Recent Understanding of the Biosynthesis of Capsaicinoids and Low-pungent Analogs towards Quality Improvement of Chili Pepper

Abstract

Chili pepper (Capsicum spp.) is an economically important crop used as a spice and vegetable. The striking feature of chili pepper fruits is their pungency, which is attributed to capsaicin and its analogs (collectively called capsaicinoids). Capsaicinoids are unique compounds synthesized only in Capsicum fruits, and their biosynthesis exhibits temporal- and spatial-specific patterns in the placental septum tissue. Capsaicinoids are biosynthesized through the condensation of vanillylamine (produced in the phenylpropanoid pathway) and a fatty acid moiety (produced from branched amino acids). Capsaicinoids have beneficial bioactivities, such as thermogenetic and anti-obesity properties, and thus they are regarded as health-promoting compounds. Furthermore, low-pungent capsaicinoid analogs (capsinoids and capsiconinoids) have been discovered in chili pepper fruits. They exhibit similar physiological activities to pungent capsaicinoids and are promising compounds with high economic value because of their low-pungency. Huge variation in capsaicinoid and analog contents among Capsicum accessions has been observed. Research using various Capsicum accessions has identified important genetic factors in the biosynthesis of capsaicinoids and low-pungent analogs. This review provides an overview regarding the biosynthesis of capsaicin and its analogs. Furthermore, future options to enrich the quality of chili pepper fruits are discussed.

Introduction

Chili pepper (Capsicum spp.) is an important fruit crop used as a spice and vegetable. The genus Capsicum is a member of the Solanaceae family and includes over 30 species (Carrizo Garcia et al., 2016). There are five cultivated species in the Capsicum genus: C. annuum, C. chinense, C. baccatum, C. frutescens, and C. pubescens (Bosland and Votava, 2000). C. annuum is the most economically important species, and it includes pungent and bell-type sweet peppers. C. frutescens is semi-wild species used as an important spice in Southeast Asia and Micronesia (Jarret et al., 2007; Yamamoto and Nawata, 2004). C. chinense includes highly pungent accessions such as ‘Habanero’ and ‘Jolokia’ (Bosland and Baral, 2007; Bosland et al., 2012; Canto-Flick et al., 2008). C. baccatum and C. pubescens are called aji and rocoto, respectively, and are cultivated mainly in South America (Kollmannsberger et al., 2011).

The prominent characteristic of chili pepper fruits is a burning sensation when eaten, which is caused by capsaicin and its analogs, collectively known as capsaicinoids (Aza-Gonzalez et al., 2011). The capsaicinoid content determines the pungency intensity, and there is huge variation in capsaicinoid content among Capsicum accessions, ranging from non-pungent to extremely pungent (Bosland and Votava, 2000; Wahyuni et al., 2011). Capsaicinoids have beneficial bioactivity in humans, such as the promotion of thermogenesis and an anti-obesity effect, and thus they are regarded as health-promoting compounds (Wang et al., 2022). Furthermore, capsinoid, a low-pungent capsaicinoid analog, was discovered through research on the low-pungent strain ‘CH-19 Sweet’ (Kobata et al., 1998; Yazawa et al., 1989). Capsinoid has similar physiological activities to pungent capsaicinoids. Capsinoids are promising components with high economic value because of their low pungency (Luo et al., 2011; Uarrota et al., 2021). If we can genetically control the composition and content of capsaicinoids and analogs, we can increases the quality and value of chili pepper fruits and their health-promoting properties. Basic knowledge of capsaicinoid biosynthesis will contribute to breeding cultivars with high added value, and expand the potential to develop new food products using chili pepper fruits.

This article provides an overview of the current situation regarding the biosynthesis of pungent components, capsaicinoids and low-pungent analogs, and future options to improve the quality of chili pepper fruits are discussed.

Pungent capsaicinoid components and their biosynthesis

Capsaicinoids are an acid amide of vanillylamine with a branched fatty acid moiety (C9–11). Although capsaicinoids have been studied since the 1800s, their chemical structure was only established in 1919 (Nelson, 1919). Approximately 20 capsaicinoid analogs with different fatty acids have been isolated in chili pepper (Mazourek et al., 2009). Capsaicin and dihydrocapsaicin are dominant in chili pepper fruits, accounting for 90% of total capsaicinoids (Aza-Gonzalez et al., 2011; Fig. 1A).

