Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes
Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes
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DOI：
10.5511/plantbiotechnology.19.1030a
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发表时间：
2019-12-01
影响因子：
1.6
通讯作者：
Mizutani, Masaharu
中科院分区：
文献类型：
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作者：
Akiyama, Ryota;Lee, Hyoung Jae;Mizutani, Masaharu
α-番茄碱和脱氢番茄碱是甾体配糖生物碱（SGA），其在番茄（Solanumlycopersicum）的成熟绿色果实、叶和花中积累，并作为抵抗病原体和捕食者的防御性化合物起作用。α-番茄素和去氢番茄素的苷元是番茄苷和去氢番茄苷（5，6-去氢番茄苷），番茄苷是由去氢番茄苷通过四个反应步骤衍生而来：C3氧化、异构化、C5 α还原和C3还原。我们之前的研究（Lee et al. 2019）表明，除了C5 α还原外，S13 β HSD参与了三种反应，在本研究中，我们旨在阐明脱氢番茄替丁转化为番茄替丁中负责C5 α还原步骤的基因。我们表征了两个基因，SlS 5 α R1和SlS 5 α R2，其与拟南芥的油菜素类固醇5 α还原酶DET 2显示出高度同源性。S1 S5 α R2的表达模式与SGA生物合成基因的表达模式相似，而S1 S5 α R1广泛表达，表明S1 S5 α R2参与SGA生物合成。重组蛋白的生化分析显示，SlS 5 α R1和SlS 5 α R2都催化番茄-4-烯-3-酮在C5 α的还原以产生番茄-3-酮。然后，使用CRISPR/Cas9介导的基因组编辑构建SlS 5aR 1-或SlS 5aR 2-敲除的毛状根。在SlS 5 α R2敲除的毛状根中，α-番茄碱水平显著降低，并且脱氢番茄碱积累。另一方面，在SlS 5 α R1敲除的毛状根中没有观察到α-番茄碱的量的变化。这些结果表明，S1 S5 α R2负责α-番茄碱生物合成中的C5 α还原，而S1 S5 α R1对α-番茄碱生物合成没有显著贡献。
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.