Ramón Serrano

In vivo glucose activation of the yeast plasma membrane ATPase

The addition of glucose to yeast cells activates proton efflux mediated by the plasma membrane ATPase. Accordingly, the ATPase activity of purified plasma membranes is increased up to 10‐fold. The activated ATPase has a more alkaline pH optimum, better affinity for ATP and greater sensitivity to vanadate than the non‐activated enzyme. All these changes are reversed by washing the cells free of glucose. This suggests two states of the ATPase which are interconverted by a covalent modification. As glucose does not affect the phosphorylation of plasma membrane polypeptides, other type of covalent modification may be involved.

A plant genetically modified that accumulates Pb is especially promising for phytoremediation.

From a number of wild plant species growing on soils highly contaminated by heavy metals in Eastern Spain, Nicotiana glauca R. Graham (shrub tobacco) was selected for biotechnological modification, because it showed the most appropriate properties for phytoremediation. This plant has a wide geographic distribution, is fast-growing with a high biomass, and is repulsive to herbivores. Following Agrobacterium mediated transformation, the induction and overexpression of a wheat gene encoding phytochelatin synthase (TaPCS1) in this particular plant greatly increased its tolerance to metals such as Pb and Cd, developing seedling roots 160% longer than wild type plants. In addition, seedlings of transformed plants grown in mining soils containing high levels of Pb (1572 ppm) accumulated double concentration of this heavy metal than wild type. These results indicate that the transformed N. glauca represents a highly promising new tool for use in phytoremediation efforts.

Enhancement of Abscisic Acid Sensitivity and Reduction of Water Consumption in Arabidopsis by Combined Inactivation of the Protein Phosphatases Type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.

Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump

A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system2–5; in fungi and plants an H+-pumping ATPase6 is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal2. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past2. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase7 in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.

Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters.

ABSTRACT The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1(YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H+-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

Deletion analysis of yeast plasma membrane H+ ‐ATPase and identification of a regulatory domain at the carboxyl‐terminus

The function of the amino‐ and carboxyl‐terminal domains of the yeast plasma membrane H+‐ATPase have been investigated by constructing deletions in vitro and selectively expressing the mutant enzymes in vivo. The first 27 amino acids are dispensable but deletion of a further 33 amino acids greatly decreases the appearance of the enzyme in the plasma membrane. Membrane localization is also prevented by carboxyl‐terminal deletions which include the last hydrophobic stretch, but the last 46 amino acids of the ATPase are not required. Removal of the last 11 amino acids produces an enzyme in glucose‐starved cells with the kinetic parameters of the wild‐type ATPase activated by glucose fermentation. This region seems to constitute a regulatory domain.

Immunolocalization of the plasma membrane H+-ATPase in minor veins of Vicia faba in relation to phloem loading

The immunolocalization of the plasma membrane H+ -ATPase, which generates a proton motive force energizing the uptake of inorganic and organic solutes, was studied by electron microscopy. The cells studied were in minor veins of Vicia faba L. exporting leaves, where photosynthates are supposed to be absorbed from the apoplast by phloem transfer cells. Immunologically detectable H+ -ATPase varied among the different cell types and was considerably denser in the transfer cells than in the other cell types, particularly in the sieve tube. Moreover, the distribution of the H+ -ATPase was not homogeneous in transfer cells, that pump being more concentrated in the region adjacent to the bundle sheath, phloem parenchyma, and xylem vessels than along the smooth part of the wall bordering the sieve tube. These results show that the plasma membrane infoldings of transfer cells possess the proton-pumping machinery required to energize an efficient uptake of photosynthates from the phloem apoplast and an efficient retrieval of nitrogenous compounds from the vascular sap.

Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences

SummaryIn order to probe the physiological role of the yeast plasma membrane ATPase we have replaced the constitutive promoter of its gene by a galactose-dependent promoter. The resulting cells stop growing on glucose medium when the preformed ATPase is diluted to 20% of normal. There is a correlation between ATPase activity and both proton efflux from the cells and amino acid transport. A large proportion of growth-arrested cells appear enlarged and with several buds containing nuclei.

Analysis of the regulatory domain of yeast plasma membrane H+-ATPase by directed mutagenesis and intragenic suppression

The yeast plasma membrane H+‐ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C‐terminus of the enzyme to be involved in this regulation. Site‐directed mutagenesis demonstrates that Arg909 and Thr912 at the C‐terminus are important for the increase in V max of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these ammo acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin‐dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547 → Val) at the catalytic domain, providing genetic evidence for domain interaction.

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-β-glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. ‘Pharo’. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 g l−1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants