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 (Solanum lycopersicum) 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, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 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, SlS5αR1- or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-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 SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.