Functional analysis of stress-responsive transporter genes in plants

植物在缺水条件下通过一系列生理、细胞和分子过程对生存做出反应，最终形成逆境耐受性。植物在渗透胁迫过程中积累渗透物，如糖、氨基酸和钾。我们发现，在渗透胁迫下，拟南芥的多种转运蛋白基因被上调。我们分析了来自拟南芥的渗透胁迫诱导糖转运蛋白ESL1。ESL1主要表达于中柱鞘和木质部薄壁细胞中。在转基因植株的叶绿体上检测到esl1 - gfp融合蛋白的荧光。丙氨酸扫描突变显示，ESL1的n端LXXXLL基序对其在细胞质上的定位至关重要。表达突变ESL1的转基因烟草BY-2细胞对单糖具有吸收能力，ESL1定位于质膜。在转基因BY-2细胞中，突变ESL1的葡萄糖摄取活性K(m)值非常高，并且运输活性不依赖于质子梯度。这些结果表明，ESL1是一种低亲和力的促进扩散转运蛋白。我们还分析了拟南芥胁迫诱导的钾转运蛋白KUP6。在质膜上观察到KUP6-GFP的定位。过表达kup6的转基因植株蒸腾失水减少，对干旱胁迫的耐受性增强。此外，我们还分析了在冷胁迫下受DREB1A调控的拟南芥胁迫诱导转运蛋白Cor413家族。COR413-IM1和COR413-IM2.1定位于叶绿体膜，而COR413-PM1定位于内质网。此外，我们使用了组氨酸激酶AHK1的功能增益和功能损失分析，并表明它在拟南芥渗透胁迫信号传导中起着积极的调节作用。过表达AHK1提高了转基因植株的抗旱性。

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.