Queirós Filipa, Fontes Natacha, Silva Paulo, Almeida Domingos, Maeshima Masayoshi, Gerós Hernâni, Fidalgo Fernanda
Departamento de Botânica, Faculdade de Ciências, Universidade do Porto, Ed. FC4, Rua do Campo Alegre, s/n masculine, 4169-007 Porto, Portugal.
J Exp Bot. 2009;60(4):1363-74. doi: 10.1093/jxb/erp011. Epub 2009 Feb 12.
The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.
将过量的 Na 从细胞质中有效排出以及液泡中 Na⁺ 的积累是植物适应盐胁迫的主要机制。这通常由跨膜转运蛋白来完成，这些蛋白将 Na⁺ 从细胞质中排出以交换 H⁺，这是一个依赖能量的次级转运过程，由质膜和液泡膜质子泵产生的质子动力驱动。来自对照和 150 mM NaCl 耐受愈伤组织系的富含液泡膜的囊泡被用作模型系统，以研究 V-H⁺-焦磷酸酶（V-H⁺-PPase）和 V-H⁺-ATP 酶的活性，以及 Na⁺ 区室化进入液泡作为马铃薯耐盐机制的参与情况。来自耐盐系的液泡膜囊泡中依赖 ATP 和焦磷酸（PP(i)）的 H⁺ 转运都高于对照细胞的囊泡。液泡膜蛋白的 Western 印迹证实，V-H⁺-PPase 活性的变化与蛋白量的增加相关。相反，V-H⁺-ATP 酶 A 亚基的免疫检测表明可能涉及翻译后调控机制。利用预先建立的 pH 梯度的 Na⁺ 依赖性耗散来测量液泡膜囊泡中的 Na⁺/H⁺ 交换。质子外流的初始速率遵循米氏动力学，与对照相比，耐 NaCl 愈伤组织中质子耗散的 V(max) 高出 2 倍。H⁺ 偶联交换对 Na⁺ 和 Li⁺ 具有特异性，而对 K⁺ 不具有特异性。响应盐胁迫时，液泡膜上 pH 梯度和 Na⁺/H⁺ 反向转运活性的增加强烈表明，Na⁺ 隔离到液泡中有助于马铃薯的耐盐性。