Fig. 1

Pungent capsaicinoids and their biosynthesis in chili pepper fruit. (A) Chemical structure of capsaicin and dihydrocapsaicin. (B) Transection of chili pepper fruit. (C) Tissue section of the placental septum. Capsaicinoid-producing slender cells are distributed throughout the epidermis. bl: capsaicinoid blister.

Capsaicinoids are unique compounds synthesized only in the Capsicum genus, and their biosynthesis exhibits temporal- and spatial-specific patterns (Arce-Rodríguez and Ochoa-Alejo, 2019; Aza-Gonzalez et al., 2011). Their biosynthesis occurs actively in the mature green fruit stage (approximately 30 days after flowering [DAF]). Chili pepper fruits grow over 30 DAF, and then the fruits mature up to 40 DAF. Capsaicinoid biosynthesis begins around 15 DAF, and the capsaicinoid content increases during fruit development (Iwai et al., 1979). As the fruit matures, capsaicinoid biosynthesis is downregulated. Biosynthesis is also locally restricted to the placental septum within the fruit (Fig. 1B). Slender epidermal cells develop in the placental septum, and these are thought to be the site of capsaicinoid biosynthesis (Stewart et al., 2007; Suzuki et al., 1980; Fig. 1C). In epidermal cells, the accumulation of capsaicinoids in vacuoles has been observed (Fujiwake et al., 1980). Capsaicinoids are transported extracellularly and accumulate in the inter-cuticular space, resulting in the formation of blisters on the surface of the placental septum.

Although the placental septum is the main location of capsaicinoid production, recent studies on highly pungent accessions of C. chinense such as ‘Jolokia’ and ‘Moruga Scorpion’ revealed that biosynthesis occurs not only in the placental septum, but also in the pericarp (Bosland et al., 2015; Tanaka et al., 2017). In the pericarp of the highly pungent accessions, the transcriptome profile and morphological characteristics are like that of the placental septum, such as upregulation of capsaicinoid biosynthesis-related genes and development of slender epidermal cells and spongy-like parenchymal tissue (Sugiyama, 2017; Tanaka et al., 2021). This suggests that capsaicinoid biosynthesis in the pericarp arises with an ambiguous boundary at the placental septum and pericarp. Although the placental septum accounts for only 15% of the whole fruit, the pericarp makes up the major portion of chili pepper fruit. In the most pungent cultivars, even though capsaicinoids are highly accumulated in the placental septum, the concentration in the whole fruit is diluted by other tissues, such as the pericarp and seeds. In extremely pungent accessions, biosynthesis in both the placental septum and pericarp achieve high capsaicinoid concentrations in the whole fruit (Tanaka et al., 2017). Capsaicinoid biosynthesis in the pericarp may be an efficient strategy to enhance capsaicinoid concentration in whole fruit.

