Juan Pedro Donaire

Changes induced by NaCl in lipid content and composition, lipoxygenase, plasma membrane H+-ATPase and antioxidant enzyme activities of tomato (Lycopersicon esculentum. Mill) calli

Tomato (Lycopersicon esculentum Mill. cv. Pera) calli tolerant to 50 mM NaCl were obtained by successive subcultures in NaCl supplemented medium. Salt-tolerant calli showed an increase of fresh and dry weight respective to control calli. When control and 50 mM NaCl-tolerant calli were stressed with 100 mM NaCl for 48 h, a decrease in respiration rate of 32 and 9%, respectively, was observed. Relative proportions of phospholipid fatty acids and free-sterol molecular species were the same in both control and NaCl tolerant calli. While the content of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased in salt-tolerant calli, the free sterol content was similar in both cases. A substantial increase of vanadate-sensitive ATP-dependent H+ pumping activity without any modification in specific phosphohydrolytic activity and in passive H+ conductance was detected in microsomes from salt-tolerant calli, which could be explained by an increased coupling between H+ pumping and ATP hydrolysis. The higher lipoxygenase and antioxidant enzyme activities such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione-S-transferase in 50 mM NaCl-tolerant calli as compared to controls also suggest that salt-tolerant calli has a high capacity of polyunsaturated fatty acid hydroperoxide formation and active oxygen species scavenging.

Regulation of root plasma membrane H+-ATPase in sunflower seedlings

Abstract We have developed specific assays for the levels of mRNA, antigenic protein and catalytic activity under standard conditions of the root plasma membrane H + -ATPase of sunflower ( Helianthus annuus L. var. HSE) seedlings. During the first 6 days of root development there is a parallel increase in the levels of mRNA, antigenic protein and activity. No significant changes were induced by alkaline pH or NaCl. Acetic acid at pH 4 reduces the level of mRNA more than the levels of antigenic protein and activity (to 30% and 50–60% of controls, respectively). Illumination of the aerial part of the seedlings reduces the antigenic protein and activity in the roots to 50–60% of controls without affecting the level of mRNA. These results suggest that there is regulation of the expression of root plasma membrane H + -ATPase by development and environmental conditions.

Effects of salt stress on H+-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots

It has previously been shown that mild and severe salt-stress increased both ATP- and PPi-dependent H+ transport and induced a Na+/H+ exchange activity in tonoplast vesicles from sunflower seedling roots. In this study, the vanadate-sensitive H+-ATPase activity was characterized in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots exposed for 3 days to 0, 75 or 150 mM NaCl. Blots immunoassayed with an Arabidopsis thaliana plasma membrane ATPase polyclonal antibody revealed the existence of a band of about 100 kDa, which was highly enriched in the 32/43% sucrose interface. ATP hydrolyzing activity in this fraction was mostly inhibited by vanadate, and scarcely by azide, oligomycin, nitrate and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Properties of vanadate-sensitive ATPase activity, such as inhibitor sensitivity, pH optimum, substrate specificity, ion effects and kinetic data were consistent with those of other plasma membrane ATPases. Although the pH profile, ion dependence, apparent Km and the amount of antigenic protein were unaffected by salt-stress, the Vmax of the vanadate-sensitive ATPase activity (measured in the presence of detergent) was reduced as a function of salt treatments. Likewise, while mild and severe salt-stress reduced the basal ATP hydrolyzing activity (measured in the absence of detergent) about a 35 and 55%, respectively, the ATP-dependent H+ transport activity sensitive to vanadate remained unchanged. As a consequence, the ratio of H+ pumping to the basal phosphohydrolase activity was proportionally increased. These results and those previously reported at the sunflower tonoplast suggest that the activity of H+ pumps under salt stress can form part of a tolerance mechanism in sunflower roots, which could regulate the ion fluxes (Na+ and Cl−) across the tonoplast and plasma membrane.

Metabolic changes in fruit and leaf during ripening in the olive

Abstract Changes in the content of several metabolites (fat, sugars, protein nitrogen, uronic acids and Krebs cycle acids) were studied in fruits and leaves of Olea europaea cv. Marteno during fruit development. Fat accumulation parallels fruit development, and fat and sugar levels in both fruit and leaf follow similar patterns until maturation. Then, while the fruit fat content remains at its previous high level, the leaf fat drops to its low initial level. By contrast, the sugar content of both fruit and leaf returns to its low starting value. The total Krebs cycle acids seems to be in an inverse relationship in leaf and fruit during its development. Also a high level in uronic acids was observed in fruit during ripening. Finally, the protein nitrogen content slowly drops in the fruit during development with a small increase at maturity.

