title: Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper.
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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. kana9999102@yahoo.co.jp.
authors.forename: Kana
authors.initials: K
authors.lastname: Ogawa
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. murota@abs.agr.hokudai.ac.jp.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Murota
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. hana@res.agr.hokudai.ac.jp.
authors.forename: Hanako
authors.initials: H
authors.lastname: Shimura
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. fam.ggb.836@gmail.com.
authors.forename: Misaki
authors.initials: M
authors.lastname: Furuya
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. ytogawa@res.agr.hokudai.ac.jp.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Togawa
authors.affiliation: Plant Molecular Technology Research Group, Research Institute of Bioproduction, National Institute of Advanced Industrial Science and Technology, Sapporo, 062-8517, Japan. matsumura-t@aist.go.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Matsumura
authors.affiliation: Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan. masuta@res.agr.hokudai.ac.jp.
authors.forename: Chikara
authors.initials: C
authors.lastname: Masuta
mesh_terms: D002211:Capsaicin / Q000378:metabolism*; D002212:Capsicum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D002211:Capsaicin