It has been established that capsaicinoids are biosynthesized through condensation of vanillylamine (produced in the phenylpropanoid pathway) and a fatty acid moiety (produced from branched amino acids), and numerous genes related to their biosynthesis have been reported (Arce-Rodríguez and Ochoa-Alejo, 2019; Venkatesh et al., 2023; Fig. 2). Early radiotracer studies showed that vanillylamine was synthesized from phenylalanine, and branched fatty acids are produced from valine or leucine (Bennett and Kirby, 1968; Leete and Louden, 1968; Suzuki et al., 1981). The phenylpropanoid pathway provides the sequential production of phenylalanine, shikimic acid, p-coumaric acid, caffeic acid, and ferulic acid. These reactions are mediated by phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), coumaric acid 3-hydroxylase (C3H), and caffeic acid O-methyl transferase (COMT) (Curry et al., 1999; Fujiwake, 1982a, b; Mazourek et al., 2009). Then, vanillin is synthesized from ferulic acid. Hydroxycinnamoyl-CoA hydratase/lyase (HCHL) has been proposed to be involved in vanillin synthesis from ferulic acid. Putative aminotransferase (pAMT) catalyzes the production of vanillylamine, a direct precursor of capsaicinoids, from vanillin (Curry et al., 1999). A recent study shown that pAMT is a member of the Solanaceae cytoplasmic GABA-Ts, and it appears to have evolved as a specific gene that can contribute to efficient capsaicinoid production in chili pepper fruit, by developing specific expression patterns and high catalysis efficiency for vanillin (Kusaka et al., 2024). A detailed biochemical study demonstrated that pAMT has substrate specificity for vanillin (Kato and Nomura, 2024; Nakaniwa et al., 2024; Sano et al., 2023). It has been proposed that pAMT should be named as vanillin aminotransferase (VAMT) (Nakaniwa et al., 2024; Weber et al., 2014). In the branched chain fatty acid pathway, branched-chain amino acid transferase (BCAT), branched-chain α-ketoacid dehydrogenase (BCKDH), ketoacyl-ACP synthase (KAS), ketoacyl-ACP reductase (KR), acyl-ACP thioesterase (FatA), and acyl-CoA synthetase (ACS) are involved in the production of C9–11 branched chain fatty acid from valine or leucine (Aluru et al., 2003; Mazourek et al., 2009). Finally, capsaicinoids are synthesized by the condensation of vanillylamine and a fatty acid. Pun1 encodes an acyltransferase called AT3 (also known as Capsaicin synthase (CS)) to form different capsaicinoid analogs depending on branched fatty acid moiety (Stewart et al., 2005). Capsaicin is formed by the combination of vanillylamine and 8-methyl-6-nonenoyl-CoA. Focusing on the correlation between capsaicinoid accumulation and gene expression pattern, numerous genes have been reported as capsaicinoid biosynthesis-related genes (Liu et al., 2013; Zhang et al., 2016). However, only a few of them have been validated to be involved in biosynthesis using mutants and Virus Induced Gene Silencing (VIGS) (Abraham-Juárez et al., 2008; Arce-Rodríguez et al., 2015, 2017; Han et al., 2019; Koeda et al., 2019; Lang et al., 2009; Stewart et al., 2005). Further research to validate the function of each candidate gene will lead to a clearer overview of the capsaicinoid biosynthesis pathway.

Fig. 2

Schematic overview of the capsaicinoid biosynthesis pathway. PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; HCT, hydroxycinnamoyl transferase; C3H, coumaric acid 3-hydroxylase; COMT, caffeic acid O-methyl transferase; HCHL, hydroxycinnamoyl-CoA hydratase/lyase; pAMT, putative aminotransferase (VAMT: vanillin aminotransferase); BCAT, branched-chain amino acid transferase; BCKDH, branched-chain α-ketoacid dehydrogenase; KAS III, ketoacyl-ACP synthase III; KR, ketoacyl‐ACP reductase; DH, hydroxyacyl-ACP dehydratase; ENR, enoyl-ACP reductase; KASI, ketoacyl-ACP synthase I; FAT, acyl-ACP thioesterase; ACS, acyl-CoA synthetase; AT3, acyltransferase3.

Low-pungent capsaicinoid analogs in chili pepper fruit

Pungent capsaicinoids have various beneficial health effects such as anti-obesity and thermogenic actions (Wang et al., 2022). These physiological effects are reported to be due to the activation of transient receptor potential vanilloid 1 (TRPV1) (Caterina et al., 1997; Srinivasan, 2016). In order to activate TRPV1 and obtain physiological effects, it is necessary to ingest a sufficient amount of capsaicinoids. However, it is not favorable to consume capsaicinoids in large amounts, because they have strong pungency and induce nociceptive stimulus.

