Identification of a 3b-hydroxysteroid dehydrogenase/ 3-ketosteroid reductase involved in a-tomatine biosynthesis in tomato

Abstract

a-Tomatine and dehydrotomatine are major steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves and flowers of tomato (Solanum lycopersicum), and function as defensive compounds against bacteria, fungi, insects and animals. The aglycone of dehydrotomatine is dehydrotomatidine (5,6-dehydrogenated tomatidine, having the 5,6 double bond; the dehydro-type). The aglycone of a-tomatine is tomatidine (having a single bond between C5 and C6; the dihydro-type), which is believed to be derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5 reduction and C3 reduction; however, these conversion processes remain uncharacterized. In the present study, we demonstrate that a short-chain alcohol dehydrogenase/reductase designated Sl3bHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3HSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of a-tomatine are accumulated. Biochemical analysis of the recombinant Sl3bHSD1 protein revealed that Sl3bHSD1 catalyzes the C3 oxidation of dehydrotomatidine to form tomatid-4-en-3-one and also catalyzes the NADH-dependent C3 reduction of a 3-ketosteroid (tomatid-3-one) to form tomatidine. Furthermore, during co-incubation of Sl3bHSD1 with SlS5aR1 (steroid 5a-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3HSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in a-tomatine content. Furthermore, the constitutive expression of Sl3HSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3bHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in a-tomatine biosynthesis.