Role of ENA ATPase in Na + efflux at high pH in bryophytes Ana Fraile-Escanciano Æ Blanca Garciadebla ´s Æ Alonso Rodrı ´guez-Navarro Æ Begon ˜a Benito Received: 6 May 2009 / Accepted: 13 August 2009 / Published online: 12 September 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Potassium or Na ? efflux ATPases, ENA ATP- ases, are present in all fungi and play a central role in Na ? efflux and Na ? tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na ? efflux in Saccharomyces cerevisi- ae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na ? efflux in planta and that they exist in more bryophytes than P. patens. For these demonstra- tions (1) we cloned a third ATPase from P. patens, PpE- NA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Dppena1 and Dppena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na ? efflux at high pH depended on PpENA1. The ENA1 ATPase of M. poly- morpha suppressed the defective growth of a S. cerevisiae mutant at high K ? or Na ? concentrations, especially at high K ? . Keywords ENA ATPases Á Na ? efflux Á Physcomitrella Á Marchantia Introduction As a general rule, Na ? is a toxic cation to all living cells, which keep high K ? and low Na ? concentrations in their cytoplasm. To maintain this cytoplasmic environment, all cells have transport systems that mediate K ? accumulation and Na ? extrusion. The number of these transport systems may be low in cells that are exposed to constant K ? and Na ? concentrations but are numerous in cells that can thrive in different types of environments or that are exposed to environments that can drastically change their ionic composition. This variability of environments applies to plants, which use different types of K ? and Na ? trans- porters, depending on environmental conditions (Rodrı ´- guez-Navarro 2000; Pardo and Quintero 2002; Ve ´ry and Sentenac 2003). In addition to cellular extrusion, Na ? may be sequestered into the vacuole or other organelles by means of specific organelle transport systems, thus allevi- ating its toxic effect in the cytoplasm (Pardo et al. 2006; Munns and Tester 2008). In summary, the level of Na ? toxicity exerted on plants by a certain ionic environment depends on the cationic steady states established by the transporters that the plant expresses in each condition. Among these transporters those which mediate Na ? efflux play a central role (Haro et al. 1993; Zhu 2003; Munns and Tester 2008). In general terms plant and fungal cells use transporters of the same families for transmembrane movements of K ? and Na ? and for plasma membrane energization (Rodrı ´- guez-Navarro 2000; Serrano and Rodrı ´guez-Navarro 2001). An apparent exception to this general rule is the fungal K ? or Na ? efflux ATPase (ENA ATPase). This ATPase is universal in fungi (Benito et al. 2002) but has not been found in plants (Garciadeblas et al. 2001), with the exception of the moss Physcomitrella patens (Benito A. Fraile-Escanciano Á B. Garciadebla ´s Á A. Rodrı ´guez-Navarro Á B. Benito (&) Centro de Biotecnologı ´a y Geno ´mica de Plantas, Universidad Polite ´cnica de Madrid, Campus Montegancedo, carretera M-40, km 37.7, 28223 Pozuelo de Alarco ´n, Madrid, Spain e-mail: begona.benito@upm.es 123 Plant Mol Biol (2009) 71:599–608 DOI 10.1007/s11103-009-9543-5