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- Title
Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper.
- Authors
Kana Ogawa; Katsunori Murota; Hanako Shimura; Misaki Furuya; Yasuko Togawa; Takeshi Matsumura; Chikara Masuta
- Abstract
Background: 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. Results: To verify that the Pun1 protein actually plays a role in capsaicin production, we generated anti-Pun1 antibodies against an -synthesized Pun1 protein and used them to antagonize endogenous Pun1 Escherichia coli activity. To confirm the anti-Pun1 antibodies' specificity, we targeted mRNA using virus-induced gene silencing. In Pun1 the -down-regulated placental tissues, the accumulated levels of the Pun1 protein, which was identified on a Pun1 western blot using the anti-Pun1 antibodies, were reduced, and simultaneously, capsaicin accumulations were reduced in the same tissues. In the capsaicin synthesis cell-free assay, which uses protoplasts isolated from de novo in vitro placental tissues, capsaicin synthesis was inhibited by the addition of anti-Pun1 antibodies. We next analyzed the expression profiles of and in various pepper cultivars and found that high levels of capsaicin accumulation pAMT Pun1 always accompanied high expression levels of both and, indicating that both genes are important for pAMT Pun1 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. Conclusions: Using a newly developed protoplast-based assay for capsaicin synthesis and the anti-Pun1 de novo antibodies, we successfully demonstrated that the gene and its gene product are involved in capsaicin Pun1 synthesis. The analysis of the vanillylamine accumulation relative to that of capsaicin indicated that Pun1 was the primary determinant of their accumulation levels.
- Subjects
CAPSAICIN; PEPPER research; PLANT proteins; PUNGENCY; GENE silencing
- Publication
BMC Plant Biology, 2015, Vol 15, Issue 1, p1
- ISSN
1471-2229
- Publication type
Article
- DOI
10.1186/s12870-015-0476-7