Capsaicin Biosynthesis Research Articles

The CaABCG14 transporter gene regulates the capsaicin accumulation in Pepper septum

Capsaicin (CAP), a crucial compound found in chili peppers, not only contributes to their spicy flavor but also possesses several industrial applications. CAP biosynthetic pathway is well known, while its transport mechanism remains elusive. Herein, we performed a comparative transcriptome analysis conducted on pepper fruit tissues at three different stages of development. Four important CAP transporter genes, including one MATE and three ABCs, were identified by differential expression and WGCNA analysis. Specifically, the expression patterns of three ABC genes were assessed in the septum of fruits from nine distinct genotypes of peppers with high capsaicin levels. Interestingly, CaABCG14 was associated with variations in CAP concentration and co-expressed with genes involved in CAP biosynthesis. Transient expression assay revealed that CaABCG14 is localized to the membrane and nucleus. Silencing of CaABCG14 resulted in a notable reduction in the levels of CAP contents and the expression of its biosynthetic genes in the septum of pepper. The overexpression of CaABCG14 greatly intensified the cytotoxic effects of CAP on the yeast cells. Taken together, we for the first time identified a new transporter gene CaABCG14, regulating the CAP accumulation in pepper septum. These findings offer a fresh molecular theoretical framework for CAP transport and accumulation.

Negative regulation of CcPAL2 gene expression by the repressor transcription factor CcMYB4-12 modulates lignin and capsaicin biosynthesis in Capsicum chinense fruits

Peppers globally renowned for their distinctive spicy flavor, have attracted significant research attention, particularly in understanding spiciness regulation. While the activator MYB's role in spiciness regulation is well-established, the involvement of repressor MYB factors remains unexplored. This study identified the MYB4 transcription factor through RNA-seq and genome-wide analysis as being associated with spiciness. Consequently, CcMYB4-2 and CcMYB4-12 were cloned from Hainan Huangdenglong peppers, both exhibiting nuclear subcellular localization. qRT-PCR analysis revealed that CcMYB4-2/4-12 had high expression levels during the accumulation period of capsaicin, but there were differences in their peak expression levels, which may be related to the formation of pepper spiciness. Heterologous expression in Arabidopsis thaliana resulted in significantly elevated CcMYB4-2/4-12 expression levels and reduced lignin content. In CcMYB4-2 silenced plants, PAL expression remained unchanged, while PAL expression significantly increased in CcMYB4-12 silenced plants, leading to elevated lignin content and reduced capsaicin content. Yeast one-hybrid (Y1H) and dual luciferase reporter assays (DLR) demonstrated that CcMYB4-2/4-12 inhibited the transcription of CcPAL2 by binding to its promoter. Notably, CcMYB4-12 exhibited more pronounced inhibition. Therefore, it is hypothesized that CcMYB4-12 plays a pivotal role in regulating lignin and capsaicin biosynthesis. This study elucidates the molecular mechanism of MYB4 binding to the PAL promoter, providing a foundational understanding for analyzing phenylpropanoid metabolism and its diverse branches. Key messageThrough functional verification analysis of the repressor CcMYB4, transcriptional regulation experiments revealed that CcMYB4 can bind to the CcPAL2 promoter, negatively regulating the capsaicin biosynthesis in Capsicum chinense fruits.

Full-length transcriptome sequencing of pepper fruit during development and construction of a transcript variation database.

Chili pepper is an important spice and a model plant for fruit development studies. Large-scale omics information on chili pepper plant development continues to be gathered for understanding development as well as capsaicin biosynthesis. In this study, a full-spectrum transcriptome data of eight chili pepper tissues at five growth stages using the Oxford Nanopore long-read sequencing approach was generated. Of the 485 351 transcripts, 35 336 were recorded as reference transcripts (genes), while 450 015 were novel including coding, lnc, and other non-coding RNAs. These novel transcripts belonged to unknown/intergenic (347703), those retained introns (26336), and had multi-exons with at least one junction match (20333). In terms of alternative splicing, retained intron had the highest proportion (14795). The number of tissue-specific expressed transcripts ranged from 22 925 (stem) to 40 289 (flower). The expression changes during fruit and placenta development are discussed in detail. Integration of gene expression and capsaicin content quantification throughout the placental development clarifies that capsaicin biosynthesis in pepper is mainly derived from valine, leucin, and isoleucine degradation as well as citrate cycle and/or pyrimidine metabolism pathways. Most importantly, a user-friendly Pepper Full-Length Transcriptome Variation Database (PFTVD 1.0) (http://pepper-database.cn/) has been developed. PFTVD 1.0 provides transcriptomics and genomics information and allows users to analyse the data using various tools implemented. This work highlights the potential of long-read sequencing to discover novel genes and transcripts and their diversity in plant developmental biology.

Spatiotemporal analysis of microstructure, sensory attributes, and full-spectrum metabolomes reveals the relationship between bitterness and nootkatone in Alpinia oxyphylla miquel fruit peel and seeds

The Alpinia oxyphylla fruit (AOF) is a popular condiment and traditional Chinese medicine in Asia, known for its neuroprotective compound nootkatone. However, there has not been a comprehensive study of its flavor or the relationship between sensory and bioactive compounds. To address this issue, we examined AOF’s microstructure, flavor, and metabolomic profiles during fruit maturation. The key markers used to distinguish samples included fruit expansion, testa pigmentation, aril liquefaction, oil cell expansion, peel spiciness, aril sweetness, and seed bitterness. A full-spectrum metabolomic analysis, combining a nontargeted metabolomics approach for volatile compounds and a widely targeted metabolomics approach for nonvolatile compounds, identified 1,448 metabolites, including 1,410 differentially accumulated metabolites (DAMs). Notably, 31 DAMs, including nootkatone, were associated with spicy peel, sweet aril, and bitter seeds. Correlational analysis indicated that bitterness intensity is an easy-to-use biomarker for nootkatone content in seeds. KEGG enrichment analysis linked peel spiciness to phenylpropanoid and capsaicin biosynthesis, seed bitterness to terpenoid (especially nootkatone) biosynthesis, and aril sweetness to starch and sucrose metabolism. This investigation advances the understanding of AOF’s complex flavor chemistry and underlying bioactive principle, encapsulating the essence of the adage: “no bitterness, no intelligence” within the realm of phytochemistry.

Fertilizing the Flame: Effects of AB Fertilizer Concentration on Vegetative Growth, Fruit Yield, and Capsaicin Biosynthesis in Capsicum frutescens

The Capsicum crops, known for their diverse phytochemical composition and pungency, are significantly influenced by agricultural practices. This study presents research on the 'cili padi' variety of Capsicum frutescens, a relatively unexplored species, grown hydroponically on a cocopeat medium using a high-quality AB fertilizer with varied nutrient concentrations. The research focused on analysing plant growth patterns, fruit yield, and capsaicinoid levels throughout the growth cycle. This involved detailed morphological measurements, productivity assessments, and the use of cyclic voltammetry for capsaicinoid quantification. The findings revealed that vegetation growth, yield, and metabolite parameters were closely linked to fertilizer concentration. Notably, an increase in nutrient availability led to improvements in plant growth metrics, fruit yield, and capsaicin levels. However, these benefits peaked within a specific fertilizer concentration range (1.5-2.1 E.C.-mS/cm) before declining, indicating the existence of toxicity thresholds. The study highlights the critical importance of balanced fertilization in hydroponic cultivation of C. frutescens, as it simultaneously enhances productivity and pungency quality. Interestingly, the research uncovered a threshold in nutrient concentration that optimizes nitrate conversion into capsaicin, beyond which overall plant growth continues to increase with higher fertilizer quantities. This finding necessitates further investigation into the metabolic and transport pathways that limit phytochemical production in these plants. Additionally, the capsaicinoid concentrations achieved in this study resulted in only moderately pungent Scoville ratings, suggesting a variance in biosynthetic capacity between this specific local species and more commonly studied pepper types. In summary, this research underscores the importance of a well-calibrated fertilization strategy to effectively boost both yield and pungency quality in C.frutescens . The insights gained from this study contribute significantly to our understanding of optimal hydroponic cultivation practices for enhancing the desirable traits of this unique chili variety.

