María Pilar Rodríguez-Rosales

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.

Expression of LeNHX isoforms in response to salt stress in salt sensitive and salt tolerant tomato species

In general, wild tomato species are more salt tolerant than cultivated species, a trait that is related to enhanced Na(+) accumulation in aerial parts in the wild species, but the molecular basis for these differences is not known. Plant NHX proteins have been suggested to be important for salt tolerance by promoting accumulation of Na(+) or K(+) inside vacuoles. Therefore, differences in expression or activity of NHX proteins in tomato could be at the basis of the enhanced salt tolerance in wild tomato species. To test this hypothesis, we studied the expression level of four NHX genes in the salt sensitive cultivated species Solanum lycopersicum L. cv. Volgogradskij and the salt tolerant wild species Solanum pimpinelifolium L in response to salt stress. First, we determined that in the absence of salt stress, the RNA abundance of LeNHX2, 3 and 4 was comparable in both species, while more LeNHX1 RNA was detected in the tolerant species. LeNHX2 and LeNHX3 showed comparable expression levels and were present in all tissues, while LeNHX4 was expressed above all in stem and fruit tissues. Next, we confirmed that the wild species was more tolerant and accumulated more Na(+) in aerial parts of the plant. This correlated with the observation that salt stress induced especially the LeNHX3 and LeNHX4 isoforms in the tolerant species. These results support a role of NHX genes as determinants of salt tolerance in tomato, inducing enhanced Na(+) accumulation observed in the wild species when grown in the presence of NaCl.

Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K+/H+ antiporter with a chloroplast transit peptide

KEA genes encode putative K(+) efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K(+) efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(+)(K(+))/H(+) antiporter Nhx1p to confer hygromycin resistance and tolerance to Na(+) or K(+) stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H(+) exchange with preference for K(+)=Cs(+)>Li(+)>Na(+). When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K(+)/H(+) antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.

Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes

Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H(+) with K(+) and, to lesser extent, Na(+) . Here, we investigated the response to NaCl supply and K(+) deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K(+) the opposite was found. Analysis of mineral composition showed a higher K(+) content in roots, shoots and xylem sap of transgenic plants and no differences in Na(+) content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na(+)/H(+) and, above all, K(+)/H(+) transport activity in root intracellular membrane vesicles. Under K(+) limiting conditions, transgenic plants enhanced root expression of the high-affinity K(+) uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K(+) depletion rates and half cytosolic K(+) activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K(+) and lower cytosolic K(+) activity than untransformed plants. These results indicate the fundamental role of K(+) homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.

Envelope K+/H+ Antiporters AtKEA1 and AtKEA2 Function in Plastid Development

Antiporters localized to polar microdomains of dividing and developing plastids affect thylakoid membrane formation and chloroplast differentiation. It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K+/H+ antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation.

Involvement of SlSOS2 in tomato salt tolerance.

The Ca2+-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na+ and K+ under saline conditions. We recently identified and functionally characterized by complementation studies in yeast and Arabidopsis the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2.1 We also show evidences on the biotechnological potential of SlSOS2 conferring salt tolerance to transgenic tomato. The increased salinity tolerance of SlSOS2 overexpressing plants is associated with higher sodium content in stems and leaves. SlSOS2 overexpression upregulates the Na+/H+ exchange at the plasma membrane (SlSOS1) and K+,Na+/H+ antiport at the endosomal and vacuolar compartments (LeNHX2 and LeNHX4). Therefore, SlSOS2 seems to be involved in tomato salinity tolerance through regulation of Na+ extrusion from the root, active loading of Na+ into the xylem and Na+ and K+ compartmentalization.

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...