Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes.

α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes () and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, α and α, which show high homology with , a brassinosteroid 5α reductase of . The expression pattern of α is similar to those of SGA biosynthetic genes, while α is ubiquitously expressed, suggesting the involvement of α in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, α or α-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the α-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the α-knockout hairy root. These results indicate that α is responsible for the C5α reduction in α-tomatine biosynthesis and that α does not significantly contribute to α-tomatine biosynthesis.