Functional characterization of Capsicum chinense vanillin aminotransferase: Detection of vanillylamine-forming activity from vanillin

In capsaicin biosynthesis, vanillin aminotransferase (VAMT; EC 2.6.1.119) catalyzes the conversion of vanillin (V) to vanillylamine (VA). In vitro analysis of the recombinant VAMT enzyme has been reported; however, this enzyme catalyzed only the V-forming reaction and not the VA-forming reaction, which is inconsistent with the postulated pathway for capsaicin biosynthesis. In this study, we expressed, purified, and characterized functional recombinant VAMT of Capsicum chinense cv. Habanero from an Escherichia coli strain. The enzyme catalyzed reversible transamination between V and VA, and its VA-forming activity was high when γ-aminobutyric acid (GABA) was used as an amino donor. The enzyme exhibited maximum activity at pH 8.0 and 55 °C, and was stable up to 60 °C over a pH range from 4.5 to 8.0. The enzyme was stable in the presence of various chemicals and metal ions. The enzyme accepted several 4–8-carbon long primary amines and ω-amino acids with carbon chains longer than 4 as amino donors despite the narrow specificity of the amino acceptor. Based on its kinetic attributes and localization, VAMT appears to have evolved from GABA-aminotransferase to catalyze reversible transamination between V and VA, and is responsible for VA biosynthesis using GABA as an amino donor in the cytosol of capsicum fruit cells.

CAT Bridge: an efficient toolkit for gene-metabolite association mining from multiomics data.

With advancements in sequencing and mass spectrometry technologies, multiomics data can now be easily acquired for understanding complex biological systems. Nevertheless, substantial challenges remain in determining the association between gene-metabolite pairs due to the nonlinear and multifactorial interactions within cellular networks. The complexity arises from the interplay of multiple genes and metabolites, often involving feedback loops and time-dependent regulatory mechanisms that are not easily captured by traditional analysis methods. Here, we introduce Compounds And Transcripts Bridge (abbreviated as CAT Bridge, available at https://catbridge.work), a free user-friendly platform for longitudinal multiomics analysis to efficiently identify transcripts associated with metabolites using time-series omics data. To evaluate the association of gene-metabolite pairs, CAT Bridge is a pioneering work benchmarking a set of statistical methods spanning causality estimation and correlation coefficient calculation for multiomics analysis. Additionally, CAT Bridge features an artificial intelligence agent to assist users interpreting the association results. We applied CAT Bridge to experimentally obtained Capsicum chinense (chili pepper) and public human and Escherichia coli time-series transcriptome and metabolome datasets. CAT Bridge successfully identified genes involved in the biosynthesis of capsaicin in C. chinense. Furthermore, case study results showed that the convergent cross-mapping method outperforms traditional approaches in longitudinal multiomics analyses. CAT Bridge simplifies access to various established methods for longitudinal multiomics analysis and enables researchers to swiftly identify associated gene-metabolite pairs for further validation.

Surviving mutations: how an Indonesian Capsicum frutescens L. cultivar maintains capsaicin biosynthesis despite disruptive mutations

Surviving mutations: how an Indonesian Capsicum frutescens L. cultivar maintains capsaicin biosynthesis despite disruptive mutations

Genome-Wide Identification of WRKY Gene Family and Functional Characterization of CcWRKY25 in Capsicum chinense.

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.

Capsaicin derived from endemic chili landraces combats Shigella pathogen: Insights on intracellular inhibition mechanism

Ethnic tribals in northeast India have been growing and maintaining local chili landraces for ages. These chilies are known for their characteristic pungency and immense therapeutic properties. Capsaicin, a significant chili metabolite, is recognized as a natural drug for pain relief, diabetic neuropathy, psoriasis, arthritis, etc. In this study, we tried to observe the influence of locality factors on the pungency and bioactive features of Capsicum annuum L. landraces. We also checked the gastro-protective ability of these chilies, especially in the cure of shigellosis. Phytometabolite characterization and estimation were done through spectrophotometric methods. Preparative and analytical HPLC techniques were employed for extracting and purifying capsaicin-enriched fractions. Shigella flexneri growth retardation was determined through the broth dilution method. Gentamicin protection assay and ELISA were done to assess the intracellular invasion and IL-1β inflammasome production by S.flexneri. The correlation analyses postulated that phenols, flavonoids, chlorophylls, β-carotene, and capsaicin synthase upregulation strongly influenced capsaicin biosynthesis in chili cultivars. Correspondingly, the inhibitory efficacy of the HPLC-purified Balijuri-derived capsaicin was more effective than the Raja-derived capsaicin in inhibiting intracellular Shigella growth. Reduced levels of pro-inflammatory cytokine (IL1β) in capsaicin-treated Shigella-infected cells probably reduced inflammation-mediated intestinal damage, limiting bacterial spread. This investigation advocates the unique potential of local chilies in curing deadly ‘shigellosis’ with mechanistic evidence. Our observation justifies the traditional healing practices of the ethnic people of NE India.

