Comunicação em painel no congresso "14th Congress of the Federation of European Societies of Plant Biology". August 23-27. 2004. Cracow. Poland.Fundação para a Ciência e a Tecnologia (FCT

Façanha, Arnoldo

Gerós, H.

Silva, Paulo

Tavares, R. M.

Acta Physiologiae Plantarum

Universidade do Minho: RepositoriUM

INTRODUCTION+ +Efficient exclusion of Na  excess from the cytoplasm and vacuolar Na  accumulation are the main mechanisms for the adaptation of plants to salt stress. P. euphratica is the only tree species that occurs naturally in semiarid areas. It is a halophytic plant, tolerating salt and drought stress, and has been used as a model to study plant defense mechanisms against salt stress. The aim of this work was to contribute to the elucidation of mechanisms underlying salt tolerance in P. euphratica using heterotrophic cell suspensions as a biological model. Tonoplast vesicles were purified from cells grown in medium containing different salt concentrations and used for the determination + +of the activity of proton pumps and Na /H  exchanger.RESULTSFigure 2 depicts the growth of P. euphratica cell suspensions in MS medium with 2.5% sucrose in the presence and absence of 150 mM NaCl. Cells grown with 150 mM NaCl exhibited the same -1maximal specific growth rate (µ = 0.2 day ) than those grown without salt, however, the lag phase maxwas longer.Growth in batch cultures with NaClReferences+ +- Façanha, A.R., de Meis, L. 1998. Reversibility of H -ATPase and H -Pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds. Plant Physiol 116: 1487-1495.+ + +- Venema, K., Quintero, F. J., Pardo, J. M. and Donaire, J. P. 2002. The Arabidopsis Na /H  exchanger AtNHX1 catalizes low affinity Na  +and K  transport in reconstituted liposomes. J Biol Chem 277: 2413-2418.Acknowledgements: Paulo Silva is supported by FCT, grant ref. SFRH/BD/13460/2003Carlos Conde is supported by FCT, grant ref. SFRH/BD/10689/2002Preparation of tonoplast vesiclesCells were homogenised in a Polytron at 4ºC. After removal of the mitochondrial fraction, microsomal fraction was obtained by centrifugation at 100,000xg, resuspended and layered onto a discontinuous sucrose gradient (32/46%). After centrifugation, tonoplast fraction was collected from the top of 32% layer of the gradient (Figure 3) and sedimented at 180,000xg.Activity of tonoplast proton pumpsIn P. euphratica suspension cultured cells, V-PPase (pyrophosphatase) seems to be the main + + -1tonoplast H  pump (Figure 4). Activity was as follows: H  pumping K , 3.4 µM and V , 960 ∆%F min  m max-1 -1 -1mg  protein; PPi hydrolysis K , 44 µM and V , 94 nmol PPi min  mg  protein (Figure 5 and Figure 6). m maxSaturating substrate concentration of V-PPase (0.1 mM PPi) is about 10-fold lower than V-ATPase (1 mM ATP).+ +Activity of Na /H  exchanger+ + +The activity of the tonoplast Na /H  exchanger, monitored as the ability of Na  to dissipate an established pH gradient, was negligible in cells grown without salt, however, the increase of salt in the culture medium up to 150 mM promoted a 8-fold increase of the exchanger activity (Figure 8).Concluding remarks+! Two electrogenic H  pumps, V-ATPase and V-PPase, generate and maintain the electrochemical gradient across the vacuolar membrane of P. euphratica. + +! V-PPase is the predominant H  pump in vacuoles of P. euphratica, since its H  pumping activity is much higher than that of V-ATPase. Also, the V  of its activity is achieved at 10-fold lower substrate maxconcentration when compared to that of V-ATPase.+ +! Na /H  exchange activity is negligible in cells grown without salt; activity is induced abruptly when +suspension cells were grown with salt, indicating the important role of the antiporter in Na  detoxification.Figure 7. Initial velocities of proton pumping of P. euphratica V-PPase in the absence (¢) and presence (£) of 100 mM NaCl (A). Inhibition of V  of proton pumping by NaCl in the assay medium (B).max+ + +Role of tonoplast H -pyrophosphatase and Na /H  antiporter in salt tolerance of Populus euphratica Oliv.P. Silva , C. Conde , A. R. Façanha , R. M. Tavares  and H. Gerós1 1 2 1 11  | | |  |  | Centro de Biologia  Departamento de Biologia Universidade do Minho Campus de Gualtar Braga Portugal2 | | |Centro de Biociências e Biotecnologia  Universidade Estadual do Norte Fluminense Darcy Ribeiro  Campos dos Goytacazes  BrasilUENFUniversidade Estadual do Norte Fluminense Darcy RibeiroFigure 3. Populus euphratica plasma membranes (←) and vacuolar membranes (⇐) after the fractionation procedure.⇐←Figure 4. Proton pumping activity of V-PPase (A) and V-ATPase (B) in tonoplast vesicles from P. euphratica cultured cells. The accumulation of protons inside the vesicles was determined by measuring the fluorescence quenching of ACMA (Façanha and de Meis, 1998).CFigure 2. Growth of P. euphratica cultured cells in the presence and absence of NaCl.+Figure 5. Kinetic analysis of P. euphratica V-PPase. Initial velocities of PPi hydrolysis (A) and H  pumping (B).A B+Figure 9. Ion specificity of tonoplast H -coupled exchange activity in salt treated P. euphratica suspension cultured cells.+ +Figure 8. Plasma membrane Na /H  exchange activity in tonoplast vesicles isolated from P. euphratica suspension cells cultivated in the absence (A) and presence of 50 mM (B) and 150 mM (C) of NaCl.Figure 1. Populus euphratica in the deserts of western China (A) and cell suspension culture from calli as a model system to study salt stress defence mechanisms (B).A BFigure 6. Hydrolysis activity (V ) of V-PPase and maxV-ATPase in tonoplast vesicles from P. euphratica suspension cells cultivated in the absence and presence of salt.When cells were cultivated in the presence of up to 150 mM salt, V-ATPase hydrolysis activity increased but not V-PPase activity (Figure 6).+NaCl behaved as an uncompetitive inhibitor of V- PPase (Figure 7). V  of H  pumping decreased max-1according to an exponential inhibition kinetics: k  = 4.6 M ; C  = 158 mM.i 50+ +As the exchanger AtNHX1 from Arabidopsis (Venema et al., 2002), P. euphratica vacuolar Na /H  + +exchanger catalyses low affinity Na  transport (K  ≈ 100 mM); Li  is also a substrate for this antiporter m(Figure 9).-As shown in Figure 9, Cl  is also able to accumulate in the vacuole following its electrochemical gradient, the depolarizing effect causing an increase of proton pumping.A BUniversidade do Minho

Role of tonoplast H+ pyrophosphatase and Na+/H+ antiporter in salt tolerance of populus euphratica Oliv.

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Role of tonoplast H+ pyrophosphatase and Na+/H+ antiporter in salt tolerance of populus euphratica Oliv.

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