Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.Potato (Solanum tuberosum) is the world’s fourth most important food crop after maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). Potato steroidal glycoalkaloids (SGAs) are abundant poisons in tuber sprouts and green tubers and are described as bitter tasting, burning, scratchy, or acrid (Friedman, 2006; Ginzberg et al., 2009; Taylor et al., 2007). SGAs in the tuber are induced by exposure to light, low temperature, and mechanical injury (Valkonen et al., 1996). Producers and consumers have called for the removal of SGAs from potatoes. Only potato with SGAs of major food crops has such broad industry consensus on the need to solve this important worldwide problem. Controlling the SGA content is also important for potato breeding. Wild germplasm has been used in potato breeding programs as a source of pest and disease resistance. Since high SGA concentrations are found in most wild tuber-bearing species, introgression of wild germplasm may increase the risk for high SGA levels. Although the initial concentrations are still low in new breeding lines, dangerous levels of SGAs can accumulate due to environmental factors, pathogen infections, and postharvest treatments (Valkonen et al., 1996).SGAs are biosynthesized from a common precursor, cholesterol (CHR; Sawai et al., 2014), but little is known about intermediates and enzymes in the SGA biosynthetic pathway. To change a biosynthetic flow to CHR and decrease SGA contents, transgenic potatoes overexpressing a heterologous soybean sterol methyltransferase gene were produced (Arnqvist et al., 2003). Three genes responsible for glycosylating potato SGA have been identified (McCue et al., 2005, 2006, 2007). However, changing the expression of the sterol methyltransferase or glycosyltransferase genes does not effectively decrease SGA levels. To control the SGA content of potato, we focused on the biosynthetic steps from CHR to the aglycone, solanidine. Few details about the biosynthetic pathway are verified; however, the pathway is hypothesized to require at least three oxidization steps at positions C16, C22, and C26 of CHR structure and the addition of one nitrogen atom at the position C26 (Fig. 1; Kaneko et al., 1977; Erich, 1983; Eich, 2008). The later step was shown to be another oxidation and an amination reaction at the position C26 (Ohyama et al., 2013). Here, we identified two cytochrome P450 monooxygenase (CYPs) genes, POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2 that encode enzymes mediating two oxidation steps. Silencing PGA1 and PGA2 resulted in a significant reduction in SGA composition and the creation of novel phenotypes, including the suppression of flower development and tuber sprouting. Sprouting reduces the quality and yield of potato tubers in storage. Suppression of tuber sprouting is of significant benefit to the industry for the long-term storage of tubers. Thus, controlling tuber sprouting is another important objective in potato breeding (Sonnewald and Sonnewald, 2014).Open in a separate windowFigure 1.Biosynthetic pathway for SGAs in potato. The structures of CHR and solanidine, two biosynthetic intermediates of potato SGAs, are shown with the structures of the SGA products. Circles indicate putative carbon positions that are oxidized in the hypothesized pathway.