Salt Toxicity Research Articles

Genotype-Dependent Differential Responses of Three Forage Species to Calcium Supplement in Saline Conditions

ABSTRACT Salt toxicity comprises of osmotic and ionic components both of which can severely affect root and shoot growth. In many crop species, supplemental calcium (Ca) reduces the inhibition of growth typical of exposure to salt stress. The objective of this study was to compare whole plant growth and physiological responses to interactive effect of salinity and Ca level on three forage species [African millet (AM), tall wheat grass (TW), and perennial ryegrass (PR)] differing in tolerance to sodium chloride (NaCl) salinity. Plants were grown under glasshouse condition and supplied with nutrient solution containing 0, 100, and 250 mM NaCl supplemented with 0.5, 5, or 10 mM calcium chloride (CaCl2). Plant growth, ionic concentration, water relations, and solute (proline and glycinebetaine) concentrations of the plants were determined two weeks after the salinity treatments. At 100 mM NaCl, there was a moderate reduction in dry matter (DM) production of all three species. A drastic decrease in DM occurred at 250 mM NaCl. Supplemental Ca reduced the adverse effects of salinity on all three species. The TW showed higher shoot and root growth in 100 and 250 mM NaCl than AM and PR. It also showed the highest DM at 5 and 10 mM Ca supplement. The shoot and root DM of TW increased by about 45 and 15%, respectively compared to the control. Chemical analysis indicated that in TW, Ca restricted both uptake and transport of sodium (Na) from root to shoot. It also increased Ca and potassium (K) concentrations in both organs. The transport of K and Ca from root to shoot of AM and PR were decreased by NaCl, but were restored with increasing Ca in the medium. The opposite occurred for Na. In PR, more K uptake was observed in shoot at 250 mM NaCl with 10 mM Ca supplement. The sap osmotic potential (ΨS) was the highest in TW at 10 mM Ca in the presence of 250 mM NaCl. Contribution of various solutes to the difference in ΨS among the species from the control and 250 mM salt treatment differed greatly. Supplemental Ca induced decline in the leaf ΨS of TW which was predominately due to K, glycinebetaine, Na and proline accumulation. Addition of 10 mM Ca to the growth medium maintained a low Na and a high K level. Accumulation of glycinebetaine and proline in leaf contributed the NaCl tolerance of TW. The presented results suggest that supplement Ca, not only improved ionic relations but also induced plant ability in production of compatible solutes (glycinebetaine and proline) and osmotic adjustment. Accordingly, genotype dependent capacity could be found using supplemental Ca.

The role of hull in germination and salinity tolerance in some sunflower ( Helianthus annuus L.) cultivars

Six different NaCl levels (0, 5, 10, 20, 30 and 40 dS m-1) and two seed types (hulled and dehulled) of sunflower seeds of Opal, Shelly (Confectionary) and Pactol (Oily) were tested to determine the effects of the hull on salinity tolerance during germination. Germination percentage (%), mean germination time (day), root and shoot length (cm) were investigated. Kernel type seeds exhibited higher germination percentage, longer root and shoot length and lower mean germination time compared to the achene type seeds at all NaCl levels. Both achene and kernel type seeds of cv. Pactol gave the highest germination percentage, root and shoot length. Hull was responsible for delaying germination and water uptake by seeds while it had no role in lowering the toxic effects of NaCl during germination. Therefore, it was concluded that inhibition in germination at all levels of NaCl was mainly due to osmotic barriers created by the presence of hulls rather than salt toxicity.

The role of hull in germination and salinity tolerance in some sunflower (Helianthus annuus L.) cultivars

Six different NaCl levels (0, 5, 10, 20, 30 and 40 dS m-1) and two seed types (hulled and dehulled) of sunflower seeds of Opal, Shelly (Confectionary) and Pactol (Oily) were tested to determine the effects of the hull on salinity tolerance during germination. Germination percentage (%), mean germination time (day), root and shoot length (cm) were investigated. Kernel type seeds exhibited higher germination percentage, longer root and shoot length and lower mean germination time compared to the achene type seeds at all NaCl levels. Both achene and kernel type seeds of cv. Pactol gave the highest germination percentage, root and shoot length. Hull was responsible for delaying germination and water uptake by seeds while it had no role in lowering the toxic effects of NaCl during germination. Therefore, it was concluded that inhibition in germination at all levels of NaCl was mainly due to osmotic barriers created by the presence of hulls rather than salt toxicity. Key words: Sunflower, Helianthus annuus L., hull, NaCl, cultivar, germination.

