BE-61 Hot or not? A link between peroxidases, Pun1 expression, and hotness of Capsicum sp.

Capsicum sp. peppers are known for their spice, which comes from nonvolatile alkaloid compounds called capsaicinoids. Capsaicinoids differ in pungency; for example, the most common capsaicinoid, capsaicin, is nearly 10x as pungent as other capsaicinoids. Capsaicinoids are made through the phenylpropanoid and branched-chain fatty acid pathways, and an important aspect in that process is the Pun1 gene (also called AT3). Pun1 codes for an enzyme, capsaicin synthase, which is active in the phenylpropanoid pathway for synthesis of capsaicin. According to prior studies, expression and length of the Pun1 gene can be correlated with spice levels in peppers as it corresponds to accumulation of capsaicinoids. Further, peroxidase enzymes are involved in breaking down capsaicinoids through oxidation, and peroxidases have been found to generally increase with increasing pungency of peppers. However, peroxidases can also increase due to outside stressors to the plant. Therefore, we hypothesized that pepper pungency could be assayed through testing of Pun1 expression and gene size as well as through peroxidase levels except when plants endured stressors, as stressors would increase peroxidase activity, catalyzing the breakdown of capsaicinoids. Our study investigates the peroxidase content and Pun1 expression and gene length of a wide variety of peppers in different conditions; mild peppers, like green bell peppers, cubano, and cowhorn, as well as some of the hottest varieties, such as jalapeño, habanero, scorpion, dragon breath, death spiral, ghost pepper, mutant X, and Carolina reaper were used. The peppers were grown in a single grow bed at the USC Upstate community garden, and fruits were harvested at varying levels of ripeness, during different average temperatures, and pre- and post- hurricane. Once peppers were picked, they were sealed in a plastic bag and immediately frozen at -80°C. Proteins, RNA, and DNA were extracted from the fruit tissue for further study. Peroxidase activity was measured using chloronaphthol and spectrophotometry, and expression of Pun1 was analyzed using qPCR after extracting and reverse-transcribing the RNA. This study connects Pun1 gene expression and peroxidase enzyme activity to capsaicinoid levels in various conditions, giving insight into what controls heat in peppers. Newer peppers, such as Carolina Reaper and Mutant X, have never been investigated for these qualities before now. Understanding these mechanisms could help with breeding new pepper varieties tailored for specific levels of spice and flavor.