Chuang Huang

Pepper is renowned worldwide for its distinctive spicy flavor. While the gene expression characteristics of the capsaicinoid biosynthesis pathway have been extensively studied, there are already a few reports regarding transcriptional regulation in capsaicin biosynthesis. In this study, 73 WRKYs were identified in the genome of Capsicum chinense, and their physicochemical traits, DNA, and protein sequence characteristics were found to be complex. Combining RNA-seq and qRT-PCR data, the WRKY transcription factor CA06g13580, which was associated with the accumulation tendency of capsaicinoid, was screened and named CcWRKY25. CcWRKY25 was highly expressed in the placenta of spicy peppers. The heterologous expression of CcWRKY25 in Arabidopsis promoted the expression of genes PAL, 4CL1, 4CL2, 4CL3, CCR, and CCoAOMT and led to the accumulation of lignin and flavonoids. Furthermore, the expression of the capsaicinoid biosynthesis pathway genes (CBGs) pAMT, AT3, and KAS was significantly reduced in CcWRKY25-silenced pepper plants, resulting in a decrease in the amount of capsaicin. However, there was no noticeable difference in lignin accumulation. The findings suggested that CcWRKY25 could be involved in regulating capsaicinoid synthesis by promoting the expression of genes upstream of the phenylpropanoid pathway and inhibiting CBGs’ expression. Moreover, the results highlighted the role of CcWRKY25 in controlling the pungency of pepper and suggested that the competitive relationship between lignin and capsaicin could also regulate the spiciness of the pepper.

Aroma is a crucial attribute affecting the quality of pepper and its processed products, which has significant commercial value. However, little is known about the composition of volatile aroma compounds (VACs) in pepper fruits and their potential molecular regulatory mechanisms. In this study, HS-SPME-GC-MS combined with transcriptome sequencing is used to analyze the composition and formation mechanism of VACs in different kinds and development stages of pepper fruits. The results showed that 149 VACs, such as esters, alcohols, aldehydes, and terpenoids, were identified from 4 varieties and 3 development stages, and there were significant quantitative differences among different samples. Volatile esters were the most important aroma components in pepper fruits. PCA analysis showed that pepper fruits of different developmental stages had significantly different marker aroma compounds, which may be an important provider of pepper’s characteristic aroma. Transcriptome analysis showed that many differential genes (DEGs) were enriched in the metabolic pathways related to the synthesis of VACs, such as fatty acids, amino acids, MVA, and MEP in pepper fruits. In addition, we identified a large number of differential transcription factors (TFs) that may regulate the synthesis of VACs. Combined analysis of differential aroma metabolites and DEGs identified two co-expression network modules highly correlated with the relative content of VACs in pepper fruit. This study confirmed the basic information on the changes of VACs in the fruits of several Chinese spicy peppers at different stages of development, screened out the characteristic aroma components of different varieties, and revealed the molecular mechanism of aroma formation, providing a valuable reference for the quality breeding of pepper.

The wide application of pepper is mostly related to the content of capsaicin, and phenylpropanoid metabolism and its branch pathways may play an important role in the biosynthesis of capsaicin. The expression level of MYB24, a transcription factor screened from the transcriptome data of the pepper fruit development stage, was closely related to the spicy taste. In this experiment, CcMYB24 was cloned from Hainan Huangdenglong pepper, a hot aromatic pepper variety popular in the world for processing, and its function was confirmed by tissue expression characteristics, heterologous transformation in Arabidopsis thaliana, and VIGS technology. The results showed that the relative expression level of CcMYB24 was stable in the early stage of pepper fruit development, and increased significantly from 30 to 50 days after flowering. Heterologous expression led to a significant increase in the expression of CcMYB24 and decrease in lignin content in transgenic Arabidopsis thaliana plants. CcMYB24 silencing led to a significant increase in the expression of phenylpropanoid metabolism pathway genes PAL, 4CL, and pAMT; lignin branch CCR1 and CAD; and capsaicin pathway CS, AT3, and COMT genes in the placenta of pepper, with capsaicin content increased by more than 31.72% and lignin content increased by 20.78%. However, the expression of PAL, pAMT, AT3, COMT, etc., in the corresponding pericarps did not change significantly. Although CS, CCR1, and CAD increased significantly, the relative expression amount was smaller than that in placental tissue, and the lignin content did not change significantly. As indicated above, CcMYB24 may negatively regulate the formation of capsaicin and lignin by regulating the expression of genes from phenylpropanoid metabolism and its branch pathways.