Capsiate is a low-pungent capsaicinoid analog, which was first discovered from a low-pungent mutant strain ‘CH-19 Sweet’ (Kobata et al., 1998; Yazawa et al., 1989). Capsiate has an ester bond instead of an amide bond in capsaicin. Similar to capsaicinoids, capsiate analogs with different fatty acids have been identified from ‘CH-19 Sweet’, and the group has been named capsinoids (Kobata et al., 1999; Fig. 3). Capsinoids have about 1000 times lower pungency levels compared with capsaicinoids. Despite their low pungency, they have similar physiological effects as capsaicinoids. They exhibit similar agonistic activity to capsaicinoids for TRPV1 receptors (Iida et al., 2003), and various beneficial health effects have been reported including thermogenic, antioxidant, anti-inflammatory, anti-diabetic, and anti-obesity activities (Luo et al., 2011; Uarrota et al., 2021). Capsinoids are more palatable as an ingredient in vegetables and in supplements compared with capsaicinoids. Low pungency in the oral cavity can be explained by capsinoid not reaching the nerves under the epithelial cell layer because of its high lipophilicity or hydrolysis (Iida et al., 2003; Sasahara et al., 2010). Capsiate activates TRPV1 receptors in the gut, but not in the oral cavity, and the activation of TRPV1 in the gut triggers physiological effects such as thermogenesis and the promotion of fat metabolism in humans (Ohyama et al., 2016; Zsiborás et al., 2017). It has also been reported that capsinoids are non-toxic or less toxic than capsaicinoids (Watanabe et al., 2011). Although capsaicinoids are stable in any solvent independent of polarity, capsinoids are hydrolyzed in polar solvents such as water and alcohol (Sutoh et al., 2001). Prior to capsinoids, several capsaicinoid analogs with low pungency such as olvanil, palvanil, and arvanil were developed as artificial anti-nociceptive agents (Alsalem et al., 2016). Capsiate was the first naturally occurring capsaicin analog with low pungency. Capsinoids are considered to have high economic value and greater utility in foods and medicines, so they are used commercially in supplements for people who desire anti-obesity effects.

Fig. 3

Capsaicinoid and its low-pungent analogs.

Although most pungent accessions contain small amounts of capsinoid, ‘CH-19 Sweet’ contains predominantly capsinoid with traces of capsaicinoid (Kobata et al., 1998; Yazawa et al., 1989). Molecular genetic analysis of ‘CH-19 Sweet’ revealed that capsinoid production is caused by loss-of-function of pAMT (Lang et al., 2009), which suppresses the production of vanillylamine from vanillin, and vanillyl alcohol is produced instead. This results in a significant accumulation of capsinoids (Kobata et al., 2013; Sutoh et al., 2006; Fig. 4). Pun1 is involved in the condensation of vanillyl alcohol and a branched fatty acid moiety to form capsinoid (Han et al., 2013). In addition to ‘CH-19 Sweet’, several low-pungent accessions, such as ‘Himo’ and ‘Aji Dulce’, have been reported to accumulate capsinoids and possess loss-of-functional pAMT alleles (Tanaka et al., 2010a, b, 2015). Such a loss-of-function mutation may be useful for chili pepper breeding programs aimed at increasing capsinoid content (Jeong et al., 2015; Tanaka et al., 2014). A recent study demonstrated that cinnamyl-alcohol dehydrogenase (CAD) is responsible in the biosynthesis of vanillyl alcohol from vanillin in capsinoid biosynthesis (Sano et al., 2022). CAD is an enzyme gene involved in lignin biosynthesis and is expressed ubiquitously in chili pepper plants. On the contrary, pAMT is specifically expressed in the placental septum, and its expression pattern is closely related to capsaicinoid biosynthesis (Curry et al., 1999; Tanaka et al., 2017). Capsinoids may be regarded as byproducts of the capsaicinoid biosynthesis pathway, resulting from the metabolism of vanillin by the ubiquitous enzyme CAD (Fig. 4).

Fig. 4

Capsaicinoid and capsinoid biosynthesis in chili pepper fruit. pAMT, putative aminotransferase; VAMT, vanillin aminotransferase; CAD, cinnamyl alcohol dehydrogenase. Mutations in pAMT (VAMT) reduce the content of vanillylamine and capsaicinoid, simultaneously causing the accumulation of low-pungent capsaicinoid analogs, termed capsinoids, instead of capsaicinoids. CAD catalyzes the conversion of vanillin to vanillyl alcohol.

Capsiconinoids constitute another group of low-pungent analogs. Capsiconiate (coniferyl (E)-8-methyl-6-nonenoate) and dihydrocapsiconiate (coniferyl 8-methylnonanoate) were isolated from fruits of ‘CCB’ (Capsicum baccatum var. praetermissum), and collectively named capsiconinoids (Kobata et al., 2008; Fig. 3). Capsiconinoids are esters of coniferyl alcohol and a branched chain fatty acid; its fatty acid chain side is common to capsaicinoids and capsinoids. Capsiconinoids also have almost no pungency, but they have the effect of activating TRPV1 receptors. The antagonistic activity of capsiconinoids is weaker than those of capsaicinoids and capsinoids (Kobata et al., 2008). Capsiconinoids can be found in many chili pepper accessions, but they are present in lower amounts compared with capsaicinoids and capsinoids. Particularly pungent accessions such as ‘CCB’ and ‘Charapita’ contain exceptionally high levels of capsiconinoids (Tanaka et al., 2009). Coniferyl alcohol is considered to be a precursor, but the biosynthetic mechanism is not well understood.

