.

Silencing of CaMET1-like1 in pepper fruit leads to premature ripening and DNA hypomethylation. (A) Infection efficiency of the virus-induced gene silencing (VIGS). ‘pTRV1/2M-RA’ indicates injected plants that carried both the pTRV1 and pTRV2-CaMET1-like1 plasmids and which showed accelerated fruit ripening. ‘pTRV1/2M’ indicates injected plants that carried both the pTRV1 and pTRV2-CaMET1-like1 plasmids and which showed no effect on fruit ripening. ‘ND’ indicates injected plants that only carried one of the above plasmids. (B) Boxplot of the time of onset of ripening of fruit injected with pTRV1 + pTRV2-CaMET1-like1 (pTRV2M) and pTRV1 + pTRV2. Data are shown from two replicates (I, II). Student’s t-test was used to determine the P-values. (C–E) Plants injected with pTRV1 + pTRV2 (C), pTRV1 + pTRV2-CaMET1-like1 (D), and pTRV1 + pTRV2-CaPDS (E). The white arrowhead in (D) indicates the coloration on a premature-ripe fruit. The yellow and white arrows in (E) indicate a light-bleached peduncle and pericarp, respectively. Light-bleached leaves are also shown in (E). (F) PCR-based detection of pTRV1 and pTRV2-CaMET1-like1 plasmids in three heterogeneously colored precocious fruits, which were collected from three replicate injected plants (V11, V49, and V72). ‘R’ and ‘G’ indicate premature-ripe and green pericarps, respectively, collected from the same precocious fruit. (G) Relative expression of CaMET1-like1 in the premature-ripe (red bars) and green pericarps (green bars). Significant differences were determined using Student’s t-test: **P<0.01. (H) Relative expression of CaMET1-like1 in leaves collected from three replicate plants injected with pTRV1 + pTRV2 and pTRV1 + pTRV2-CaMET1-like1 (VIGS11, VIGS49, and VIGS72). Significant differences were determined using ANOVA followed by Tukey’s HSD test (P<0.01). (I–L) 5mC levels of the upstream region of the transcriptional start site (UROT) of CaPSY1 (I), CaCEL1 (J), and CaNOR (K, L) in pericarps of premature-ripe (shaded red) and green fruit (shaded green) of V11 (top), V49 (middle), and V72 (bottom) plants. (K) and (L) are two different regions of the UROT of CaNOR.

Fig. 4.

Transcriptome analysis of premature-ripe (PR) and green (G) pericarps of CaMET1-like1-silenced pepper fruit and of red-ripe pericarp (CR) of the negative control. (A) Statistics of pathway enrichment of the differentially expressed genes (DEGs) between the premature-ripe and green pericarps. (B) DEGs of interest between the premature-ripe and green pericarps of the CaMET1-like1-silenced fruits. (C) DEGs of interest between the premature-ripe and the red-ripe pericarps. The numerals indicate genes involved in (I) ‘DNA methylation’, (II) ‘carotenoids biosynthesis’, (III) ‘cell wall degradation’, (IV) ‘ABA metabolism and signaling’, (V) ‘transcriptional regulation’, (VI) ‘ethylene biosynthesis and signaling’, (VII) ‘auxin biosynthesis and signaling’, (VIII) ‘gibberellin biosynthesis and signaling’, (IX) ‘capsaicin biosynthesis’, and (X) ‘cytokinin biosynthesis and signaling’.

Fig. 5.

Responses of genes to phytohormones in mature-green (MG) pericarps and dynamic changes in endogenous ABA and IAA during pepper fruit ripening. (A) Changes in gene expression in response to exogenous auxin (IAA), ABA, gibberellin (GA₃), cytokinin (6-BA), and ethephon in pericarp discs at the MG stage. Data are log₂-transformed. ‘T/M’ indicates the treatment value divided by the mock control value. ‘*’ indicates that the log₂ fold-change (FC) value is either greater than 1 or less than –1. (B) Dynamic changes of ABA and IAA levels during pepper fruit ripening. ‘IM’, immature-green; ‘M’, mature-green; ‘T’, turning; ‘R’, red-ripening. Different letters indicate significant differences as determined using ANOVA followed by Tukey’s HSD test (P<0.01): IAA, lowercase letters; ABA, uppercase letters.

Fig. 6.

A proposed model of the interactions between DNA methylation and phytohormones during ripening of pepper fruit. Arrows indicate promotion, blocked lines indicate inhibition. Solid lines indicate that the interaction is supported by data obtained from this study. Dashed lines indicate that the interaction is deduced from the gene function.