Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

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 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M 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 m M 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 m M 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 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M 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.     

The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea

Exposure of the hyperaccumulator Alyssum lesbiacum to nickel (Ni) is known to result in a dose-dependent increase in xylem sap concentrations of Ni and the chelator free histidine (His). Addition of equimolar concentrations of exogenous L-His to an Ni-amended hydroponic rooting medium enhances Ni flux into the xylem in the nonaccumulator Alyssum montanum, and, as reported here, in Brassica juncea L. cv Vitasso. In B. juncea, reducing the entry of L-His into the root by supplying D-His instead of L-His, or L-His in the presence of a 10-fold excess of L-alanine, did not affect root Ni uptake, but reduced Ni release into the xylem. Compared with B. juncea, root His concentrations were constitutively about 4.4-fold higher in A. lesbiacum, and did not increase within 9 h of exposure to Ni. Cycloheximide did not affect root His or Ni concentrations, but strongly decreased the release of His and Ni from the root into the xylem of A. lesbiacum, whereas xylem sap concentrations of Ca and Mg remained unaffected. Near-quantitative chelation of Ni with nitrilotriacetate in the rooting medium did not enhance Ni flux into the xylem of A. lesbiacum and B. juncea, suggesting the absence of a significant apoplastic pathway for Ni entry into the xylem. The data suggest that in B. juncea roots, Ni(2+) uptake is independent of simultaneous uptake of His. In both species, enhanced release of Ni into the xylem is associated with concurrent release of His from an increased root free His pool.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.