Mei Hui, Zhao Jian, Pittman Jon K, Lachmansingh Jinesh, Park Sunghun, Hirschi Kendal D
Molecular and Environmental Plant Science, Texas A&M University, College Station, Texas 77843, USA.
J Exp Bot. 2007;58(12):3419-27. doi: 10.1093/jxb/erm190. Epub 2007 Sep 26.
Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.
液泡定位的Ca(2+)/H(+)交换体，如拟南芥阳离子交换体1（CAX1），在Ca(2+)稳态中发挥重要作用。当在酵母中表达时，CAX1通过N端自抑制结构域进行调节。在酵母表达试验中，CAX1的36个氨基酸N端截短体，称为sCAX1，以及该N端具有特定突变的变体，显示出CAX1介导的Ca(2+)/H(+)反向转运活性。此外，表达sCAX1的转基因植物表现出Ca(2+)积累增加和液泡Ca(2+)/H(+)反向转运活性增强。本文比较并对比了植物和酵母表达系统中N端CAX1变体的特性，以确定CAX1在植物中是否发生自抑制。最初，通过离子组分析表明，只有表达活化CAX1转运体的酵母细胞总钙含量发生改变，锌和镍出现波动。表达活化CAX1变体的烟草植物对离子失衡表现出超敏反应，钙积累增加，钾、镁和锰等其他矿质营养元素浓度升高，液泡膜富集的Ca(2+)/H(+)转运活性增强。尽管在植物中基因表达水平很高，但在酵母中无活性的CAX1及其N端变体均未表现出上述表型。虽然几种植物转运体似乎含有N端自抑制结构域，但这项工作首次明确记录了转基因植物中Ca(2+)转运体的N端依赖性调节。因此，对自抑制结构域进行工程改造提供了一种增强转运功能以影响农艺性状的策略。