Genetic factors controlling pungency

A mutation for non-pungency

Classical genetic studies have demonstrated that pungency is qualitatively controlled by a single dominant locus C (=Pun1). Genetic mapping has shown that Pun1 encodes the acyltransferase gene AT3 (Stewart et al., 2005). AT3 is the enzyme responsible for the condensation of vanillylamine and fatty acids in the final step of the capsaicinoid biosynthetic pathway. Pun1 is composed of two exons. A recessive Pun1 allele (pun1¹) has a large deletion spanning from the promoter region to exon 1, resulting in the loss of gene function (Stewart et al., 2005; Table 1). The recessive homozygote (pun1¹/pun1¹) exhibited complete absence of capsaicinoids. The pun1 allele is widely distributed among many bell-type and giant sweet pepper cultivars (Lee et al., 2005). DNA marker-assisted selection based on Pun1 is useful to select non-pungent individuals in breeding programs. In Japan, the pun1¹ allele is utilized to stabilize non-pungency in breeding programs of unstable non-pungent strains such as ‘Shishito’, which occasionally bear pungent fruits (Minamiyama et al., 2012; Tanaka et al., 2022). In addition to pun1¹, several loss-of-function alleles of Pun1 (pun1^2–5) have been reported (Kirii et al., 2017; Stellari et al., 2010; Stewart et al., 2007; Yamaguchi et al., 2024; Table 1). Although pun1¹ is widely present in non-pungent strains of C. annuum, pun1^2–5 are found only in specific accessions. Thus, the distribution of pun1^2–5 appears to be limited among chili pepper genetic resources.

Table 1

Mutant alleles affecting capsaicinoid biosynthesis and their structural features.

Another genetic factor for non-pungency is loss-of-function mutations in pAMT. Such mutations in pAMT cause a dramatic decrease in capsaicinoid content and lead to the accumulation of capsinoids (Lang et al., 2009). It has been noted that loss-of-function pamt mutants still contain very low amounts of capsaicinoid and exhibit low-pungency, whereas the pun1 mutation lacks pungency completely. The first loss-of-function allele of pAMT was derived from ‘CH-19 Sweet’, and pamt¹ has a frameshift mutation due to a single nucleotide insertion in exon 16 (Lang et al., 2009). Another pamt allele was found in ‘Himo’, which is a landrace in Nara prefecture, Japan (Tanaka et al., 2010a). A single nucleotide polymorphism in exon 8 leads to an amino acid substitution in the PLP binding domain, which is important for aminotransferase activity. To date, 11 loss-of-function alleles have been reported (Park et al., 2015; Tsurumaki and Sasanuma, 2019; Table 1). The Pun2 locus was found to be the locus related to non-pungency in C. chacoense, and recently it was revealed that pun2 encodes a loss-of-function allele of pAMT (Yi et al., 2022). It is notable that C. chinense includes diverse mutant alleles of pamt (Koeda et al., 2014; Tanaka et al., 2010b, 2015, 2018). Although C. chinense is characterized by high pungency, low-pungent accessions called ‘Aji Dulce’ (‘Sweet chili pepper’ in Spanish) are consumed in the Caribbean. The low-pungent C. chinense accessions commonly produce capsinoids, but they have different loss-of-function pamt alleles. Tcc family transposon insertion and excision are involved in the generation of loss-of-function alleles of pamt in C. chinense (Jang et al., 2015; Koeda et al., 2014; Tanaka et al., 2010b, 2015, 2018). In addition to loss-of-function alleles, two leaky pamt alleles (pamt^L1 and pamt^L2) with different levels of pAMT activity have been found (Tanaka et al., 2019). Notably, both alleles have a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron are different. pamt^L1, pamt^L2, and loss-of-function pamt alleles reduce capsaicinoid levels to about 50%, 10%, and less than 1%, respectively. The intronic transposons disrupt splicing in intron 3, which results in simultaneous expression of functional pAMT mRNA and non-functional splice variants containing partial sequences of Tcc. The difference in position of the intronic transposons may alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid contents to different levels (Tanaka et al., 2019). The allelic variation in pAMT can be utilized to adjust pungency levels quantitatively in Capsicum breeding.

The third factor is a loss-of-function mutation in KR1, which was identified in the non-pungent strain ‘No. 3341’ (Koeda et al., 2019). KR1 encodes ketoacyl-ACP reductase and is thought to be involved in branched chain fatty acid biosynthesis. The loss-of-function mutation of KR1 is caused by a transposon insertion in intron 1, and this allele has been recognized in several non-pungent C. chinense accessions, such as ‘No. 3327’, ‘No. 4026’, and ‘No. 4028’ (Koeda et al., 2020).

MYB31 has been reported as a transcription factor that activates capsaicin biosynthesis, and genetic analysis of the Pun3 locus in ‘YCM334’ revealed that loss-of-function in MYB31 leads to non-pungency (Han et al., 2019). Compared with pungent accessions, multiple capsaicin biosynthesis-related genes were down-regulated in ‘YCM334’.

As described above, mutations in four genes (Pun1, pAMT, KR1, and MYB31) are known to be responsible for non-pungency. In the future, the identification of novel genes responsible for non-pungency will lead to a more comprehensive understanding of the capsaicinoid biosynthesis pathway.

QTLs related to capsaicinoid content

Among the pungent accessions, pungency levels can vary, ranging from low varieties that exhibit only slight pungency to extremely pungent varieties. Capsaicinoid content is said to be determined by multiple quantitative genes and environmental factors. More than 20 QTLs associated with capsaicinoid content have been reported on 8 chromosomes (1, 2, 3, 4, 6, 7, 10, and 11) (Ben-Chaim et al., 2006; Blum et al., 2003; Han et al., 2018; Hill et al., 2017; Nimmakayala et al., 2016; Park et al., 2019; Yarnes et al., 2013). One of the most well-studied major QTLs is the QTL located on chromosome 7 (Cap1/cap7.2) (Ben-Chaim et al., 2006). Cap1/cap7.2 has been detected in an interspecific hybrid population of C. annuum ‘Maor’ and C. frutescens BG2816 (Ben-Chaim et al., 2006; Blum et al., 2003), which also corresponds to the non-pungency causative locus Pun3 (Han et al., 2019). The gene responsible for Cap1 has been identified as MYB31 (Zhu et al., 2019). In C. chinense, the MYB31 promoter region has a W- box sequence that is not found in other domesticated species. Binding of the transcription factor WRKY9 to the W-box increases the expression level of MYB31, which contributes to the higher capsaicinoid content in C. chinense compared with other domesticated species (Zhu et al., 2019). In crossing populations using C. chinense ‘Jolokia’, QTLs related to capsaicinoid levels were found on chromosomes 2, 3, 6, and 11 (Lee et al., 2016; Park et al., 2019). Among them, the QTL on chromosome 6 is the major QTL related to higher capsaicinoid content in the pericarp (Park et al., 2019). The QTL region includes C4H, KR, FAT, and an ankyrin-repat-containing protein. Han et al. (2018) found 5 common QTLs in chromosomes 1, 2, 3, 4, and 10 by QTL mapping using two recombinant inbred line (RIL) populations. Genome-wide association study (GWAS) analysis using 208 accessions identified 69 QTLs, and 10 of them were colocalized with QTLs detected in the RIL populations (Han et al., 2018). The QTLs include genes involved in capsaicinoid biosynthesis, such as pAMT, C4H, caffeoyl shikimate esterase (CSE), 4CL, and FatA. Genetic analysis of an intraspecific crossing population of C. annuum ‘Takanotsume’ × ‘Shishito’ detected QTLs related to capsaicinoid content on chromosomes 3 and 7 (Kondo et al., 2023). The G2P-SOL project has constructed a core collection of 423 accessions from over 10,000 strains derived from 10 gene banks, and GWAS analysis detected loci involved in the presence or absence of pungency in chromosomes 2 and 7 (McLeod et al., 2023), suggesting that MYB63 and MYB61 are associated with pungency. Overall, although QTLs have been reported, the causative gene mutations have not yet been analyzed in detail with the exception of a few cases. Determining the mechanism of quantitative variation in capsaicinoid content remains an area for future studies.