Yeast-Based Whole-Cell Factory for the Ecofriendly Synthesis of Vanillylamine as a Critical Step toward Capsaicin Production

Capsaicin is a biologically active alkaloid with important pharmaceutical, food, and cosmetic applications. As capsaicin production is dependent on its extraction from plants, its industrial separation and purification capacity is greatly limited. The chemical synthesis of capsaicin via the Schotten–Baumann reaction requires vanillylamine and 8-methyl-6-nonenoic acid as reaction substrates. Currently, petrochemical products obtained by the catalysis of precious metals are used to produce vanillylamine; however, this approach does not meet the requirements advocated by the green chemical industry. Herein, we developed an efficient yeast-based whole-cell factory for the de novo biosynthesis of vanillylamine and capsaicin. A heterologous recombinant vanillylamine biosynthetic pathway in yeast yielded 6.47 g/L vanillylamine. Enhancing the phenylalanine pool by upregulating the shikimic acid pathway led to an 11% increase in vanillylamine production. Optimization of methyl transfer efficiency via a reconstituted S-adenosylmethionine (SAM) circulating cycle resulted in an 84% increase in vanillylamine production. Switching the yeast strain from Z2 to Z6 and rewiring the precursor pool and SAM circulating cycle increased vanillylamine production to 14.89 g/L (125%)─the highest reported level in the current literature. Based on the biosynthesis of vanillylamine via the heterologous recombinant fatty acid pathway, capsaicin was synthesized de novo in yeast. We also revealed that protocatechuic acid can be produced from 3-dehydroshikimic acid by phenylalanine ammonium lyase or tyrosine ammonia lyase. In summary, this study provides a feasible strategy for developing yeast-based whole-cell factories for the eco-friendly synthesis of vanillylamine, which is a critical step toward capsaicin production in yeast.

Integrated transcriptomic and metabolomic analysis of chili pepper fruits provides new insight into the regulation of the branched chain esters and capsaicin biosynthesis

Chili pepper (Capsicum spp.) is one of the world’s most popular vegetables and spices. Aroma is an important quality indicator of pepper, but the nature of the related volatiles is still not clear. In this study, we investigated the fruit of two pepper varieties, one with strong fruity aroma ‘CC’ Capsicum chinense and one without ‘TJ’ Capsicum annuum at four different developmental stages using transcriptomic and metabolomic analysis. The results showed that the content of green leaf volatiles (GLVs) was higher in TJ than in CC and was higher in the young fruit stage in both varieties. GLVs content was positively correlated with the expression of 13-LOX1, 2, 5 and HPL. But the levels of branched-chain (BC) esters and capsaicin were higher in CC, and were positively correlated with the expression of IMPS4 and DADH1. Our findings shed light on the molecular mechanism of aroma biosynthesis in pepper and provide a theoretical basis for the molecular breeding of high-quality pepper fruits.

Post-Harvest Red- and Far-Red-Light Irradiation and Low Temperature Induce the Accumulation of Carotenoids, Capsaicinoids, and Ascorbic Acid in Capsicum annuum L. Green Pepper Fruit.

Environmental factors, such as light of different spectral compositions and temperature, can change the level of activated photoreceptors which, in turn, can affect the biosynthesis of secondary metabolites in the cells of green fruit. By briefly irradiating the harvested fruit of Capsicum annuum L. hot peppers with red light (RL, maximum 660 nm) and far-red light (FRL, maximum 730 nm) and by keeping them at a low temperature, we attempted to determine whether the state of phytochromes in fruit affects the biosynthesis of secondary metabolites. Using HPLC, we analysed the qualitative composition and quantitative content of the main carotenoids and alkaloids and the chlorophylls and ascorbate, in pepper fruit exposed to the above factors. We measured the parameters characterising the primary photochemical processes of photosynthesis and the transcript levels of genes encoding capsaicin biosynthesis enzymes. The total carotenoids content in the fruit increased most noticeably after 24 h of RL irradiation (more than 3.5 times compared to the initial value), and the most significant change in the composition of carotenoids occurred when the fruit was irradiated with FRL for 72 h. The capsaicin alkaloid content increased markedly after 72 h of FRL irradiation (more than 8 times compared to the initial value). It was suggested that decrease in the activity of phytochromes due to a low temperature or FRL may result in an increase in the expression of the PAL and CAM genes.

MYB24 Negatively Regulates the Biosynthesis of Lignin and Capsaicin by Affecting the Expression of Key Genes in the Phenylpropanoid Metabolism Pathway in Capsicum chinense.

