Consuelo Montesinos

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

Expression of the yeast HAL2 gene in tomato increases the in vitro salt tolerance of transgenic progenies

Abstract Agrobacterium -mediated transformation has been used to introduce the yeast halotolerant HAL2 as well as the npt II and uid A marker genes into tomato ( Lycopersicon esculentum ) cv. UC82B. Five to six percent of the explants produced transgenic plants. HAL2 expressing transformants were allowed to self-pollinate and salt tolerance assays were performed in vitro on progenies from two independent transgenic plants with different levels of expression of the transgene. In vitro salt tolerance was evaluated according to the level of growth of hypocotyl-derived calli as well as the rooting capability of isolated shootlets on MS-modified medium supplemented with NaCl. Under salt stress, callus formation from hypocotyl explants was higher on both transgenic-derived progenies than in the control. In addition, progenies from the plant with the highest expression of the transgene (2H20b), also showed a higher level of root production on NaCl-supplemented medium. These results suggested a positive effect of the yeast HAL2 gene on the level of salt tolerance in progenies derived from transgenic plants.

A novel role for protein kinase Gcn2 in yeast tolerance to intracellular acid stress

Intracellular pH conditions many cellular systems, but its mechanisms of regulation and perception are mostly unknown. We have identified two yeast genes important for tolerance to intracellular acidification caused by weak permeable acids. One corresponded to LEU2 and functions by removing the dependency of the leu2 mutant host strain on uptake of extracellular leucine. Leucine transport is inhibited by intracellular acidification, and either leucine oversupplementation or overexpression of the transporter gene BAP2 improved acid growth. Another acid-tolerance gene is GCN2, encoding a protein kinase activated by uncharged tRNAs during amino acid starvation. Gcn2 phosphorylates eIF2α (eukaryotic initiation factor 2α) (Sui2) at Ser51 and this inhibits general translation, but activates that of Gcn4, a transcription factor for amino acid biosynthetic genes. Intracellular acidification activates Gcn2 probably by inhibition of aminoacyl-tRNA synthetases because we observed accumulation of uncharged tRNAleu without leucine depletion. Gcn2 is required for leucine transport and a gcn2-null mutant is sensitive to acid stress if auxotrophic for leucine. Gcn4 is required for neither leucine transport nor acid tolerance, but a S51A sui2 mutant is acid-sensitive. This suggests that Gcn2, by phosphorylating eIF2α, may activate translation of an unknown regulator of amino acid transporters different from Gcn4.

Lithium treatment induces a hypersensitive-like response in tobacco

Treatment of tobacco (Nicotiana tabacum L.) plants with lithium induces the formation of necrotic lesions and leaf curling as in the case of incompatible pathogen interactions. Further similarities at the molecular level include accumulation of ethylene and of salicylic and gentisic acids, and induced expression of pathogenesis-related PR-P, PR5 and PR1 genes. With the exception of PR1 induction, lithium produced the same effects in transgenic tobacco plants that do not accumulate salicylate because of overexpression of the bacterial hydroxylase gene nahG. On the other hand, inhibition of ethylene biosynthesis with aminoethoxyvinylglycine prevented lithium-induced cell death and PR5 expression. These results suggest that lithium triggers a hypersensitive-like response where ethylene signalling is essential.

Domains of yeast plasma membrane and ATPase-associated glycoprotein

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

The role of K+ and H+ transport systems during glucose‐ and H2O2‐induced cell death in Saccharomyces cerevisiae

Glucose, in the absence of additional nutrients, induces programmed cell death in yeast. This phenomenon is independent of yeast metacaspase (Mca1/Yca1) and of calcineurin, requires ROS production and it is concomitant with loss of cellular K+ and vacuolar collapse. K+ is a key nutrient protecting the cells and this effect depends on the Trk1 uptake system and is associated with reduced ROS production. Mutants with decreased activity of plasma membrane H+‐ATPase are more tolerant to glucose‐induced cell death and exhibit less ROS production. A triple mutant ena1‐4 tok1 nha1, devoid of K+ efflux systems, is more tolerant to both glucose‐ and H2O2‐induced cell death. We hypothesize that ROS production, activated by glucose and H+‐ATPase and inhibited by K+ uptake, triggers leakage of K+, a process favoured by K+ efflux systems. Loss of cytosolic K+ probably causes osmotic lysis of vacuoles. The nature of the ROS‐producing system sensitive to K+ and H+ transport is unknown. Copyright

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton‐pumping H+‐ATPase Pma1 (which drives nutrient and K+ uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K+ uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K+ transport.

A fungal transcription factor gene is expressed in plants from its own promoter and improves drought tolerance

AbstractMain conclusionA fungal gene encoding a transcription factor is expressed from its own promoter inArabidopsisphloem and improves drought tolerance by reducing transpiration and increasing osmotic potential. Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the ArabidopsisUBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacteriumtumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with β-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology.

Crucial Reactions for Salt Tolerance in Yeast

Random over-expression of genes in multicopy plasmids and gene disruptions have uncovered rate-limiting steps for salt tolerance in the model organism Saccharomyces cerevisiae (baker’s yeast). In cells growing in glucose media intracellular sodium toxicity is the major component of salt stress. In minimal synthetic media sodium toxicity primarily affects methionine biosynthesis. This is due to sodium inhibition of the 3′,5′-bisphosphate nucleotidase encoded by the HAL2/MET22 gene. The sodium-extrusion ATPase encoded by the ENA1/PMR2 gene is the major determinant of sodium homeostasis, together with its complex regulatory system, which includes the calcineurin and HAL3 (HAL1) pathways.