Perspectives

Capsaicinoid content determines the fruit quality of chili peppers, and the preferred capsaicinoid level can vary greatly depending on region, individual, and utilization. Adjusting the capsaicinoid content to meet needs is important when breeding chili peppers. Low-pungent analogs contain desirable health-promoting ingredients, and their enrichment will lead to further improvements in fruit quality. Genetic analysis of non-pungent accessions has identified pun1 and additional genes responsible for non-pungency, which has made it easy to breed non-pungent chili peppers using marker-assisted selection (Wyatt et al., 2012). Regarding capsinoids, the content can be increased by using loss-of-functional pAMT alleles (Jeong et al., 2015; Seki et al., 2020; Tanaka et al., 2014). However, it is still challenging to control the content quantitatively in a tailored manner (Venkatesh et al., 2023). Identification of potential genetic resources and important new QTLs related to capsaicinoid level, and detailed analysis to identify causative genes will contribute to tailor-made breeding related to capsaicinoid and capsinoid levels. Another attempt has also been made to estimate pungency levels using a genome prediction method, which has achieved a certain degree of accuracy (Kim et al., 2022).

Capsaicinoid production is restricted to placental septum tissues (Stewart et al., 2007; Sugiyama et al., 2006), and the content can fluctuate depending on the fruit development stage and cultivation environment, even in for identical genotypes (Estrada et al., 1999; Kondo et al., 2021a, b; Murakami et al., 2006; Zewdie and Bosland, 2000). Numerous genes in the capsaicinoid biosynthesis pathway have been isolated, but their regulation is not well understood. The mechanisms underlying spatial and temporal specificity of capsaicinoid production remain an unresolved issue. The biochemical characterization and promoter analysis of each structural gene, and interactions between transcription factors and enzyme genes will contribute to a better understanding of the capsaicinoid biosynthetic pathway and will help stabilize or increase the content.

Another unresolved issue is an evolutionary aspect of capsaicinoid biosynthesis. Although comparative analyses between Capsicum and tomato have provided some insights into the evolutionary process of several structural genes (Kim et al., 2014; Qin et al., 2014), it remains uncertain how Capsicum established the capsaicinoid biosynthesis pathway during the evolution of Solanaceae (Naves et al., 2019). The available Solanaceae genomes have been updated, and it is possible to conduct comparative genomic analysis between Capsicum and multiple Solanaceae plants to investigate the evolutionary process of the capsaicinoid biosynthesis pathway. Better understanding of the mechanism underlying species-specific capsaicinoid biosynthesis may bring about innovation and the introduction of capsaicinoid biosynthesis to other plant species in the future (Naves et al., 2019).

The network between capsaicinoid synthesis and other metabolites is also of interest to improve overall fruit quality. The ester volatile and lignin contents tend to be higher in pungent fruits, compared with non-pungent fruits (Sukrasno and Yeoman, 1993; Wahyuni et al., 2013). Mutations for non-pungency also seem to affect other chemical compositions in fruit. The volatile ester content decreases in pun1 or kr1 mutants, but the pamt mutation did not influence volatile ester levels (Koeda et al., 2023). Further omics analyses, including transcriptomic and metabolomic analyses, will provide a clearer overview of the network between capsaicinoid biosynthesis and other metabolite production mechanisms in chili pepper fruits.

In future studies, various experimental approaches such as genetic analysis, enzyme characterization, promoter analysis, and omics analysis will enable us to deepen our basic understanding of capsaicinoid biosynthesis in chili pepper. Further research in the field may enable easier manipulation in capsaicinoids and low-pungent analogs production. This will lead to breeding of new cultivars with high market value and the development of new products, contributing to the development of horticultural and food industries related to chili peppers.

Acknowledgements

I would like to thank Dr. Kenji Kobata, Dr. Karoi Sano (Josai University), Dr. Ryuji Sugiyama (Tokyo University of Agriculture), Dr. Fumiya Kondo (JSPS Research Fellow (PD)) for their helpful comments to the manuscript. A series of research studies related to low-pungent analogs began from ‘CH-19 Sweet’ which Prof. Susumu Yazawa originally found. I would like to offer my gratitude and respect to him and for his work.

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