Endogenous Bile Salts Are Associated With Bile Duct Injury in the Rat Liver Transplantation Model

Nonanastomotic biliary strictures are a serious complication after orthotopic liver transplantation (OLT) and are difficult to cure. We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. A total of 90 Sprague-Dawley rats were divided into three groups: Sham-operated group (Sham, n=30), OLT group with 1 hr donor liver preservation (OLT-1h, n=30), and OLT group with 12 hr donor liver preservation (OLT-12h, n=30). Bile was collected and analyzed biochemically. The histopathologic study was performed to determine the intrahepatic bile duct morphologic changes. Hepatic expressions of bile transporters Ntcp, Bsep, Mdr2, and Oatps were detected. During the first 2 weeks after transplantation, bile salt secretion was not parallel with phospholipid secretion, resulting in high biliary bile salt-to-phospholipid (BS:PL) ratio. The expression of bile transporters was consistent with the change of bile composition. Bile duct injury correlated significantly with bile salt secretion and BS:PL ratio. Moreover, OLT group with longer donor liver preservation time (OLT-12h) had significantly lower Mdr2 messenger RNA/protein level, higher BS:PL ratio, and better correlation between BS:PL ratio and bile duct injury compared with those of OLT-1h. The unparallel secretion of bile salts and phospholipids results in cytotoxic bile formation with high BS:PL ratio after liver transplantation. Longer donor liver preservation time will increase graft bile cytotoxicity. The results of this study suggest that endogenous bile salts play a role in the pathogenesis of bile duct injury after OLT.

Salicylic acid improves acclimation to salt stress by stimulating abscisic aldehyde oxidase activity and abscisic acid accumulation, and increases Na + content in leaves without toxicity symptoms in Solanum lycopersicum L.

Pre-treatment with 10(-4)M salicylic acid (SA) in hydroponic culture medium provided protection against salinity stress in tomato plants (Solanum lycopersicum L. cv. Rio Fuego). The effect of 10(-7) or 10(-4)M SA on the water status of plants was examined in relation to the biosynthesis and accumulation of abscisic acid (ABA) in order to reveal the role of SA in the subsequent response to salt stress. Both pre-treatments inhibited the K+(86Rb+) uptake of plants, reduced the K+ content of leaves, and caused a decrease in leaf water potential (psi(w)). Due to the changes in the cellular water status, SA triggered the accumulation of ABA. Since the decrease in psi(w) proved to be transient, the effect of SA on ABA synthesis may also develop via other mechanisms. In spite of osmotic adaptation, the application of 10(-4)M, but not 10(-7)M SA, led to prolonged ABA accumulation and to enhanced activity of aldehyde oxidase (AO1, EC.1.2.3.1.), an enzyme responsible for the conversion of ABA-aldehyde to ABA, both in root and leaf tissues. AO2-AO4 isoforms from the root extracts also exhibited increased activities. The fact that the activities of AO are significantly enhanced both in the leaves and roots of plants exposed to 10(-4)M SA, may indicate a positive feedback regulation of ABA synthesis by ABA in this system. Moreover, during a 100mM NaCl treatment, higher levels of free putrescine or spermine were found in these leaves or roots, respectively, than in the salt-stressed controls, suggesting that polyamines may be implicated in the protection response of the cells. As a result, Na+ could be transported to the leaf mesophyll cells without known symptoms of salt toxicity.

High expression of the bile salt-homeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis #

