The soybean F3'H protein is localized to the tonoplast in the seed coat hilum
The soybean F3'H protein is localized to the tonoplast in the seed coat hilum
复制标题
大豆 F3H 蛋白定位于种皮门的液泡膜
作者：
Toda;K.;et al
我们之前从两个近等基因系 (NIL) To7B (TT) 和 To7G (tt) 中分离出一个与控制柔毛和种皮颜色的 Tlocus 相对应的大豆 (Glycine max(L.) Merr.) 类黄酮 3′-羟化酶 (F3′H) 基因 (sf3′h1)。 TheTallele 还与耐寒性有关。在这里，Western-blot 分析表明，sf3'h1 蛋白主要在 To7B 未成熟种皮的种门和珠柄中检测到，而在 To7G 中未检测到 sf3'h1。仅在通过免疫沉淀富集后才检测到从 To7G 中分离出的截短的 sf3'h1 蛋白。使用二苯硼酸 2-氨基乙酯 (DBPA) 染色进行的分析表明，类黄酮在 To7B 和 To7G 的肺门和索中积累。此外，To7B 的柄和脐的甲醇提取物中 1,1-二苯基-2-三硝基苯肼（DPPH）自由基的清除活性高于 To7G。此外，使用来自 To7B 而非 To7G 的 sf3'h1 转化的酵母的微粒体级分检测了 F3'H 的酶活性。这些结果表明 sf3'h1 参与种皮中的类黄酮生物合成并影响这些组织的抗氧化特性。免疫荧光显微镜显示，sf3'h1蛋白主要在To7B的肺门实质细胞的液泡周围检测到。进一步免疫电镜检测到液泡膜结构上有sf3'h1蛋白。基于这些观察，我们得出结论，F3'H 是一种细胞色素 P450 单加氧酶，已被发现位于其他植物系统的 ER 中，而 F3'H 位于大豆的液泡膜中。
We previously isolated a soybean (Glycine max(L.) Merr.) flavonoid 3′-hydroxylase (F3′H) gene (sf3′h1) corresponding to theTlocus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). TheTallele 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 withsf3′h1from To7B, but not from To7G. These results indicate thatsf3′h1is 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.