Functional analysis of stress-responsive transporter genes in plants

植物胁迫响应转运蛋白基因的功能分析

基本信息

批准号：
17078003
负责人：
SHINOZAKI Kazuko
金额：
$ 40.32万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2009
项目状态：
已结题

项目摘要

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.

植物通过一系列生理，细胞和分子过程在缺乏水分的条件下生存，最终在胁迫耐受性方面。植物在渗透胁迫期间积累渗透矿石，例如糖，氨基酸和钾。我们确定了拟南芥中渗透应激期间各种转运蛋白基因的上调。我们分析了拟南芥的渗透应激诱导糖转运蛋白ESL1。 ESL1主要在周围和木质部实质细胞中表达。在转基因植物的块状物质上检测到ESL1-GFP融合蛋白的荧光。丙氨酸扫描诱变表明，ESL1中的N末端LXXXLL基序对于其在块状体中的定位至关重要。位于质膜上的突变ESL1表达突变ESL1的转基因烟草BY-2细胞显示单糖的摄取能力。转基因BY-2细胞中突变的ESL1葡萄糖吸收活性的K（M）值非常高，并且转运活性独立于质子梯度。这些结果表明ESL1是低亲和力促进扩散转运蛋白。我们还分析了拟南芥诱导的钾转运蛋白KUP6。在质膜上观察到KUP6-GFP的定位。过表达KUP6的转基因植物显示出较小的蒸腾性水损失和对干旱胁迫的耐受性的增加。此外，我们还分析了拟南芥诱导型转运蛋白的cor413家族，其表达在冷应激期间受到Dreb1a的调节。 COR413-IM1和COR413-IM2.1位于叶绿体膜，而COR413-PM1位于ER。此外，我们使用了组氨酸激酶AHK1的功能和功能丧失分析，并表明它是拟南芥中渗透应力信号的阳性调节剂。 AHK1的过表达提高了转基因植物的干旱耐受性。