Fibroblast growth factor 19 (FGF19) is an endocrine factor produced by the small intestine in response to uptake of luminal bile salts. In the liver, FGF19 binds to FGF receptor-4, resulting in down-regulation of cytochrome P (CYP) 7A1 and reduced bile salt synthesis. Down-regulation of CYP7A1 under cholestatic conditions has been attributed to bile salt-mediated induction of the transcriptional repressor short heterodimer partner (SHP), because the interrupted enterohepatic cycle of bile salts is thought to abrogate intestinal FGF19 production and thus result in lowering of plasma FGF19 levels. Unexpectedly, we observed marked elevation of plasma FGF19 in patients with extrahepatic cholestasis caused by a pancreatic tumor (2.3 +/- 2.3 in cholestatic versus 0.40 +/- 0.25 ng/mL and 0.29 +/- 0.12 ng/mL in postcholestatic patients who received preoperative drainage by biliary stenting, P = 0.004, and noncholestatic control patients, P = 0.04, respectively). Although FGF19 messenger RNA (mRNA) is virtually absent in normal liver, FGF19 mRNA was strongly increased (31-fold to 374-fold, P < 0.001) in the liver of cholestatic patients in comparison with drained and control patients. In the absence of changes in SHP mRNA, CYP7A1 mRNA was strongly reduced (7.2-fold to 24-fold, P < 0.005) in the liver of cholestatic patients in comparison with drained and control patients, indicating an alternative regulatory pathway. Alterations in transcripts encoding hepatobiliary transporters [adenosine triphosphate-binding cassette, subfamily C, member 3 (ABCC3)/multidrug resistance protein 3 (MRP3), organic solute transporter alpha/beta (OSTalpha/beta), organic anion-transporting polypeptide (OATP1B1)] further suggest that bile salts are secreted via a nonbiliary route in patients with extrahepatic cholestasis. The liver expresses FGF19 under conditions of extrahepatic cholestasis. This is accompanied by a number of adaptations aimed at protecting the liver against bile salt toxicity. FGF19 signaling may be involved in some of these adaptations.

Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures

Nonanastomotic biliary strictures are troublesome complications after liver transplantation. The pathogenesis of NAS is not completely clear, but experimental studies suggest that bile salt toxicity is involved. In one hundred and eleven adult liver transplants, bile samples were collected daily posttransplantation for determination of bile composition. Expression of bile transporters was studied perioperatively. Nonanastomotic biliary strictures were detected in 14 patients (13%) within one year after transplantation. Patient and donor characteristics and postoperative serum liver enzymes were similar between patients who developed nonanastomotic biliary strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter expression. Although patients who develop nonanastomotic biliary strictures are initially clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary strictures.

Effect of salt and osmotic stresses on the germination of pepper seeds of different maturation stages

Germination percentage, seedling fresh weight and sensitivity index of the pepper (Capsicum annuum L. cv. Sera Demre) seed lots harvested 50, 60 and 70 days after the anthesis (daa) in 2001 and 2002 were determined under the salt (NaCl) and osmotic (PEG) stresses at the same water potentials of -0.3, -0.6 and -0.9 MPa. The objective was to study whether the germination and seedling growth were inhibited by the salt toxicity or osmotic effect during the development. The higher the salt and osmotic stress concentrations the lower was the germination percentage and seedling fresh weight. The seed germination was higher and sensitivity index were lower in NaCl than in PEG at the same water potential and harvest in both the years. They were able to germinate at all the concentrations of NaCl but at -0.9 MPa of PEG, none of the seeds of any harvest germinated in both the years. The seeds harvested 70 daa showed the highest germination and seedling weight in all the NaCl and PEG concentrations in both the years. Results showed that the inhibition of the germination at the same water potential of NaCl and PEG resulted from the osmotic effect rather than the salt toxicity. Moreover, the seeds harvested 70 daa appeared to be more tolerant to the salt and osmotic water stress conditions than the other two lots.

Dissection of genetic overlap of salt tolerance QTLs at the seedling and tillering stages using backcross introgression lines in rice

QTLs for salt-tolerance (ST) related traits at the seedling and tillering stages were identified using 99 BC(2)F(8) introgression lines (IL) derived from a cross between IR64 (indica) as a recurrent parent and Binam (japonica) from Iran as the donor parent. Thirteen QTLs affecting survival days of seedlings (SDS), score of salt toxicity of leaves (SST), shoot K(+) concentration (SKC) and shoot Na(+) concentration (SNC) at the seedling stage and 22 QTLs underlying fresh weight of shoots (FW), tiller number per plant (TN) and plant height (PH) at the tillering stage were identified. Most QTLs detected at the tillering stage showed obvious differential expression to salt stress and were classified into three types based on their differential behaviors. Type I included 11 QTLs which were expressed only under the non-stress condition. Type II included five QTLs expressed in the control and the salt stress conditions, and three of them (QPh5, QPh8 and QTn9) had similar quantity and the same direction of gene effect, suggesting their expression was less influenced by salt stress. Type III included six QTLs which were detectable only under salt stress, suggesting that these QTLs were apparently induced by the stress. Thirteen QTLs affecting trait difference or trait stability of ILs between the stress and non-stress conditions were identified and the Binam alleles at all loci except QPh4, QTn2 and QFw2a decreased trait difference. The three QTLs less influenced by the stress and 13 QTLs affecting trait stability were considered as ST QTLs which contributed to ST. Comparing the distribution of QTLs detected at the seedling and tillering stages, most (69%) of them were genetically independent. Only four were the same or adjacent regions on chromosomes 1, 2, 8 and 11 harboring ST QTLs detected at the two stages, suggesting that partial genetic overlap of ST across the two stages occurs. It is likely, therefore, to develop ST rice variety for both stages by pyramiding of ST QTLs of different stages or selection against the overlapping QTLs between the two stages via marker-assisted selection (MAS).

