Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

Cellular and whole plant responses ofVigna radiata to NaCl stress

The effect of different NaCl regimes was examined on the growth and ion accumulation in whole plants and callus cultures ofVigna radiata. Whole plants grown in sand culture were watered with Hoagland's solution supplemented with 0–350 mol m⁻³ of NaCl. Callus cultures were initiated from leaves of 7-d old seedlings of the same seed stock and grown in modified PC-L2 medium containing the same levels of NaCl as in Hoagland's solution. Callus showed the same tolerance to salt as did the whole plant suggesting thatV. radiata appears to have a mechanism(s) for salt tolerance which operates at the cellular level. Ion analysis of whole plant showed that root sodium concentrations of the tolerant cultivar G-65 was much higher while shoot sodium was much less than those of salt sensitive cultivar ML-1. Callus cultures of cv. G-65 also accumulated higher Na⁺ levels. Thus, the greater salt tolerance of cv. G-65 was associated with the control of sodium accumulation at the shoot or cellular level. Communicated by J. POSPíŠILOVá     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Distinct Cellular and Organismic Responses to Salt Stress

We have compared metabolic effects of high salinity betweenplants and cell suspension cultures from the facultative halophyteMesembryanthemum crystallinum (common ice plant). This plantshows developmentally-programmed inducibility for a switch fromC₃-photosynthesis to CAM (Crassulacean Acid Metabolism). Themetabolic switch is enhanced by environmental factors such asdrought, low temperature, and, most effectively, soil salinity.CAM induction is dependent on organized leaf tissue and cannotbe elicited by salt stress in suspension culture cells. In contrast,the accumulation of proline [Thomas et al. (1992) Plant Physiol.98: 626] is induced by NaCl in cultured cells as well as inplants and must be considered a cellular response to stress.We have extended our observations to include another trait ofsalt- and low-temperature-stress responses in the ice plant,the accumulation of putative osmoprotective sugars and sugaralcohols. In whole plants the cyclic sugar alcohol, pinitol,accumulates to amounts that approach 1 M during stress, whilein suspension cells no increase in sugar alcohols is observed.The distribution of carbon to different sugars is markedly differentbetween cells and plants under stress. Particularly obviousis the distinction between cell types in the different compositionof sugars and polyols, as exemplified by the epidermal bladdercells of ice plants. Ion contents and the content of sugarsand sugar alcohols of bladder cells indicate that Na⁺, Cl^–,pinitol and an unknown carbohydrate compound provide osmoticpressure in these cells, while organic anion concentrationsare low. With the ice plant, we conclude that cells in culturemimic only partly the stress response mechanisms of intact plantsand we hypothesize that communication between different tissuesis required to mount a complete environmental stress response. ⁴ Present address: Department of Botany Oklahoma State University,Stillwater, OK, 74078, U.S.A.     

Improved yield,fruit quality,and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes

The K⁺, Na⁺/H⁺ antiporter LeNHX2 and the regulatory kinase SlSOS2 are important determinants of salt tolerance in tomato plants and their fruit production ability. In this work, we have analyzed the effects of LeNHX2 and SlSOS2 co-overexpression on fruit production, quality in tomato plants (Solanum lycopersicum L. cv. MicroTom), and analyzed physiological parameters related to salt tolerance. Plants overexpressing LeNHX2, SlSOS2 or both were grown in greenhouse. They were treated with 125 mM NaCl or left untreated and their salt tolerance was analyzed in terms of plant biomass and fruit yield. Under NaCl cultivation conditions, transgenic tomato plants overexpressing either SlSOS2 or LeNHX2 or both grew better and showed a higher biomass compared to their wild-type plants. Proline, glucose and protein content in leaves as well as pH and total soluble solid (TSS) in fruits were analyzed. Our results indicate that salinity tolerance of transgenic lines is associated with an increased proline, glucose and protein content in leaves of plants grown either with or without NaCl. Salt treatment significantly reduced yield, pH and TSS in fruits of WT plants but increased yield, pH and TSS in fruits of transgenic plants, especially those overexpressing both LeNHX2 and SlSOS2. All these results indicate that the co-overexpression of LeNHX2 and SlSOS2 improve yield and fruit quality of tomato grown under saline conditions.     

Characterization of a salt-responsive 24-kilodalton glycoprotein in Mesembryanthemum crystallinum.

A concanavalin A (Con A)-binding polypeptide with a molecular mass of 24 kD (termed "SRgp24") was associated with the intercellular space of Mesembryanthemum crystallinum L. callus. When callus was grown in medium containing between 0 and 100 mM NaCl, SRgp24 was detected by Con A binding. Increasing the NaCl concentration to 200 mM caused a reduction in the amount of SRgp24 within 3 d, and returning the callus to medium without salt resulted in an accumulation of SRgp24. Immunoblot analysis showed that appreciable amounts of SRgp24 accumulated in the leaves when plants were grown under sodium-limiting conditions. Unlike most of the cell-wall Con A-binding proteins in M. crystallinum callus, the carbohydrate moiety of SRgp24 was resistant to endoglycosidase H digestion. After purification of SRgp24, the N terminus was sequenced and found to share 55 to 60% identity with the N terminus of osmotin, a group 5 pathogenesis-related protein (PR-5) that accumulates in salt-adapted tobacco cell suspension. Immunocytochemical assays, with affinity-purified antibodies to SRgp24, indicated that SRgp24 preferentially accumulated in the cell-wall region. We conclude that SRgp24 is a salt-responsive glycoprotein related to the PR-5 family in M. crystallinum.     

Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin

Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCl contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs.     

The influence of salinity on phosphate uptake and distribution in lupin roots

Treeby, M. T. and van Steveninck, R. F. M. 1988. The influence of salinity on phosphate uptake and distribution in lupin roots. - Physiol. Plant. 72: 617–622.
The uptake and distribution of phosphate in lupin ( Lupinus luteus L. cv. Weiko III) roots under moderate salt (NaCl) stress was studied. Vacuolar inorganic phosphate (PJ concentrations in high phosphate plants were decreased by salt, although whole root P| was unaffected. In low phosphate plants, vacuolar P_i was unaffected by salt while whole root P_i was increased. Phosphate uptake was not altered by salt in high phosphate plants, but was depressed in low phosphate plants. These observations lead to the conclusion that in high phosphate plants P_i accumulates in cytoplasm and/or stele, ultimately giving rise to phosphate toxicity in shoots. Increasing phosphate supply had no effect on Na⁺ accumulation in root cell vacuoles in the epidermis or cortex, but the concentration of Cl⁻ in endodermal vacuoles was lowered.     

The effects of salt on the pattern of protein synthesis in barley roots

The effect of salt stress on the incorporation of [³⁵S]methionine into protein was examined in roots of barley (Hordeum vulgare L. cv California Mariout 72). Plants were grown in nutrient solution with or without 200 millimolar NaCl. Roots of intact plants were labeled in vivo and proteins were extracted and analyzed by fluorography of two-dimensional gels. Although the protein patterns for control and salt-stressed plants were qualitatively similar, the net synthesis of a number of proteins was quantitatively changed. The most striking change was a significant increase of label in two protein pairs that had pIs of approximately 6.3 and 6.5. Each pair consisted of proteins of approximately 26 and 27 kilodaltons (kD). In roots of control plants, the 27-kD proteins were more heavily labeled in the microsomal fraction relative to the 26-kD proteins, whereas the 26-kD proteins were enriched in the post 178,000 g supernatant fraction; in roots of salt treated plants, the 26- and 27-kD proteins were more intensely labeled in both fractions. Labeling of the 26- and 27-kD proteins returned to control levels when salt-stressed plants were transferred to nutrient solution without NaCl. No cross-reaction was detected between the antibody to the 26-kD protein from salt-adapted tobacco cells and the 26- and 27-kD proteins of barley.     

Physiology of acclimation to salinity stress in pea (Pisum sativum)

Pea (Pisum sativum L.) seedlings were grown in half strength Hoagland solution and exposed to 0, 10, 25 mM NaCl and 2.5% PEG 6000 for 1 week (pre-treatment). Thereafter plants were exposed to 0 and 80 mM NaCl for 2 weeks (main treatment). The control plants were maintained in half strength Hoagland solution without NaCl. Various physiological parameters were recorded from control, pretreated and non-pretreated plants. There was no negative effect of the pre-treatments on growth (total fresh and dry matter production), and plants pre-treated with 10 mM NaCl had biomass accumulation equal to control plants. The beneficial effect of salt acclimation was also evident in the prevention of K⁺ leakage and Na⁺ accumulation, primary in roots, suggesting that here the physiological processes play the major role. 2.5% PEG 6000 was not as efficient as salt in enhancing salt tolerance and acclimation appears to be more related to ion-specific rather than osmotic component of stress. We also recorded an increase of the xylem K/Na in the salt acclimated plants. Therefore, the present study reveals that short-term exposure of the glycophyte P. sativum species activates a set of physiological adjustments enabling the plants to withstand severe saline conditions, and while acclimation takes place primary in the root tissues, control of xylem ion loading and efficient Na⁺ sequestration in mesophyll cells are also important components of this process.     

Neutral lipid production in <Emphasis Type="Italic">Dunaliella salina</Emphasis> during osmotic stress and adaptation

The salt-tolerant green microalga Dunaliella salina can survive both hyper- and hypo-osmotic shock. Upon osmotic shock, the cells transiently and rapidly decreased or increased in size within minutes and slowly over hours acquired their original cell size and volume. Cell size distribution differs significantly in the cultures grown in the salinity range from 1.5 to 15 % NaCl. By using Nile Red fluorescence to detect neutral lipids, it became clear that only hyper-osmotic shock on cells induced transient neutral lipid appearance in D. salina, while those transferred from 9 to 15 % NaCl stimulated the most neutral lipid accumulation. These cells grew well in 9 % NaCl, but they cannot recover a shift to 15 % NaCl and cell division is accordingly slowed down. The transient appearance of neutral lipid could be dependent on the inhibition of cell division experiencing the NaCl shift. Moreover, the effect of nutrient limitation slows down cell division and photosynthesis as a secondary result, which triggers the cells to accumulate neutral storage lipids when they entered the stationary phase, which is seen in all the batch cultures of D. salina grown in the salinity range of 3–15 %. The changes in salt concentration did not significantly influence the overall fatty acid composition in D. salina cells. Although there shows both increased amounts of total lipids and neutral lipids in the cells grown in salinity higher than 9 % NaCl, lipid productivity is however compromised by the slower cell growth rate and lower cell density under this condition.     

