Functional analysis of stress-responsive transporter genes in plants

植物在水分亏缺条件下通过一系列生理、细胞和分子过程来响应生存，最终导致胁迫耐受性。植物在渗透胁迫过程中积累渗透调节物质，如糖、氨基酸和钾。我们确定了多种转运蛋白基因在拟南芥渗透胁迫过程中上调。我们分析了一个渗透胁迫诱导的糖转运蛋白，ESL 1，从拟南芥。ESL 1主要表达于周壁和木质部薄壁细胞。在转基因植株的液泡膜中检测到ESL 1-GFP融合蛋白的荧光。丙氨酸扫描突变显示，在ESL 1的N-末端LXXXLL基序是必不可少的定位在液泡膜。转基因烟草BY-2细胞表达定位于质膜的突变型ESL 1，表现出对单糖的摄取能力。转基因BY-2细胞中突变的ESL 1的葡萄糖摄取活性的K（m）值非常高，并且转运活性与质子梯度无关。这些结果表明，ESL 1是一种低亲和力的易化扩散转运蛋白。我们还分析了拟南芥胁迫诱导钾转运蛋白KUP 6。KUP 6-GFP定位于细胞质膜。KUP 6过表达的转基因植株表现出较少的蒸腾失水和对干旱胁迫的耐受性增加。此外，我们还分析了拟南芥逆境诱导转运蛋白类蛋白Cor 413家族，其表达受DREB 1A调控。COR 413-IM 1和COR 413-IM2.1定位在叶绿体膜上，而COR 413-PM 1定位在内质网上。此外，我们使用的组氨酸激酶，AHK 1的增益和损失的功能分析，并表明它作为一个积极的调节剂在拟南芥中的渗透胁迫信号。AHK 1的过表达提高了转基因植株的耐旱性。

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Plants accumulate osmolites, such as sugars, amino acids and potassium, during osmotic stress. We identified various transporter genes were upregulated during osmotic stress in Arabidopsis. We analyzed an osmotic stress-inducible sugar transporter, ESL1, from Arabidopsis. ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-GFP-fused protein was detected at tonoplast in transgenic plants. Alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic tobacco BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed the uptake ability for monosaccharides. The value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. We also analyzed an Arabidopsis stress-inducible potassium transporter, KUP6. The localization of KUP6-GFP was observed at plasma membrane. KUP6-overexpressing transgenic plants showed less transpirational water loss and increased tolerance to drought stress. Furthermore, we also analyzed Arabidopsis stress-inducible transporter-like proteins, Cor413 family, whose expression was regulated by DREB1A during cold stress. COR413-IM1 and COR413-IM2.1 were localized at the chloroplast membrane, while COR413-PM1 was localized at ER. Furthermore, we used both gain- and loss-of-function analysis of a histidine kinase, AHK1, and showed that it acts as a positive regulator in osmotic stress signaling in Arabidopsis. Overexpression of AHK1 improved the drought tolerance of transgenic plants.