The Role of Bile Salt Toxicity in the Pathogenesis of Bile Duct Injury After Non-Heart-Beating Porcine Liver Transplantation

Intrahepatic bile duct strictures are a serious complication after non-heart-beating (NHB) liver transplantation. Bile salt toxicity has been identified as an important factor in the pathogenesis of bile duct injury and cholangiopathies. The role of bile salt toxicity in the development of biliary strictures after NHB liver transplantation is unclear. In a porcine model of NHB liver transplantation, we studied the effect of different periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed for 0 min (group A), 15 min (group B), or more or equal to 30 min (group C). Livers were subsequently transplanted after 4 hr of cold preservation. In the recipients, bile flow was measured, and bile samples were collected daily to determine the bile salt-to-phospholipid ratio. Severity of bile duct injury was semiquantified by using a histologic grading scale. Posttransplantation survival was directly related to the duration of warm ischemia in the donor. The bile salt-to-phospholipid ratio in bile produced early after transplantation was significantly higher in group C, compared with group A and B. Histopathologic condition showed the highest degree of bile duct injury in group C. Prolonged warm ischemia in NHB donors is associated with the formation of toxic bile after transplantation, with a high biliary bile salt-to-phospholipid ratio. These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation.

Creation of salt-insensitive 3′(2′),5′-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg 2+-dependent Li +-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure–function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.

Role of phosphatidylcholine saturation in preventing bile salt toxicity to gastrointestinal epithelia and membranes

The mechanism which protects the biliary and intestinal mucosa from the detergent properties of bile acids is not fully understood. We employed three contrasting in vitro model systems (human red blood cells, polarized intestinal [Caco-2] cells, and synthetic liposomes), to compare the efficacy of saturated and unsaturated phosphatidylcholine (PC) to protect cells and membranes from bile salt injury. Hemolysis of red blood cells, electrical resistance across confluent monolayers of Caco-2 cells, and disruption of synthetic PC liposomes were assessed after incubation with varying concentrations of bile salt (sodium deoxycholate) alone or in the presence of saturated or unsaturated PC. The hemolytic activity of deoxycholate on red blood cells was observed at > or =2 mM, and could be blocked by equimolar concentration or greater of both saturated or unsaturated PC. In contrast, exposure of Caco-2 cells to deoxycholate at > or =0.8 mM induced a maximal decrease in resistance, which was reversed by > or =0.8 mM unsaturated PC or 5 mM saturated PC. Similarly, synthetic liposomes were permeabilized by 0.8 mM deoxycholate and were protected by a lower concentration of unsaturated PC (2 mM) than saturated (5 mM). Cells can show variable resistance to bile salt toxicity. Extracellular PC, especially in the unsaturated state, can directly protect cell and artificial membranes from bile salt injury. These findings support a role for biliary PC in the formation of mixed micelles that have low cytotoxic properties.

