Rodríguez-Rosales María Pilar, Jiang Xingyu, Gálvez Francisco Javier, Aranda María Nieves, Cubero Beatriz, Venema Kees
Department of Biochemistry and Molecular and Cell Biology of Plants, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain.
Department of Plant Biology, Instituto de Recursos Naturales y Agrobiología, CSIC, 41012 Sevilla, Spain.
New Phytol. 2008 Jul;179(2):366-377. doi: 10.1111/j.1469-8137.2008.02461.x.
Here, the function of the tomato (Solanum lycopersicon) K+/H+ antiporter LeNHX2 was studied using 35S-driven gene overexpression of a histagged LeNHX2 protein in Arabidopsis thaliana and LeNHX2 gene silencing in tomato. Transgenic A. thaliana plants expressed the histagged LeNHX2 both in shoots and in roots, as assayed by western blotting. Transitory expression of a green fluorescent protein (GFP) tagged protein showed that the antiporter is present in small vesicles. Internal membrane vesicles from transgenic plants displayed enhanced K+/H+ exchange activity, confirming the K+/H+ antiporter function of this enzyme. Transgenic A. thaliana plants overexpressing the histagged tomato antiporter LeNHX2 exhibited inhibited growth in the absence of K+ in the growth medium, but were more tolerant to high concentrations of Na+ than untransformed controls. When grown in the presence of NaCl, transgenic plants contained lower concentrations of intracellular Na+, but more K+, as compared with untransformed controls. Silencing of LeNHX2 in S. lycopersicon plants produced significant inhibition of plant growth and fruit and seed production as well as increased sensitivity to NaCl. The data indicate that regulation of K+ homeostasis by LeNHX2 is essential for normal plant growth and development, and plays an important role in the response to salt stress by improving K+ accumulation.
在此,通过在拟南芥中利用35S驱动的带组氨酸标签的LeNHX2蛋白基因过表达以及在番茄中沉默LeNHX2基因,对番茄(Solanum lycopersicon)K⁺/H⁺逆向转运蛋白LeNHX2的功能进行了研究。通过蛋白质免疫印迹分析可知,转基因拟南芥植株的地上部分和根部均表达带组氨酸标签的LeNHX2。绿色荧光蛋白(GFP)标签蛋白的瞬时表达表明,该逆向转运蛋白存在于小泡中。来自转基因植株的内膜小泡显示出增强的K⁺/H⁺交换活性,证实了该酶的K⁺/H⁺逆向转运蛋白功能。过表达带组氨酸标签的番茄逆向转运蛋白LeNHX2的转基因拟南芥植株在生长培养基中无K⁺时生长受到抑制,但比未转化的对照更耐高浓度的Na⁺。当在NaCl存在的条件下生长时,与未转化的对照相比,转基因植株细胞内Na⁺浓度较低,但K⁺含量更高。番茄植株中LeNHX2的沉默显著抑制了植株生长、果实和种子产量,并增加了对NaCl的敏感性。数据表明,LeNHX2对K⁺稳态的调节对于正常植物生长发育至关重要,并且通过改善K⁺积累在盐胁迫响应中发挥重要作用。