Salinity and drought tolerance of mannitol-accumulating transgenic tobacco

Tobacco plants (Nicotiana tabacum L.) were transformed with a mannitol-1-phosphate dehydrogenase gene resulting in mannitol accumulation. Experiments were conducted to determine whether mannitol provides salt and/or drought stress protection through osmotic adjustment. Non-stressed transgenic plants were 20–25% smaller than non-stressed, non-transformed (wild-type) plants in both salinity and drought experiments. However, salt stress reduced dry weight in wild-type plants by 44%, but did not reduce the dry weight of transgenic plants. Transgenic plants adjusted osmotically by 0.57 MPa, whereas wild-type plants did not adjust osmotically in response to salt stress. Calculations of solute contribution to osmotic adjustment showed that mannitol contributed only 0-003-0-004 MPa to the 0.2 MPa difference in full turgor osmotic potential (π_o) between salt-stressed transgenic and wild-type plants. Assuming a cytoplasmic location for mannitol and that the cytoplasm constituted 5% of the total water volume, mannitol accounted for only 30–40% of the change in π_o of the cytoplasm. Inositol, a naturally occurring polyol in tobacco, accumulated in response to salt stress in both transgenic and wild-type plants, and was 3-fold more abundant than mannitol in transgenic plants. Drought stress reduced the leaf relative water content, leaf expansion, and dry weight of transgenic and wild-type plants. However, π_o was not significantly reduced by drought stress in transgenic or wild-type plants, despite an increase in non-structural carbohydrates and mannitol in droughted plants. We conclude that (1) mannitol was a relatively minor osmolyte in transgenic tobacco, but may have indirectly enhanced osmotic adjustment and salt tolerance; (2) inositol cannot substitute for mannitol in this role; (3) slower growth of the transgenic plants, and not the presence of mannitol per se, may have been the cause of greater salt tolerance, and (4) mannitol accumulation was enhanced by drought stress but did not affect π_o or drought tolerance.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Calcium signaling-mediated and differential induction of calmodulin gene expression by stress in Oryza sativa L

Ca(2+)/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of Ca(2+)-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic Ca(2+)-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stressinduced expression of OsCam1 gene requires the [Ca(2+)]cyt elevation that is known to occur in response to these stimuli.     

A wound-inducible tobacco peroxidase gene expresses preferentially in the vascular system

A tobacco peroxidase gene tpoxN1 was reported to be expressed within 1 h after wounding in leaves [Hiraga et al. (2000a) Plant Cell Physiol. 41: 165]. We describe here further results on the wound-induced tpoxN1 expression. The quick tpoxN1 induction occurred preferentially in stems and petioles, but was negligible in leaf blades even 8 h after wounding. Induced GUS activity was also detected rapidly after wounding in the stem of transgenic tobacco plants carrying the tpoxN1 promoter::GUS fusion gene, localized mainly in the vascular systems where it was maintained this level for 14 d or more. Strong GUS activity was also found in the petiole and veinlet as well as the epidermal tissue in the stem. Treatment of known inducers for wound-responsive genes such as jasmonate, 1-aminocyclopropane-1-carboxylate, spermine, phytohormones and other stress treatments did not enhance wound-induced tpoxN1 gene expression in stems at all, but rather repressed it in some cases. Studies using metabolic inhibitors suggested that phosphorylation and dephosphorylation of proteins together with de novo protein synthesis are likely to be involved in the wound-induced tpoxN1 expression as well as some other wound-responsive genes. Thus, tpoxN1 is a unique wound-inducible and possible wound-healing gene which is rapidly expressed being maintained for a long time in veins via an unknown wound-signaling pathway(s).     

Osmotic regulation of prolactin secretion. Possible role of chloride

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.     

Wound- and systemin-inducible calmodulin gene expression in tomato leaves

Using a calmodulin (CaM) cDNA as a probe in northern analyses, transgenic tomato plants that overexpress the prosystemin gene were found to express increased levels of CaM mRNA and protein in leaves compared to wild-type plants. These transgenic plants have been reported previously to express several wound-inducible defense-related genes in the absence of wounding. Calmodulin mRNA and protein levels were found to increase in leaves of young wild-type tomato plants after wounding, or treatment with systemin, methyl jasmonate, or linolenic acid. CaM mRNA appeared within 0.5 h after wounding or supplying young tomato plants with systemin, and peaked at 1 h. The timing of CaM gene expression is similar to the expression of the wound- or systemin-induced lipoxygenase and prosystemin genes, signal pathway genes whose expression have been reported to begin at 0.5–1 h after wounding and 1–2 h earlier than the genes coding for defensive proteinase inhibitor genes. The similarities in timing between the synthesis of CaM mRNA and the mRNAs for signal pathway components suggests that CaM gene expression may be associated with the signaling cascade that activates defensive genes in response to wounding.     

