An R2R3-MYB Transcription Factor Regulates Capsaicinoid Biosynthesis

Abstract

Capsaicinoids are responsible for the hot taste of chili peppers. They are restricted to the genus Capsicum and are synthesized by the acylation of the aromatic compound vanillylamine (derived from the phenylpropanoid pathway) with a branched-chain fatty acid by the catalysis of the putative enzyme capsaicinoid synthase. R2R3-MYB transcription factors have been reported in different species of plants as regulators of structural genes of the phenylpropanoid pathway; therefore, we hypothesized that MYB genes might be involved in the regulation of the biosynthesis of pungent compounds. In this study, an R2R3-MYB transcription factor gene, designated CaMYB31, was isolated and characterized in Capsicum annuum 'Tampiqueño 74'. Bioinformatic analysis suggested that CaMYB31 could be involved in secondary metabolism, stress and plant hormone responses, and development. CaMYB31 expression analysis from placental tissue of pungent and nonpungent chili pepper fruits showed a positive correlation with the structural genes Ca4H, Comt, Kas, pAmt, and AT3 expression and also with the content of capsaicin and dihydrocapsacin during fruit development. However, CaMYB31 also was expressed in vegetative tissues (leaves, roots, and stems). Moreover, CaMYB31 silencing significantly reduced the expression of capsaicinoid biosynthetic genes and the capsaicinoid content. Additionally, CaMYB31 expression was affected by the plant hormones indoleacetic acid, jasmonic acid, salicylic acid, and gibberellic acid or by wounding, temperature, and light, factors known to affect the production of capsaicinoids. These findings indicate that CaMYB31 is indeed involved in the regulation of structural genes of the capsaicinoid biosynthetic pathway.

Figure 1.

Capsaicinoid biosynthetic pathway. PAL, Phe ammonia lyase; Ca4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; HCT, hydroxycinnamoyl transferase; C3H, coumaroyl shikimate/quinate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; pAMT, aminotransferase; BCAT, branched-chain amino acid transferase; KAS, ketoacyl-ACP synthase; ACL, acyl-CoA synthetase; FAT, acyl-ACP thioesterase; CS, capsaicinoid synthase; AT3, acyltransferase. The potential target genes of the CaMYB31 transcription factor are marked in red.

Figure 2.

Quantitative reverse transcription (qRT)-PCR expression assays of the capsaicinoid biosynthetic genes Kas, pAmt, AT3, Comt, Ca4H, and CaMYB31 in placental tissue from fruits of cv Tampiqueño 74 and cv California Wonder at different developmental stages. The data points represent means of three biological replicates ± sd.

Figure 3.

Structural organization of the CaMYB31 gene and phylogenetic relationship of CaMYB31 with other plant MYB transcription factors. A, Schematic diagram of CaMYB31 showing the coding regions (black boxes), 5′ and 3′ UTRs (gray boxes), introns (solid lines), R2R3-MYB domains (dotted lines), and the fragment sequence of CaMYB31 used for virus-induced gene silencing (VIGS). B, Phylogenetic tree constructed using the neighbor-joining method based on the MYB domain alignment using MEGA6 software. The bootstrap values are shown as percentages (1,000 replicates) when greater than 50%. The clades were grouped as reported previously for Arabidopsis (Stracke et al., 2001; highlighted with colored diamonds for each subgroup), while CaMYB31 (highlighted with blue letters) grouped with solanaceous sequences (marked with red circles). Accession numbers for all protein sequences are listed in Supplemental Table S2.

Figure 4.

Differential expression assays of CaMYB31 and of the capsaicinoid biosynthetic genes pAmt, Kas, Comt, and Ca4H in different tissues of cv Tampiqueño 74 and cv California Wonder and in fruits of cv Habanero BG-3821. M, Mature; I, immature; Pe, pericarp; Se, seed; Pl, placenta; Fl, flower; Le, leaf; St, stem; Ro, root. The data points are means of three biological replicates ± sd.

Figure 5.

Effects of CaMYB31 gene silencing on biosynthetic structural gene expression and on capsaicinoid content in fruits of chili pepper. A, qRT-PCR analysis of capsaicinoid biosynthetic genes and CaMYB31 in fruits of chili pepper plants infected with the viral construct pTRV2-CaMYB31 compared with uninfected control plants. B, HPLC analysis of capsaicin and dihydrocapsaicin in fruits of uninfected plants and pTRV2- and pTRV2-CaMYB31-infected plants. The data points are means of three biological replicates ± sd. Asterisks indicate significant differences between the control (uninfected) and infected plants (P ≤ 0.05; Tukey’s test).

Figure 6.

Effects of light on CaMYB31, Kas, and pAmt expression in placental tissue from chili pepper fruits at 30 DPA. Gene expression was measured at different incubation times (3, 6, 12, and 16 h). The data points are means of three biological replicates ± sd. Asterisks show significant differences between fruits incubated under dark and light conditions (P ≤ 0.05; Tukey’s test).

Figure 7.

Effects of temperature on CaMYB31, Kas, and pAmt expression in placental tissue from chili pepper fruits at 30 DPA. Gene expression was quantified at different incubation times (3, 6, 12, and 16 h) and different temperatures (4°C, 25°C, and 37°C). The data points are means of three biological replicates ± sd. Asterisks show significant differences between fruits incubated at standard temperature (25°C) and high temperature (37°C) or low temperature (4°C; P ≤ 0.05; Tukey’s test).

Figure 8.

Effects of wounding on CaMYB31, Kas, and pAmt expression in placental tissue from chili pepper fruits at 30 DPA. Gene expression was quantified at different incubation times (3, 6, 12, and 16 h). The data points are means of three biological replicates ± sd. Asterisks show significant differences between control (no injuries) and wounded fruits (P ≤ 0.05; Tukey’s test).

Figure 9.

Effects of plant hormones on CaMYB31, Kas, and pAmt expression in placental tissue from chili pepper fruits at 30 DPA. Gene expression was quantified at different incubation times (3, 6, 12, and 16 h) in fruits treated with 100 µm IAA (A), GA₃ (B), SA (C), or JA (D). The data points are means of three biological replicates ± sd. Asterisks show significant differences between control (MS medium) and treated fruits (P ≤ 0.05; Tukey’s test).