Phytoextraction and phytoexcretion of Cd by the leaves of Tamarix smyrnensis growing on contaminated non-saline and saline soils

Phytoremediation and more specifically phytoextraction, is an alternative restoration strategy for the clean up of heavy metal contaminated soils. Phytoextraction can only be successful if suitable plant species colonize the contaminated area, extract the toxic substances and accumulate them in their above ground tissues. In this study, the salt cedar Tamarix smyrnensis that is a widespread salt-tolerant plant in the Mediterranean region has been investigated. A pot experiment is conducted with T. smyrnensis grown in polluted soil with 16 ppm of cadmium and at three different salt concentrations (0.0, 0.5, 3.0% NaCl) for a 10-week period. It took place in an open-air area with natural light, at ambient temperature and humidity in an effort to keep the plants under conditions as similar as possible to those in the field. However, care was taken not to let them be rained on. Temperature ranged from 19 to 50 °C with 33 and 21 °C being the average day and night temperature, respectively. Humidity ranged from 28% to 87% with a 13–14 h photoperiod. The specific aims of this work are to investigate the accumulation of cadmium via root uptake at different saline conditions and cadmium excretion through salt glands on the surface of the leaves as a probable detoxification mechanism of the plant. Furthermore, measurements of chlorophyll content, biomass, and shoot length are used to evaluate the potential of the plant for the removal of cadmium from contaminated saline and non-saline soils. The experimental data suggest that increased soil salinity results in an increase of the cadmium uptake by T. smyrnensis. Analysis of white salt crystals taken from glandular tissue confirmed the fact that this plant excretes cadmium through its salt glands on the surface of the leaves as a possible detoxification mechanism in order to resist metal toxicity. Excreted cadmium is again released into the environment and it is redeposited on the top soil. Furthermore, increased salinity results in an increased excretion of the metal on Tamarix leaf surface. The presence of metals usually affects negatively the plant health, but T. smyrnensis developed no visible signs of metal toxicity, only salt toxicity symptoms were observed. Cadmium usually decreases the chlorophyll content in plants; however, the amount of photosynthetic pigments of T. smyrnensis was found not to be affected. All the above points to the potential of T. smyrnensis for use in phytoremediation with the metal secretion from the leaves being a unique advantage that may change current phytoextraction practices.

Na + regulation by combined nitrogen in Azolla pinnata– Anabaena azollae symbiotic association during salt toxicity

To study the regulation of Na + ion by combined-N sources in Azolla pinnata– Anabaena azollae, Na + influx, intracellular Na + and Na + efflux were investigated in the presence of different N-sources (N 2, NH 4 +, NO 3 − and urea) and various NaCl concentrations. Sodium influx by Azolla pinnata fronds was minimum in the presence of NO 3 −. Almost identical levels of intracellular sodium, although less than N 2 and NH 4 + incubated fronds were observed in the presence of NO 3 − and urea. Efflux of sodium was minimum in urea and NO 3 − grown fronds. A low residual sodium was observed in the fronds incubated in NO 3 − and urea supplemented media. Results suggest that nitrate and urea curtailed the entry of sodium, reduced salt toxicity maximally by maintaining the minimum level of sodium and also conserved energy due to slow influx and efflux of Na + within the fronds during salt shock and the process of adaptation.

SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress.

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.

Novel 3′-phosphoadenosine-5′-phosphatases from extremely halotolerant Hortaea werneckii reveal insight into molecular determinants of salt tolerance of black yeasts

The 3′-phosphoadenosine-5′-phosphatase encoded by HAL2 gene, is a ubiquitous enzyme required for the removal of the cytotoxic 3′-phosphoadenosine-5′-phosphate produced during sulfur assimilation in eukaryotes. Salt toxicity in yeast and plants results from Hal2 inhibition by sodium or lithium ions. Two novel HAL2-like genes, HwHAL2A and HwHAL2B, have been cloned from saltern-inhabited extremely halotolerant black yeast Hortaea werneckii. Expression of both HwHAL2 isoforms was differentially inducible upon salt. When the HwHAL2 genes were transferred from such a halotolerant species into the salt sensitive Saccharomyces cerevisiae, the resulting organism can tolerate 1.8 M NaCl or 0.8 M LiCl, the highest reported salt concentrations at which S. cerevisiae can grow. With genetic and biochemical validation we demonstrated the critical HwHal2B sequence motif—the META sequence—common only to Dothideales fungi, with evident effect on the HwHal2B-dependent salt tolerance. These results may have significance for biosaline agriculture in coastal environments.

