Biochemistry and molecular biology of capsaicinoid biosynthesis: recent advances and perspectives.

The most widely known characteristic of chili pepper fruits is their capacity to produce capsaicinoids, which are responsible for the pungent sensation. The capsaicinoids have several uses in different areas, such as the pharmaceutical, cosmetic and agronomic industries, among others. They are synthesized by the condensation of vanillylamine (derived from phenylalanine) with a branched-chain fatty acid (from valine or leucine precursors), and they generally accumulate in the placental tissue of the chili pepper fruits. The pungency grade depends on the genotype of the plant but is also affected by external stimuli. In recent years, new structural and regulatory genes have been hypothesized to participate in the capsaicinoid biosynthetic pathway. Moreover, the role of some of these genes has been investigated. Substantial progress has been made in discerning the molecular biology of this pathway; however, many questions remain unsolved. We previously reviewed some aspects of the biochemistry and molecular biology of capsaicinoid biosynthesis (Aza-González et al. Plant Cell Rep 30:695-706. Aza-González et al., Plant Cell Rep 30:695-706, 2011), and in this review, we describe advances made by different researchers since our previous review, including the contribution of omics to the knowledge of this pathway.

Arce-Rodríguez ML ，Ochoa-Alejo N 《-》

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.).

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.

Aza-González C ，Núñez-Palenius HG ，Ochoa-Alejo N 《-》

Difference in capsaicinoid biosynthesis gene expression in the pericarp reveals elevation of capsaicinoid contents in chili peppers (Capsicum chinense).

This research reveals that the up-regulated expression of multiple capsaicinoid biosynthetic genes in pericarp tissue leads to the elevation of total capsaicinoid content in chili pepper fruit. Capsaicinoids are health-functional compounds that are produced uniquely in chili pepper fruits. A high capsaicinoid level is one of the major parameters determining the commercial quality and health-promoting properties of chili peppers. To investigate the mechanisms responsible for its high contents, we compared an extremely pungent cultivar 'Trinidad Moruga Scorpion Yellow' (MY) with other cultivars of different pungency levels (Fushimi-amanaga, Takanotsume, Red Habanero). Capsaicinoid concentrations were markedly higher in MY fruit (23.9 mg/g DW) than in other pungent cultivars including 'Red Habanero' (HB) fruit (14.3 mg/g DW). Comparative analysis of MY and HB reveals that both cultivars accumulated similar capsaicinoid concentrations in the placental septum, with that in the HB pericarp (1.8 mg/g DW) being markedly lower than that in the placental septum (69.1 mg/g DW). The capsaicinoid concentration in HB fruit is dependent on the placental septum, as reported in other accessions. Therefore, even though placental septum tissue contains high capsaicinoid concentrations, those in the pericarp and seeds attenuated its total content. In contrast, the MY pericarp exhibited a markedly higher concentration (23.2 mg/g DW). A qRT-PCR analysis revealed that multiple capsaicinoid biosynthetic pathway genes (Pun1, pAMT, KAS, and BCAT) were strongly up-regulated in placental septum of pungent cultivars. The genes were expressed exclusively in the MY pericarp, but were barely detected in the pericarps of other pungent cultivars. Collectively, the present study indicates that the up-regulated expression of these genes not only in placental septum but also in pericarp plays an important role in driving capsaicinoid accumulation in the whole fruit.

Tanaka Y ，Nakashima F ，Kirii E ，Goto T ，Yoshida Y ，Yasuba KI ... -  《-》

The pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) provides insights regarding the relationship between pungency, the number of seeds, and gene expression involving capsaicinoid biosynthesis.

Pungent traits caused by capsaicinoids are characteristic of chili peppers (Capsicum spp.), and the pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) is unique in being known for the pungency in fruits with few seeds. In the present study, we tried to clarify the relationship between the number of seeds and pungency in 'Shishito'. First, we investigated the pungency of 'Shishito' by simple sensory evaluations and quantifications of capsaicinoids by high-performance liquid chromatography. As a result, few-seeded fruits had a larger fluctuation of capsaicinoid content than many-seeded ones. This indicates that the number of seeds, in particular a decrease of the seeds, has some sort of connection with the pungency of 'Shishito'. Then, we analyzed the relationship between pungency and gene expression involving capsaicinoid biosynthesis at the individual fruit level. We vertically separated the placental septum in which capsaicinoids are synthesized and performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 18 genes involved in capsaicinoid biosynthesis. We also used the placental septum for capsaicinoid quantification so that the gene expression levels and capsaicinoid contents in the same fruits were obtained, and their correlations were analyzed using 20 biological replicates. Among the 18 genes, expression levels of 11 genes (WRKY9, CaMYB31, AT3, BCAT, BCKDH, KAS I, KAS III, ACL, CaKR1, FAT, and pAMT) had a significant positive correlation with the capsaicinoid concentration, and they were considered to upregulate capsaicinoid biosynthesis. These results provide new insights regarding the environmental variation of the pungency traits in chili peppers and the relationship between pungency, the number of seeds, and gene expression involved in capsaicinoid biosynthesis.

Kondo F ，Hatakeyama K ，Sakai A ，Minami M ，Nemoto K ，Matsushima K ... -  《-》

Genetic control of pungency in C. chinense via the Pun1 locus.

Capsaicin, the pungent principle in hot peppers, acts to deter mammals from consuming pungent pepper pods. Capsaicinoid biosynthesis is restricted to the genus Capsicum and results from the acylation of the aromatic compound, vanillylamine, with a branched-chain fatty acid. The presence of capsaicinoids is controlled by the Pun1 locus, which encodes a putative acyltransferase. In its homozygous recessive state, pun1/pun1, capsaicinoids are not produced by the pepper plant. HPLC analysis confirmed that capsaicinoids are only found in the interlocular septa of pungent pepper fruits. Immunolocalization studies showed that capsaicinoid biosynthesis is uniformly distributed across the epidermal cells of the interlocular septum. Capsaicinoids are secreted from glandular epidermal cells into subcuticular cavities that swell to form blisters along the epidermis. Blister development is positively associated with capsaicinoid accumulation and blisters are not present in non-pungent fruit. A genetic study was used to determine if the absence of blisters in non-pungent fruit acts independently of Pun1 to control pungency. Screening of non-pungent germplasm and genetic complementation tests identified a previously unknown recessive allele of Pun1, named pun1(2). Sequence analysis of pun1(2) revealed that a four base pair deletion results in a frameshift mutation and the predicted production of a truncated protein. Genetic analysis revealed that pun1(2) co-segregated exactly with the absence of blisters, non-pungency, and a reduced transcript accumulation of several genes involved in capsaicinoid biosynthesis. Collectively, these results establish that blister formation requires the Pun1 allele and that pun1(2) is a recessive allele from C. chinense that results in non-pungency.

Stewart C Jr ，Mazourek M ，Stellari GM ，O'Connell M ，Jahn M ... -  《JOURNAL OF EXPERIMENTAL BOTANY》