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

Abstract

The participation of <i> O </i> -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 <i> Comt </i> expression caused a drastic decrease in capsaicinoid accumulation, it was presumed that a <i> Comt</i> loss-of-function mutation would cause loss of pungency in <i>Capsicum</i>. This hypothesis was tested by cloning <i>Comt </i> 1 and <i>Comt </i> 2 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 <i> Comts </i> were both expressed in placenta tissue and expressed prior to the accumulation of capsaicinoids, the two genes could be candidates for capsaicinoid biosynthesis. Second, <i> Comt </i> 1 loss-of-function mutants were screened from the germplasm. A truncated <i> Comt </i> 1 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 <i> Comt </i> 1 mutation was not related to non-pungency of No.3341, as the deletion of <i> Comt </i> 1 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 <i> Comt </i> 1 deletion mutation. Although the participation of COMT in phenylpropanoid-mediated capsaicinoid biosynthesis has long been proposed, our present study shows that <i> Comt </i> 1 can not be a target for controlling fruit pungency.