Positional differences of intronic transposons in pAMT affect the pungency level in chili pepper through altered splicing efficiency

Abstract

Summary Capsaicinoids are unique compounds that give chili pepper fruits their pungent taste. Capsaicinoid levels vary widely among pungent cultivars, which range from low pungency to extremely pungent. However, the molecular mechanisms underlying this quantitative variation have not been elucidated. Our previous study identified various loss‐of‐function alleles of the pAMT gene which led to low pungency. The mutations in these alleles are commonly defined by Tcc transposon insertion and its footprint. In this study, we identified two leaky pamt alleles (pamtL1 and pamtL2) with different levels of putative aminotransferase (pAMT) activity. Notably, both alleles had a Tcc transposon insertion in intron 3, but the locations of the insertions within the intron were different. Genetic analysis revealed that pamtL1, pamtL2 and a loss‐of‐function pamt allele reduced capsaicinoid levels to about 50%, 10% and less than 1%, respectively. pamtL1 and pamtL2 encoded functional pAMT proteins, but they exhibited lower transcript levels than the functional type. RNA sequencing analysis showed that intronic transposons disrupted splicing in intron 3, which resulted in simultaneous expression of functional pAMT mRNA and non‐functional splice variants containing partial sequences of Tcc. The non‐functional splice variants were more dominant in pamtL2 than in pamtL1. This suggested that the difference in position of the intronic transposons could alter splicing efficiency, leading to different pAMT activities and reducing capsaicinoid content to different levels. Our results provide a striking example of allelic variations caused by intronic transposons; these variations contribute to quantitative differences in secondary metabolite contents.

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird‐of‐paradise

“…chromosome-level assembly, GC content, genome assembly, Hi-C, long reads, satellite repeat, transposable element four-stranded DNA/RNA topologies that seem to be involved in numerous cellular processes, such as regulation of gene expression (Du, Zhao, & Li, 2008Raiber et al, 2011), genetic and epigenetic stability (Schiavone et al, 2014), and telomere maintenance (Biffi et al, 2012). On the repetitive element side, for example, transposable elements are a major target of epigenetic silencing (Law & Jacobsen, 2010) that may influence the epigenetic regulation of nearby genes (Chuong et al, 2016;Cowley & Oakey, 2013;Tanaka et al, 2019). The epigenetic effect of individual TE insertions may be beneficial or deleterious, but in either case it is important to acknowledge their overall effect on the evolution of gene expression (Lerat et al, 2019).…”

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise

“…Technological biases are therefore impeding the 67 complete reconstruction of genomes and substantial regions are systematically missing from genome 68 assemblies. These missing regions are often referred to as the genomic "dark matter" (Johnson et al 69 for example, transposable elements are a major target of epigenetic silencing (Law and Jacobsen 122 2010) that may influence the epigenetic regulation of nearby genes (Cowley and Oakey 2013;Chuong 123 et al 2016; Tanaka et al 2019). The epigenetic effect of transposable elements may be beneficial or 124 deleterious, but in either case it is important to acknowledge their potential involvement in the 125 evolution of gene expression (Lerat et al 2019).…”

Parthenocarpy Induced Fluctuations in Pungency and Expression of Capsaicinoid Biosynthesis Genes in a Japanese Pungency-variable Sweet Chili Pepper ‘Shishito’ (<i>Capsicum annuum</i>)

“…For example, Pun1 is the most downstream gene in the capsaicinoid biosynthesis pathway, and functional loss of Pun1 due to mutation prevents capsaicinoids synthesis (Stewart et al, 2005). As a result, functional loss of CaMYB31 and pAMT also caused the loss of, or drastic reduction in, pungency (Han et al, 2019;Tanaka et al, 2019). Moreover, it was reported that gene silencing of Pun1, pAMT, KAS, and CaMYB31 using virus-induced gene silencing (VIGS) caused dramatic decreases in capsaicinoid contents (Abraham-Juárez et al, 2008;Ochoa-Alejo, 2015, 2017).…”

“…Above all, Pun1, which encodes an acyltransferase, is one of the most critical genes in capsaicinoid biosynthesis and is responsible for the final reaction in the pathway (Stewart et al, 2005(Stewart et al, , 2007. Then, putative aminotransferase (pAMT), which produces vanillylamine from vanillin, is also important and mutation of this gene caused a drastic reduction in capsaicinoids (Koeda et al, 2014;Lang et al, 2009;Tanaka et al, 2010Tanaka et al, , 2019. Furthermore, in recent years two novel genes were discovered that induced loss of pungency.…”