Effects of NaCl stress on red raspberry (Rubus idaeus L. ‘Autumn Bliss’)

The increasing interest in the cultivation of red raspberry ( Rubus idaeus L.) in warmer climates and in the mild-Southern parts of Europe requires better understanding of the salinity effects especially in the primocane-fruiting type cultivars. The aim of this work was to study the response of the ‘Autumn Bliss’ primocane-fruiting cultivar in elevated NaCl concentrations. The experiment was carried out under natural greenhouse conditions. ‘Autumn Bliss’ cultivar was grown in pots containing perlite . The plants were irrigated with Hoagland nutrient solution containing 0, 5, 10, 15, 20 and 30 mM NaCl. Plant growth, photosynthetic parameters, water relations, chloride and sodium content, leaf chlorophyll content and fluorescence were determined. The results of this work indicate that increasing salt concentration in the nutrient solution leads to: (a) a decline in the photosynthetic rate and in stomatal conductance , although there is no reduction in intercellular CO 2 ; (b) the reduction of leaf chlorophyll content and fluorescence; (c) to a decrease in transpiration rate, leaf water potential , relative water content and plant water consumption, however, the water use efficiency is not affected; (d) the reduction of: cane length and diameter, percentage of green leaves, leaf area, leaf expansion rate and relative growth rate, while the number of leaves remain unaffected and the percentage of dead leaves increases; (e) a decrease of the plant fresh and dry weight and to an increase of the root/shoot ratio; (f) a linear increase of chloride and sodium content in the plant. Quantifying the response of raspberry in elevated NaCl concentrations provides that the reduction in photosynthesis is most probably due to damages in the photosynthetic apparatus rather than from factors affecting stomatal closure. Besides, a pronounced toxic effect of Cl was clearly demonstrated and this points towards that the salt injury effect could be attributed to Cl toxicity (uptake and translocation). As a consequence, the decrease photosynthetic capacity and salt toxicity in the plant tissues restricted plant growth under saline conditions.

Protection against salt toxicity in Azolla pinnata-Anabaena azollae symbiotic association by using combined-N sources

Protection from salt stress was observed in the terms of yield (fresh and dry weight, chlorophyll and protein) and nitrogenase activity. Azollapinnata appeared highly sensitive to 40 mM external NaCl stress. Fronds of Azolla unable to grow beyond a concentration of 30 mM NaCl and accordingly death was recorded at 40 mM NaCl on the 6th day of incubation. Yield was inhibited by various levels of NaCl (0, 10, 20 and 30 mM). Addition of combined-N to the growth medium protected the association partially from salt toxicity. Among the N-sources (NO3-, NH4+ and urea) tried, urea mitigated the salt-induced toxicity most efficiently. Reduction in nitrogenase activity was observed when intact Azolla was grown in nutrient medium either supplemented with different levels of NaCl or combined nitrogen. Only NO3- (5 mM) protected the enzymatic activity from salt toxicity while other concentrations of ammonium, nitrate and urea slowed down the salt-induced inhibition of enzyme activity in Azolla-Anabaena association. These results suggested that an optimum protection from salt stress could be obtained by using a combination of combined nitrogen sources. The reason for this protection might be due to the availability of combined nitrogen to the association, nitrogen is only available through the biological nitrogen fixation which is the most sensitive to salt stress.

The Arabidopsis Mutant stg1 Identifies a Function for TBP-Associated Factor 10 in Plant Osmotic Stress Adaptation

Plant salt tolerance is a complex trait involving many genes. To identify new salt tolerance determinants during seed germination, we have screened a population of chemically inducible activation-tagged Arabidopsis mutants. A mutant, designated stg1 (salt tolerance during germination 1), was obtained. The stg1 mutant is less sensitive than the wild type to NaCl and osmotic stress inhibition of germination in the presence of the inducer. Germination assays on media containing various salts upon inducer application indicate that the stg1 mutation enhances tolerance to Na(+) and K(+). Under salt stress, stg1 maintains a higher K(+)/Na(+) ratio and accumulates less proline than the wild-type control, suggesting that its salt tolerance mechanisms are mainly involved in the regulation of ion balance. STG1 encodes a putative Arabidopsis TATA box-binding protein (TBP)-associated factor 10 (atTAF10), which constitutes the transcriptional factor IID (TFIID) complex. Overexpression of atTAF10 under the control of the 35S promoter in Arabidopsis improves seed tolerance to salt stress during germination and the knocked-down mutant is more sensitive to salt stress, indicating the transcription initiation factor as a physiological target of salt toxicity in plants.

Extracellular Ca2+ Ameliorates NaCl-Induced K+ Loss from Arabidopsis Root and Leaf Cells by Controlling Plasma Membrane K+-Permeable Channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.