Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum.

A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC-MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

Koeda S ，Sato K ，Saito H ，Nagano AJ ，Yasugi M ，Kudoh H ，Tanaka Y ... -  《-》

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》

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 ... -  《-》

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 Pun1 gene for pungency in pepper encodes a putative acyltransferase.

Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

Stewart C Jr ，Kang BC ，Liu K ，Mazourek M ，Moore SL ，Yoo EY ，Kim BD ，Paran I ，Jahn MM ... -  《PLANT JOURNAL》