Graduate School of Agricultural Science, Kobe University, Rokkoudai 1-1, Nada-ku, Kobe, Hyogo, Japan.
RIKEN Center for Sustainable Resource Science, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanagawa, Japan.
Plant Cell Physiol. 2019 Jun 1;60(6):1304-1315. doi: 10.1093/pcp/pcz049.
α-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 Δ5,6 double bond; the dehydro-type). The aglycone of α-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 Sl3βHSD is involved in the conversion of dehydrotomatidine to tomatidine in tomato. Sl3βHSD1 expression was observed to be high in the flowers, leaves and mature green fruits of tomato, in which high amounts of α-tomatine are accumulated. Biochemical analysis of the recombinant Sl3βHSD1 protein revealed that Sl3βHSD1 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 Sl3βHSD1 with SlS5αR1 (steroid 5α-reductase) the four reaction steps converting dehydrotomatidine to tomatidine were completed. Sl3βHSD1-silenced transgenic tomato plants accumulated dehydrotomatine, with corresponding decreases in α-tomatine content. Furthermore, the constitutive expression of Sl3βHSD1 in potato hairy roots resulted in the conversion of potato SGAs to the dihydro-type SGAs. These results demonstrate that Sl3βHSD1 is a key enzyme involved in the conversion processes from dehydrotomatidine to tomatidine in α-tomatine biosynthesis.
α-茄碱和脱氧茄碱是在成熟的绿色果实、叶子和花中积累的主要甾体糖苷生物碱（SGAs），它们作为防御化合物对抗细菌、真菌、昆虫和动物。脱氧茄碱的糖苷配基是脱氧茄啶（5,6-脱氢茄啶，具有 Δ5,6 双键；脱氢型）。α-茄碱的糖苷配基是茄啶（C5 和 C6 之间有单键；二氢型），据信它是通过四个反应步骤从脱氧茄啶衍生而来的：C3 氧化、异构化、C5 还原和 C3 还原；然而，这些转化过程仍未被描述。在本研究中，我们证明了一种短链醇脱氢酶/还原酶 Sl3βHSD 参与了番茄中脱氧茄啶向茄啶的转化。Sl3βHSD1 的表达在番茄的花、叶和成熟的绿色果实中较高，在这些部位积累了大量的α-茄碱。对重组 Sl3βHSD1 蛋白的生化分析表明，Sl3βHSD1 催化脱氧茄啶的 C3 氧化形成茄啶-4-烯-3-酮，还催化 NADH 依赖性 3-酮甾体（茄啶-3-酮）的 C3 还原形成茄啶。此外，在 Sl3βHSD1 与 SlS5αR1（甾体 5α-还原酶）共同孵育时，完成了将脱氧茄啶转化为茄啶的四个反应步骤。Sl3βHSD1 沉默的转基因番茄植物积累了脱氧茄碱，相应地降低了α-茄碱的含量。此外，Sl3βHSD1 在马铃薯毛状根中的组成型表达导致马铃薯 SGA 转化为二氢型 SGA。这些结果表明，Sl3βHSD1 是参与α-茄碱生物合成中从脱氧茄啶向茄啶转化过程的关键酶。
