Kerkeb Loubna, Donaire Juan Pedro, Rodríguez-Rosales María Pilar
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain.
Physiol Plant. 2001 Apr;111(4):483-490. doi: 10.1034/j.1399-3054.2001.1110408.x.
A tomato (Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 mM NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 mM NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl- both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H+ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H+-ATPase (EC 3.6.1.35) and ATP-dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 mM NaCl was correlated with an increase in plasma membrane ATP-dependent H+ transport. The increase of ATP-dependent H+ extrusion in plasma membranes isolated from 50 mM NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl-sensitive calli, plasma membrane H+-ATPase from calli tolerant to 50 mM NaCl showed a lower Km for Mg2+-ATP. Our results indicate that tolerance of tomato calli to 50 mM NaCl increases the affinity of plasma membrane H+-ATPase for the substrate ATP and stimulates the H+-pumping activity of this enzyme without modifying its phosphohydrolytic activity.
通过在添加了50 mM NaCl的培养基中对NaCl敏感愈伤组织进行连续继代培养，获得了耐NaCl的番茄（Lycopersicon esculentum Mill. cv. Pera）愈伤组织培养物。在添加了50和100 mM NaCl的培养基中，耐NaCl愈伤组织比NaCl敏感愈伤组织生长得更好。愈伤组织离子含量分析表明，在含NaCl的培养基中培养一代的耐NaCl和敏感愈伤组织中，Na+和Cl-含量均大幅增加。耐NaCl愈伤组织的细胞比在NaCl存在下培养一代的NaCl敏感愈伤组织的细胞表现出更高的H+外排活性。NaCl敏感细胞的H+外排抑制与微粒体钒酸盐敏感的H+-ATP酶（EC 3.6.1.35）抑制及ATP依赖的H+转运抑制相关，而50 mM NaCl耐受细胞对H+外排的刺激与质膜ATP依赖的H+转运增加相关。从50 mM NaCl耐受愈伤组织分离的质膜中，ATP依赖的H+外排增加不是钒酸盐敏感的ATP水解活性刺激或对H+被动通透性增加的结果。相对于NaCl敏感愈伤组织，50 mM NaCl耐受愈伤组织的质膜H+-ATP酶对Mg2+-ATP的Km值较低。我们的结果表明，番茄愈伤组织对50 mM NaCl的耐受性增加了质膜H+-ATP酶对底物ATP的亲和力，并刺激了该酶的H+泵活性，而未改变其磷酸水解活性。
