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

Abstract

Summary 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 F2 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.

Substitution of a Dysfunctional pAMT Allele Results in Low-Pungency but High Levels of Capsinoid in Capsicum chinense ‘Habanero’

“…Biosynthesis of capsinoid is caused by mutations in the pAMT gene resulting in suppression of the formation vanillylamine from vanillin (Lang et al 2009;Tanaka et al 2010a). Dysfunction of pAMT shunts synthesis vanillylamine into vanillyl alcohol.…”

Inheritance of Non-pungency in ‘No.3341’ (<i>Capsicum chinense</i>)

“…Within C. annuum, most of the non-pungent cultivars carry the same single recessive allele of Pun1 (Stewart et al, 2005). Of late, two recessive alleles of p-AMT have been reported in two non-pungent cultivars (Lang et al, 2009;Tanaka et al, 2010a). In C. chinense, a single recessive allele of Pun1 and four recessive alleles of p-AMT have been reported to be the genetic basis of non-pungency (Koeda et al, 2014;Stewart et al, 2007;Tanaka et al, 2010b).…”

“…Given that the pungency of pepper fruit is one of its most important traits, numerous studies have been conducted on it. In C. annuum L., C. chinense Jacq., C. frutescens L., and C. chacoense Hunz., recessive mutations of acyltransferase (Pun1) and putative aminotransferase (p-AMT) have been reported to be the genetic causes of loss of pungency (Koeda et al, 2014;Lang et al, 2009;Stellari et al, 2010;Stewart et al, 2005Stewart et al, , 2007Tanaka et al, 2010a, b). Considering the long history of Capsicum as a crop, it is strange that only mutations in Pun1 and p-AMT have been used for breeding non-pungent peppers.…”

A &lt;i&gt;Comt&lt;/i&gt;1 Loss of Function Mutation Is Insufficient for Loss of Pungency in &lt;i&gt;Capsicum&lt;/i&gt;

“…Most of the non-pungent cultivars within C. annuum carry the same single Pun1 recessive allele [11]. Two recessive putative aminotransferase (p-AMT) alleles have been reported in the CH-19 Sweet and Himo nonpungent cultivars [12] [13]. In addition, a single recessive Pun1 allele has been reported for C. chinense, C. frutescens, and C. chacoense [14], and four recessive p-AMT alleles have been reported for C. chinense [15] [16].…”