Influence of cell turgor on sucrose partitioning in potato tuber storage tissues

Sucrose uptake and partitioning in potato (Solanum tuberosum L.) tuber discs were examined under a range of mannitol and ethylene-glycol concentrations. Mannitol caused the same changes in turgor over a wide range of incubation periods (90 min-6 h), indicating that it did not penetrate the tissue. In comparison, ethylene glycol reduced turgor losses but did not eliminate them, even after 6 h. Between 100 mM and 300 mM mannitol, turgor fell by 350 kPa, compared with 35 kPa in ethylene glycol. Uptake experiments in mannitol alone showed that total sucrose uptake was strongly correlated with both osmotic potential and with turgor potential. In subsequent experiments sucrose uptake and partitioning were examined after 3 h equilibration in 100 mM and 300 mM concentrations of mannitol and ethylene glycol. Total sucrose uptake and the conversion of sucrose to starch were enhanced greatly only at 300 mM mannitol, indicating an effect of turgor, rather than osmotic potential on sucrose partitioning. The inhibitors p-chloromercuribenzenesulfonic acid and carbonylcyanide m-chlorophenylhydrazone (CCCP) both reduced sucrose uptake, but in quite different ways. p-Chloromercuribenzenesulfonic acid reduced total sucrose uptake but did not affect the partitioning of sucrose to starch. By contrast, CCCP inhibited total uptake and virtually eliminated the conversion of sucrose to starch. Despite this, sucrose uptake in the presence of CCCP continued to increase as the mannitol concentration increased, indicating an increase in passive transport at higher mannitol concentrations. Increased sucrose uptake above 400 mM mannitol was shown to be the result of uptake into the free space. The data show that starch synthesis is optimised at low but positive turgors and the relation between sucrose partitioning and the changing diurnal water relations of the tuber are discussed.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid     

Enhanced salt stress tolerance in transgenic potato plants (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) expressing a bacterial <Emphasis Type="Italic">mtl</Emphasis>D gene

Bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene was introduced into potato (Solanum tuberosum L.) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants were selected on a medium containing 100 mg l⁻¹ kanamycin and confirmed by polymerase chain reaction (PCR), Southern blotting, and RT-PCR analyses. All of the selected transformants accumulated mannitol, a sugar alcohol that is not found in wildtype potato. Experiments designed for testing salt tolerance revealed that there was enhanced NaCl tolerance of the transgenic lines both in vitro and in hydroponic culture. Compared to 0 mM NaCl, the shoot fresh weight of wildtype plants was reduced by 76.5% at 100 mM NaCl under hydroponic conditions. However, under the same condition, the shoot fresh weight of transgenic plants was reduced only by 17.3%, compared to 0 mM NaCl treatment. The improved tolerance of this transgenic line may be attributed to the induction and progressive accumulation of mannitol in the roots and shoots of the plants. In contrast to in vitro experiments, the mannitol content in the transgenic roots and shoots increased at 50 mM NaCl and decreased slightly at 75 and 100 mM NaCl, respectively. Overall, the amount of accumulated mannitol in the transgenic lines was too small to act as an osmolyte; thus, it might act as an osmoprotectant. However, the results demonstrated that mannitol had more contribution to osmotic adjustment in the roots (but not in shoots). Finally, we concluded that mtlD expression in transgenic potato plants can significantly increase the mannitol accumulation that contributes to the enhanced tolerance to NaCl stress. Furthermore, although this enhanced tolerance resulted mainly from an osmoprotectant action, an osmoregulatory effect could not be ruled out.     

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.     

Effects of salt and osmotic shocks on unadapted and adapted callus lines of tobacco

Growth, viability and proline content of adapted and unadapted calluses of Nicotiana tabacum L. var. Jayasri, affected due to osmotic stresses and particularly to stress-shocks treated with different osmotica like NaCl (ionic-penetrating), mannitol (non-ionic-penetrating) and polyethylene glycol, (PEG) (non-ionic-non penetrating) were studied to evaluate the physiological differences of stress effects. The tissues adapted to a low concentration of NaCl (85 mM) showed low growth with high proline content compared to the tissues adapted to a low concentration of mannitol (165 mM). Proline content was similar in tissues adapted to high concentrations of NaCl (171 mM) and mannitol (329 mM) but growth in the latter case was relatively low. Growth and viability were subsequently correlated with the pattern of retention in or diffusion of proline out of the tissues after shock-treatments. The loss of tissue viability of the adapted calluses was comparatively less than the unadapted callus even after shock-treatments with 1282 mM NaCl and 823 mM mannitol. The former calluses retained the capability of regrowth though at a slow rate. Such adapted tissues also retained more proline. The mannitol-adapted tissues, when shocked with PEG (200 g l-1), showed low viability with more diffusion and a very little retention of proline while, in the unadapted tissue, all the proline was leached out. The results indicated that the effects of different osmotica on plant tissue varied depending upon the physico-chemical nature of the compounds used as stress-inducing-agents, and retention and diffusion of proline was altered when the tissues were shocked with high concentrations of all these compounds. This revised version was published online in June 2006 with corrections to the Cover Date.