Effect of boron on plasma membrane proton extrusion and redox activity in sunflower cells

Abstract The effect of boron on H + extrusion and FeCN reduction in sunflower cell suspensions and on vanadate-sensitive H + -ATPase and NADH-FeCN reductase activities of isolated microsomes was investigated. Boron deficiency inhibited FeCN-dependent H + extrusion and FeCN reduction in cells grown in darkness or at a light irradiance of 30 μmol · m −2 · s −1 . In addition, an inhibition of FeCN-independent H + extrusion by boron deficiency was detected when cells were maintained at a light irradiance of 30 μmol · m −2 · s −1 . FeCN-dependent and FeCN-independent H + extrusion were stimulated by FC and inhibited by vanadate and DCCD. However, FeCN reduction was not affected either by FC or by vanadate. In isolated microsomes, boron deficiency inhibited the vanadate-sensitive H + -ATPase and NADH-FeCN reductase activities. The presence of 2,4-D in the assay medium provoked an inhibition of H + extrusion and FeCN reduction in cells grown with 0.1 mM H 3 BO 3 ; in the same way, the addition of 2,4-D to the reaction medium inhibited the NADH-FeCN reductase activity of microsomes isolated from cells grown in media with boron. These data support the existence of a relationship between boron and auxins. Likewise, the present results suggest that boron could regulate H + extrusion in sunflower cells through an effect on plasma membrane H + -ATPase and redox systems.

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.

Effect of sodium chloride on growth, ion content, and hydrogen ion extrusion activity of sunflower and jojoba roots

Abstract The effect of sodium chloride (NaCl) on growth, internal ion content, and proton release of sunflower (Helianthus annuus L. cv. Enano) and jojoba [Simmondsia chinensis (Link) Schneid.] roots was studied in order to understand the relationship between the above parameters and salt tolerance. The increase in sodium (Na) ions concentration and the decrease in calcium (Ca) ion concentration in sunflower roots grown in the presence of 50 mM NaCl, relative to control without added NaCl, run parallel to the reduction in growth capacity and in hydrogen (H) ion extrusion activity. However, while the rate of growth was not affected by the addition of Ca to a culture medium containing 50 mM NaCl, H‐ion extrusion activity was reversed, reaching a similar value to that of the roots cultivated in deionized water or in 25 mM NaCl. In jojoba, a reduction of root growth at 100 and 150 mM NaCl was observed, while no significant effects on Ca and potassium (K) contents and on H‐ion extrusion activity were found. Th...

Heterologously expressed protein phosphatase calcineurin downregulates plant plasma membrane H+‐ATPase activity at the post‐translational level

To investigate the effects of calcineurin expression on cellular ion homeostasis in plants, we have obtained a transgenic cell culture of tomato, expressing constitutively activated yeast calcineurin. Transgenic cells exhibited reduced growth rates and proton extrusion activity in vivo. We show that reduction of plasma membrane H+‐ATPase activity by expression of calcineurin is the basis for the observed phenotypes. Transgenic calli and cell suspensions displayed also increased salt tolerance and contained slightly higher Ca2+ and K+ levels. This demonstrates that calcineurin can modulate ion homeostasis in plants as it does in yeast by affecting the activity of primary ion transporters.

Effect of Fusicoccin on the Early Infection Process of Legume Roots by Rhizobium spp

Nod factors, the first detectable signals produced by Rhizobium spp., were reported to induce cytosolic pH changes and plasma membrane depolarization in root hairs as specific responses. In this study, it has been found that fusicoccin inhibits nodulation of alfalfa roots. This inhibition was only observed when fusicoccin was applied in the earlier steps of the bacteria-plant interaction. The observed effect is similar to that caused by the undissociated permeant acetic acid, which decreases the cytoplasmic pH and, like fusicoccin, significantly stimulates net H+ efflux. These results suggest that the fusicoccin-induced plasma membrane H+-ATPase activity and membrane hyperpolarization could be responsible for the nodulation inhibition observed. Moreover, it was found that nodulation was inhibited by removing external calcium with EGTA. When fusicoccin is present, a lower concentration of EGTA is necessary to inhibit nodulation. Furthermore, the addition of Ca2+ ionophore A23187 was found to inhibit H+ eff...

H+ATPase activities of tonoplast-enriched vesicles from non-treated and NaCl-treated jojoba roots

Abstract Tonoplast-enriched vesicles prepared by discontinuous sucrose density gradient centrifugation of microsomal membranes from jojoba ( Simmondsia chinensis (Link) Schneid. ) roots grown with or without 100 mM NaCl were analysed for nitrate-sensitive ATPase and ATP-dependent H + -pumping activities as well as for total phospholipid and free sterol content. The inhibitor sensitivity and pH optimum of the ATP hydrolysis and H + -pumping activities match quite closely those reported for the tonoplast ATPase of other plant materials. NaCl treatment increased the V max of the nitrate-sensitive ATPase for ATP hydrolysis and of the initial rate of ATP-dependent H + -pumping but did not cause alteration of the apparent K m values. The magnitude of the increase in ATPase activity in response to NaCl is about 25%. Palmitic, oleic and linoleic acids were the three major fatty acid components of total phospholipids of tonoplast fraction, whereas the most abundant free sterols were campesterol, stigmasterol and, above all, sitosterol. The percentages of phospholipid fatty acids and free sterol molecular species were not altered whereas the content of these lipids increased when roots were grown in 100 mM NaCl. Further studies will examine the regulation by NaCl of the tonoplast ATPase, and the role of different phospholipid classes of enriched-tonoplast fraction in NaCl stress response.