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, C5alpha reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3betaHSD is involved in the three reactions except for C5alpha reduction, and in the present study, we aimed to elucidate the gene responsible for the C5alpha reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5alphaR1 and SlS5alphaR2, which show high homology with DET2, a brassinosteroid 5alpha reductase of Arabidopsis thaliana. The expression pattern of SlS5alphaR2 is similar to those of SGA biosynthetic genes, while SlS5alphaR1 is ubiquitously expressed, suggesting the involvement of SlS5alphaR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5alphaR1 and SlS5alphaR2 catalyze the reduction of tomatid-4-en-3-one at C5alpha to yield tomatid-3-one. Then, SlS5alphaR1- or SlS5alphaR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5alphaR2-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 SlS5alphaR1-knockout hairy root. These results indicate that SlS5alphaR2 is responsible for the C5alpha reduction in alpha-tomatine biosynthesis and that SlS5alphaR1 does not significantly contribute to alpha-tomatine biosynthesis.