Gene Resource Research Group, Radiation-Applied Biology Division, Japan Atomic Energy Agency (JAEA), 1233 Watanuki, Takasaki 370-1292, Japan.
Plant J. 2010 May 1;62(4):549-59. doi: 10.1111/j.1365-313X.2010.04174.x. Epub 2010 Feb 18.
Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione-S-transferase (GST)-like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar-like structures, whereas those in tt12, a mutant of the tonoplast-bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP-TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent-insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent-soluble fraction by LC-MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP-TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.
拟南芥 TRANSPARENT TESTA19（TT19）编码谷胱甘肽-S-转移酶（GST）样蛋白，该蛋白参与种皮中原花青素（PAs）的积累。在 tt19 不成熟种子中，PA 积累部位被观察到为小液泡样结构，而在 tt12（PA 质膜结合转运蛋白的突变体）和 tt12 tt19 中，PA 积累部位则位于小液泡的外周区域。我们发现 tt19 不成熟种子中存在具有独特厚形态的小球形结构，通过微分干涉对比显微镜观察到。厚结构的分布模式与 PA 积累部位重叠，并且在 tt19 中，厚结构被 GFP-TT12 蛋白勾勒出来。PA 分析表明，与野生型相比，tt19 不成熟种子中溶剂不溶性 PA 的水平更高（八倍）。通过 LC-MS 对溶剂可溶部分进行代谢物分析表明，尽管在野生型中高度积累了表儿茶素和表儿茶素低聚物等 PA 衍生物，但在 tt19 中却不存在。我们还揭示了 tt12 特异性积累了糖基化表儿茶素，这是 TT12 的推定运输底物。tt12 tt19 表现出与 tt19 相似的代谢谱。鉴于功能性 GFP-TT19 蛋白的细胞质定位，我们的结果表明，TT19 在 TT12 之前发挥作用，在细胞质中维持 PA 前体（如表儿茶素和糖基化表儿茶素）在液泡中的正常积累。讨论了拟南芥种皮中 PA 途径与 PA 代谢物的亚细胞定位之间的关系。
