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The participation of O-methyltransferase (COMT) in phenylpropanoid-mediated capsaicinoid biosynthesis has long been proposed. Ferulic acid, a phenylpropanoid intermediate, is a precursor of capsaicinoid biosynthesis and is produced from caffeic acid by the action of COMT. As previously reported that silencing Comt expression caused a drastic decrease in capsaicinoid accumulation, it was presumed that a Comt loss-of-function mutation would cause loss of pungency in Capsicum. This hypothesis was tested by cloning Comt1 and Comt2 from the placenta tissue of the pungent cultivar Habanero. The phylogenetic analysis and comparison of critical amino-acid residues for enzyme function showed that the two COMTs had high similarity with the COMTs of other plant species. Moreover, as the two Comts were both expressed in placenta tissue and expressed prior to the accumulation of capsaicinoids, the two genes could be candidates for capsaicinoid biosynthesis. Second, Comt1 loss-of-function mutants were screened from the germplasm. A truncated Comt1 transcript was expressed in non-pungent pepper No.3341 caused by deletion of the genomic region. The predicted No.3341 COMT1 lacked His-265, which was absolutely necessary for enzymatic activity. Contrary to our expectations, the Comt1 mutation was not related to non-pungency of No.3341, as the deletion of Comt1 did not co-segregate with non-pungency in the F2 population obtained from crossing No.3341 with Habanero. This result was confirmed by screening several pungent accessions harboring the same Comt1 deletion mutation. Although the participation of COMT in phenylpropanoid-mediated capsaicinoid biosynthesis has long been proposed, our present study shows that Comt1 can not be a target for controlling fruit pungency.

A <i>Comt</i>1 Loss of Function Mutation Is Insufficient for Loss of Pungency in <i>Capsicum</i>