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.

Genome-Wide Analysis of the MYB-Related Transcription Factor Family in Pepper and Functional Studies of CaMYB37 Involvement in Capsaicin Biosynthesis.

Chili pepper is an important economic vegetable worldwide. MYB family gene members play an important role in the metabolic processes in plant growth and development. In this study, 103 pepper MYB-related members were identified and grouped into nine subfamilies according to phylogenetic relationships. Additionally, a total of 80, 20, and 37 collinear gene pairs were identified between pepper and tomato, pepper and Arabidopsis, and tomato and Arabidopsis, respectively. We performed promoter cis-element analysis and showed that CaMYB-related members may be involved in multiple biological processes such as growth and development, secondary metabolism, and circadian rhythm regulation. Expression pattern analysis indicated that CaMYB37 is significantly more enriched in fruit placenta, suggesting that this gene may be involved in capsaicin biosynthesis. Through VIGS, we confirmed that CaMYB37 is critical for the biosynthesis of capsaicin in placenta. Our subcellular localization studies revealed that CaMYB37 localized in the nucleus. On the basis of yeast one-hybrid and dual-luciferase reporter assays, we found that CaMYB37 directly binds to the promoter of capsaicin biosynthesis gene AT3 and activates its transcription, thereby regulating capsaicin biosynthesis. In summary, we systematically identified members of the CaMYB-related family, predicted their possible biological functions, and revealed that CaMYB37 is critical for the transcriptional regulation of capsaicin biosynthesis. This work provides a foundation for further studies of the CaMYB-related family in pepper growth and development.

Multiomics analyses reveal high temperature-induced molecular regulation of ascorbic acid and capsaicin biosynthesis in pepper fruits

Heat stress (HS) is one of the most severe abiotic stressors that affects pepper fruit development, coloration, nutritional quality and yield. However, the molecular basis of pepper fruit response to HS has not been extensively studied. In this study, we performed integrated transcriptomic, proteomic and metabolomic analyses in pepper fruits subjected to HS (36 ℃) for 6 h, 12 h and 24 h. A total of 14513 differentially expressed genes (DEGs), 1999 differentially abundant proteins (DAPs), and 39 differentially accumulated metabolites (DAMs) were identified in response to heat treatments. Molecular function, regulatory network, and metabolic pathway analyses revealed that the DEGs and DAPs in heat-stressed fruits were mainly attributed to photosynthesis, response to stimulus, carbohydrate metabolic process, and protein folding process. Interestingly, among the DAMs, the content of capsaicin and ascorbic acid (Vitamin C), which are important phytochemical substances of pepper as food, was substantially impaired by HS. By integrated analysis of the genes, proteins and intermediate metabolites related to the biosynthesis pathway of capsaicin and ascorbic acid, we found that transcripts of most of the capsaicin biosynthesis genes are positively correlated to capsaicin content, while there is no obvious correlation between the biosynthesis genes and the content of ascorbic acid. Furthermore, by analyzing the promoter sequence, we found 3 transcription factors that could potentially regulate the expression of key genes in the capsaicin biosynthesis pathway in response to HS. Our research here provides a better understanding of the molecular mechanism of pepper fruit response to HS, and also provides a theoretical basis for the pre-harvest temperature management to harvest high-quality pepper fruits.

Vanillin reduction in the biosynthetic pathway of capsiate, a non-pungent component of Capsicum fruits, is catalyzed by cinnamyl alcohol dehydrogenase

Capsicum fruits synthesize capsaicin from vanillylamine, which is produced from vanillin in a reaction catalyzed by a putative aminotransferase (pAMT). Capsiate, a non-pungent compound that is structurally similar to capsaicin, is synthesized from vanillyl alcohol rather than vanillylamine. Vanillyl alcohol is possibly generated by the enzymatic reduction of vanillin, but the enzyme responsible for this reaction is unknown. In the present study, we revealed that the vanillin reductase in the capsiate biosynthetic pathway is cinnamyl alcohol dehydrogenase (CAD), which is an enzyme involved in lignin synthesis. The reduction of vanillin to vanillyl alcohol was greater in the mature red fruit placental extract than in the immature green fruit placental extract. This reduction was suppressed by both N-(O-hydroxyphenyl) sulfinamoyltertiobutyl acetate, a specific inhibitor of CAD, and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The CaCAD1 transcript levels in the placenta were higher in the red fruits than in the green fruits. A recombinant CaCAD1 protein obtained using an Escherichia coli expression system reduced vanillin to vanillyl alcohol. This reaction was suppressed by the CAD inhibitors. These results strongly suggest that CAD is the enzyme that catalyzes the reduction of vanillin to vanillyl alcohol during capsiate biosynthesis. Syntenic analyses indicated that genes encoding CAD and capsaicin synthase (Pun1) involved in capsiate biosynthesis were acquired before the pAMT gene during the evolution of the family Solanaceae. This raises the possibility that in the genus Capsicum, the capsiate biosynthetic pathway emerged before the pAMT-encoding gene was acquired as the final trigger for capsaicin biosynthesis.

