National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai 200032, China.
Mol Plant. 2012 Mar;5(2):387-400. doi: 10.1093/mp/ssr110. Epub 2011 Dec 26.
Anthocyanins are synthesized in the cytosolic surface of the endoplasmic reticulum (ER) but dominantly accumulate in the vacuole. Little is known about how anthocyanins are transported from the ER to the vacuole. Here, we provide evidence supporting that Transparent Testa 19 (TT19), a glutathione S-transferase (GST), functions as a carrier to transport cyanidin and/or anthocyanins to the tonoplast. We identified a novel tt19 mutant (tt19-7), which barely accumulates anthocyanins but produces a 36% higher level of flavonol than the wild-type (WT), from ethyl methanesulfonate mutagenized seeds. Expressing TT19-fused green fluorescence protein (GFP) in tt19-7 rescues the mutant phenotype in defective anthocyanin biosynthesis, indicating that TT19-GFP is functional. We further showed that TT19-GFP is localized not only in the cytoplasm and nuclei, but also on the tonoplast. The membrane localization of TT19-GFP was further ascertained by immunoblot analysis. In vitro assay showed that the purified recombinant TT19 increases water solubility of cyanidin (Cya) and cyanidin-3-O-glycoside (C3G). Compared with C3G, Cya can dramatically quench the intrinsic tryptophan fluorescence of TT19 to much lower levels, indicating a higher affinity of TT19 to Cya than to C3G. Isothermal titration calorimetry analysis also confirmed physical interaction between TT19 and C3G. Taken together, our data reveal molecular mechanism underlying TT19-mediated anthocyanin transportation.
花色素苷在细胞质内质网膜（ER）的表面合成，但主要积累在液泡中。目前尚不清楚花色素苷是如何从 ER 运输到液泡的。在这里，我们提供的证据表明，透明种皮 19（TT19），一种谷胱甘肽 S-转移酶（GST），作为一种载体将矢车菊素和/或花色素苷运输到液泡膜。我们鉴定了一个新的 tt19 突变体（tt19-7），它几乎不积累花色素苷，但类黄酮的含量比野生型（WT）高出 36%，这是从乙基甲烷磺酸诱变的种子中得到的。在 tt19-7 中表达 TT19 融合绿色荧光蛋白（GFP）可以挽救其在花色素苷生物合成缺陷中的表型，表明 TT19-GFP 是有功能的。我们进一步表明，TT19-GFP 不仅定位于细胞质和细胞核，还定位于液泡膜上。免疫印迹分析进一步证实了 TT19-GFP 的膜定位。体外实验表明，纯化的重组 TT19 增加了矢车菊素（Cya）和矢车菊素-3-O-糖苷（C3G）的水溶性。与 C3G 相比，Cya 可以显著猝灭 TT19 的固有色氨酸荧光到更低的水平，这表明 TT19 与 Cya 的亲和力比 C3G 更高。等温滴定量热法分析也证实了 TT19 与 C3G 之间的物理相互作用。综上所述，我们的数据揭示了 TT19 介导的花色素苷运输的分子机制。
