National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan.
Planta. 2012 Jul;236(1):79-89. doi: 10.1007/s00425-012-1590-5. Epub 2012 Jan 19.
We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.
我们之前从两个近等基因系（NILs）To7B（TT）和 To7G（tt）中分离出一个与 T 座位控制茸毛和种皮颜色的大豆（Glycine max（L.）Merr.）类黄酮 3'-羟化酶（F3'H）基因（sf3'h1）。T 等位基因也与耐寒性有关。在这里，Western-blot 分析表明，sf3'h1 蛋白主要在 To7B 的未成熟种皮珠孔和珠柄中检测到，而在 To7G 中未检测到 sf3'h1。从 To7G 中分离出的截短的 sf3'h1 蛋白仅在免疫沉淀富集后才被检测到。使用二苯基硼酸 2-氨乙基酯（DBPA）染色进行的分析表明，类黄酮在 To7B 和 To7G 的珠孔和珠柄中积累。此外，来自 To7B 珠柄和珠孔的甲醇提取物的 1,1-二苯基-2-苦基肼（DPPH）自由基清除活性高于 To7G。此外，使用来自转化有 To7B 的 sf3'h1 的酵母的微粒体部分检测到 F3'H 的酶活性，但来自 To7G 的则没有。这些结果表明，sf3'h1 参与种皮中类黄酮的生物合成，并影响这些组织的抗氧化特性。如免疫荧光显微镜所示，sf3'h1 蛋白主要在 To7B 的珠孔的薄壁细胞中的液泡周围检测到。进一步的免疫电子显微镜检测到 sf3'h1 蛋白在液泡的膜状结构上。基于这些观察结果，我们得出结论，F3'H 是一种细胞色素 P450 单加氧酶，在其他植物系统中已被发现定位于内质网，在大豆中定位于液泡的质膜上。
