Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper.

Capsaicinoids, including capsaicin and its analogs, are responsible for the pungency of pepper (Capsicum species) fruits. Even though capsaicin is familiar and used daily by humans, the genes involved in the capsaicin biosynthesis pathway have not been well characterized. The putative aminotransferase (pAMT) and Pungent gene 1 (Pun1) proteins are believed to catalyze the second to last and the last steps in the pathway, respectively, making the Pun1 protein the putative capsaicin synthase. However, there is no direct evidence that Pun1 has capsaicin synthase activity. To verify that the Pun1 protein actually plays a role in capsaicin production, we generated anti-Pun1 antibodies against an Escherichia coli-synthesized Pun1 protein and used them to antagonize endogenous Pun1 activity. To confirm the anti-Pun1 antibodies' specificity, we targeted Pun1 mRNA using virus-induced gene silencing. In the Pun1-down-regulated placental tissues, the accumulated levels of the Pun1 protein, which was identified on a western blot using the anti-Pun1 antibodies, were reduced, and simultaneously, capsaicin accumulations were reduced in the same tissues. In the de novo capsaicin synthesis in vitro cell-free assay, which uses protoplasts isolated from placental tissues, capsaicin synthesis was inhibited by the addition of anti-Pun1 antibodies. We next analyzed the expression profiles of pAMT and Pun1 in various pepper cultivars and found that high levels of capsaicin accumulation always accompanied high expression levels of both pAMT and Pun1, indicating that both genes are important for capsaicin synthesis. However, comparisons of the accumulated levels of vanillylamine (a precursor of capsaicin) and capsaicin between pungent and nonpungent cultivars revealed that vanillylamine levels in the pungent cultivars were very low, probably owing to its rapid conversion to capsaicin by Pun1 soon after synthesis, and that in nonpungent cultivars, vanillylamine accumulated to quite high levels owing to the lack of Pun1. Using a newly developed protoplast-based assay for de novo capsaicin synthesis and the anti-Pun1 antibodies, we successfully demonstrated that the Pun1 gene and its gene product are involved in capsaicin synthesis. The analysis of the vanillylamine accumulation relative to that of capsaicin indicated that Pun1 was the primary determinant of their accumulation levels.

Ogawa K ，Murota K ，Shimura H ，Furuya M ，Togawa Y ，Matsumura T ，Masuta C ... -  《BMC PLANT BIOLOGY》

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》

Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet.

Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH-19 Sweet is a non-pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH-19. CH-19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH-19 Sweet placenta tissue. RT-PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH-19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH-19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH-19 Sweet, and showed that the pAMT genotype co-segregated with the capsinoid or capsaicinoid fruit phenotype in the F(2) population. The T insertion was not found in other pungent and non-pungent Capsicum lines, suggesting that it is specific to CH-19 Sweet. CH-19 Sweet's pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid-containing fruits in pepper-breeding programs.

Lang Y ，Kisaka H ，Sugiyama R ，Nomura K ，Morita A ，Watanabe T ，Tanaka Y ，Yazawa S ，Miwa T ... -  《-》

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》

Assessment of Capsaicinoid and Capsinoid Accumulation Patterns during Fruit Development in Three Chili Pepper Genotypes (Capsicum spp.) Carrying Pun1 and pAMT Alleles Related to Pungency.

Quantification, using an accurate analytical approach, of capsinoids and capsaicinoids was performed on three chili pepper ( spp.) genotypes: "Chiltepı́n", "Tampiqueño 74", and "Bhut Jolokia" at various stages of fruit development. The accumulation of capsinoids, in all these peppers started between 10 to 20 days post-anthesis (dpa), increased and reached the highest capsinoid amount at 40 dpa, and then decreased until 60 dpa. Conversely, capsaicinoids could already be determined at 10 dpa in "Bhut Jolokia" and their accumulation pattern was different from that of the capsinoids in this genotype. The capsiate/dihydrocapsiate ratio presented a higher variation between genotypes and developmental stages than the capsaicin/dihydrocapsaicin ratio. Capsinoid ratios (4-24%) and / genotyping were determined. These results provide information on the progress of the accumulation of capsinoids in the aforementioned pungent and superhot cultivars and could support future breeding studies toward the understanding of the factors affecting their accumulation.

Fayos O ，Ochoa-Alejo N ，de la Vega OM ，Savirón M ，Orduna J ，Mallor C ，Barbero GF ，Garcés-Claver A ... -  《-》