Okayama University of Science, Department of Biochemistry, Faculty of Science, 1-1 Ridai-cho, Kita-ku, Okayama, 700-0005, Japan.
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
Plant Physiol Biochem. 2018 Nov;132:138-144. doi: 10.1016/j.plaphy.2018.08.034. Epub 2018 Aug 28.
The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-β-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-β-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.
这种植物菘蓝产生的次生代谢物靛苷（吲哚-β-D-葡萄糖苷），是蓝色染料靛蓝的前体。菘蓝通过 UDP-葡萄糖基转移酶（UGT）、靛苷合成酶的作用合成靛苷。在此，我们从叶片中部分纯化了一种靛苷合成酶，随后进行肽质量指纹图谱分析。结果，我们鉴定出一个与 UDP-葡萄糖基转移酶 72B（UGT72B）家族成员同源的片段。我们将其命名为 PtIgs（菘蓝吲哚-β-D-葡萄糖苷合成酶），并使用快速扩增 cDNA 末端获得全长 cDNA。PtIGS 编码的 PtIGS 的一级结构与来自 Indigofera tinctoria 的靛苷合成酶（ItUGT1 和 ItUGT2）具有高度同源性（Inoue 等人，2017 年）。此外，在菘蓝的表达分析中，PtIGS mRNA 几乎只在叶片中发现，在第 1 片叶中表达最高，随着叶片年龄的增加而降低。由于 PtIGS 表达倾向于反映靛苷含量和合成活性，我们得出结论，PtIGS 在植物细胞中作为靛苷合成酶发挥作用。为了检验 PtIGS 的细胞内定位，粗叶片提取物被分离成胞质和微粒体部分，并在胞质和微粒体部分中发现了 PtIGS。此外，存在去污剂和脲素时，微粒体 PtIGS 可溶，并与膜强烈结合。最后，我们使用蔗糖密度梯度超速离心证实了 PtIGS 的内质网（ER）膜定位。这些数据表明，PtIGS 与 ER 膜上的某些蛋白质相互作用，以确保底物的传递。
