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

alpha-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 alpha-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 alpha reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3 beta HSD is involved in the three reactions except for C5 alpha reduction, and in the present study, we aimed to elucidate the gene responsible for the C5 alpha reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5 alpha R1 and SlS5 alpha R2, which show high homology with DET2, a brassinosteroid 5 alpha reductase of Arabidopsis thaliana. The expression pattern of SlS5 alpha R2 is similar to those of SGA biosynthetic genes, while SlS5 alpha R1 is ubiquitously expressed, suggesting the involvement of SlS5 alpha R2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5 alpha R1 and SlS5 alpha R2 catalyze the reduction of tomatid-4-en-3-one at C5 alpha to yield tomatid-3-one. Then, SlS5 alpha R1- or SlS5 alpha R2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5 alpha R2-knockout hairy roots, the alpha-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of alpha-tomatine was observed in the SlS5 alpha R1-knockout hairy root. These results indicate that SlS5 alpha R2 is responsible for the C5 alpha reduction in alpha-tomatine biosynthesis and that SlS5 alpha R1 does not significantly contribute to alpha-tomatine biosynthesis.