Analyses of Pepper Cinnamoyl-CoA Reductase Gene Family and Cloning of CcCCR1/2 and Their Function Identification in the Formation of Pungency

Cinnamoyl-CoA reductases (CCR) have a possible role in pungency formation of pepper because they can convert feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA into lignin, which are also competitive precursors of capsaicin biosynthesis in phenylpropanoid metabolism. In this study, genome-wide CCR gene family, exon–intron structures, sequence homology, phylogenetic characterization, and promoters were analyzed in pepper. Two CCR genes were cloned from Capsicum chinense, their enzymic kinetic parameters and regulatory function were identified by heterologous expression, ectopic expression, and VIGS. In total, 38 genes were found as predicted CCRs or CCR-like proteins and were composed of 2–10 exons. The promoters of pepper CCRs contained growth, stress, hormone, and light-response elements. The affinity and catalytic efficiency of CcCCR1/2 to feruolyl-CoA was the highest. The analysis of metabolic substances showed that capsaicin content was negatively correlated with lignin and positively correlated with flavonoids. The highest expression of CcCCR1 was found in stems, the higher expression of CcCCR2 was found in stem and early fruit than other organs. CCR1, 2 had certain effects on capsaicin content by regulating related enzyme activity, CCR2 played a more important role in regulating pungency formation. Our results clarify the competitive mechanism between lignin and capsaicin biosynthesis and provide an explanation for spice regulation.

Identification and expression analysis of capsaicin biosynthesis pathway genes at genome level in Capsicum chinense

Capsicum chinense is one of the best ingredients for making chili sauces and extracting capsaicin. In this study, we identified 59 genes associated with shikimic acid, phenylpropane, unsaturated fatty acids, phenylalanine, valine, leucine, isoleucine and other metabolic pathways based on pepper genome. In the present study, 58 of the 59 genes involved in capsaicin synthesis were found to be unevenly distributed on 11 chromosomes, and several genes with the same number of exons and high homology were found to be clustered in a duplication region of the chromosome. The promoter regions of these genes were predicted and analyzed, and multiple gene promoters were found to contain MYB binding sites, which provided a reference for the subsequent exploration of MYB target genes. At the same time, the expression levels of genes related to capsaicin synthesis were analyzed based on different transcriptomes, and it was found that these genes showed significant differences in different plant development stages and different tissues. The results of this study will be helpful to further study the synthesis mechanism of capsaicin in Capsicum chinense and the functional relationship between genes.

Genome-wide identification, characterisation, and expression of C3HC4-type RING finger gene family in Capsicum annuum L

ABSTRACT C3HC4-type RING finger gene family is one of the largest, ubiquitous, and diverse proteins, involved in positive regulation of stress response, and enhances drought and salt stress tolerance. The genome-wide characterisation and biological functions of this family protein are well characterised in several plants and animals. However, their characterisation and functions in Capsicum and its related species are unknown. In this study, we identified 16 C3HC4-type RING finger family genes in pepper (Capsicum annuum L). Moreover, chromosome localisation, gene structure, phylogenetic relationships, conserved domain analysis, and cis-element analysis were carried out. In silico expression analysis was carried out to determine the level of expression of these genes in different tissues of pepper. Moreover, RT-qPCR analysis validated that CaRHC1, CaRHC7, and CaRHC13 are upregulated, while CaRHC3, CaRHC4, and CaRHC6 are downregulated in various tissues of pepper. Upregulation and downregulation of few candidate C3HC4-type RING finger genes in the placenta and pericarp of pepper indicates their possible role in capsaicin biosynthesis and pungency, which needs to be investigated further. Thus, the genome-wide analysis provides a comprehensive understanding of C3HC4-type RING finger gene family in Capsicum genome and it shall help to design effective strategies for functional analysis of these genes.