Mechanisms of salt stress tolerance development in barley plants under the influence of 5-aminolevulinic acid

Growing barley (Hordeum vulgare L.) plants for 7 days on NaCl solutions (20–200 mM) decreased chlorophyll (Chl) a and b content with respect to that in untreated control plants. The content of free proline and the plant ability to synthesize 5-aminolevulinic acid (ALA) started to increase in parallel at salt concentrations of 20–50 mM. The maximum amount of ALA accumulated in plants grown at 100 mM NaCl was twofold higher than in control plants grown on fresh water. In this case the proline content increased 2.8-fold. On further increase in salt concentration, the rate of ALA accumulation decreased, approaching control values at 150 mM NaCl; even lower rates were observed at 200 mM NaCl. The reduced ability to synthesize ALA was accompanied by an increase in proline content. The albino tissue of plants treated at the seed stage with the antibiotic streptomycin lost its ability to synthesize ALA needed for Chl formation. The proline content in the albino tissue was tenfold higher than in control green plants and was 30-fold higher when the plants were grown on solutions with 100 mM NaCl. No effect of NaCl on ALA-dehydratase activity was noted. As NaCl concentration was raised, there occurred the decrease in magnesium chelatase activity, accumulation of reactive oxygen species (ROS), the increase in ascorbate peroxidase activity, and a slight decrease in lipid peroxidation level. Growing plants in the presence of 150 mM NaCl and 10 or 60 mg/l exogenous ALA led to the increase in proline content (by a factor of 1.8 and 4.2, respectively) and to the decrease in ROS content, in comparison with plants grown on salt solutions without ALA. Furthermore, in the presence of exogenous ALA, the parameters of seedling growth became similar to those of NaCl-untreated plants. The role of ALA in plants as an antistress agent is considered. ALA is supposed to confer tolerance to salt stress by taking part in Chl and heme biosynthesis and also through functioning as a plant growth regulator. A hypothesis is put forward that the impairment of ALA-synthesizing ability may redirect metabolic conversions of glutamic acid from Chl and heme synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and improve salt tolerance.     

Effects of NaCl and Mycorrhizal Fungi on Antioxidative Enzymes in Soybean

The effects of different concentrations of NaCl on the activities of antioxidative enzymes in the shoots and roots of soybean (Glycine max [L.] Merr cv. Pershing) inoculated or not with an arbuscular mycorrhizal fungus, Glomus etunicatum Becker & Gerdemann, were studied. Furthermore, the effect of salt acclimated mycorrhizal fungi on the antioxidative enzymes in soybean plants grown under salt stress (100 mM NaCl) was investigated. Activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased in the shoots of both mycorrhizal (M) and nonmycorrhizal (NM) plants grown under NaCl salinity. Salinity increased SOD activity in the roots of M and NM plants, but had no effect on CAT and polyphenol oxidase activities in the roots. M plants had greater SOD, POD and ascorbate peroxidase activity under salinity. Under salt stress, soybean plants inoculated with salt pre-treated mycorrhizal fungi showed increased SOD and POD activity in shoots, relative to those inoculated with the non pre-treated fungi.     

Accumulation of gamma-aminobutyric acid in the halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> under salt and acid stress

γ-Aminobutyric acid (GABA) is known as an inhibitory neurotransmitter in human, while in plants, GABA is an intermediate for amino acid metabolism and also is accumulated in response to a wide range of environmental stress. In the present study, GABA accumulation in Aphanothece halophytica was increased 2-fold in mid-log phase cells grown under salt stress (2.0 M NaCl). When mid-log phase cells were subjected to changes in NaCl concentrations and pH for 4 h, the highest GABA accumulation was observed in cells adapted in medium that contained 2.0 M NaCl and that was adjusted to pH 4.0, respectively. The increase of GABA accumulation was accompanied by an increased glutamate decarboxylase activity. Addition of glutamate to growth medium stimulated GABA accumulation under acid stress but had no effect under salt stress. However, the highest GABA accumulation was detected in cells exposed to both high salt and acid stresses combined with the 5 mM glutamate supplementation with an approximately 3-fold increase as compared to the control. The unicellular A. halophytica showed a similarly high content of GABA to that of a filamentous Arthrospira platensis suggesting the possibility of genetic manipulation of the genes of A. halophytica involved in GABA synthesis to increase GABA yield.     

Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro

The use of in vitro shoot cultures to evaluate osmotic and salt tolerance and the effects of salt and mannitol in the medium on proline and sugar accumulation were investigated in two poplar species, P. euphratica and P. alba cv. Pyramidalis × P. tomentosa. Shoot length, leaf number, whole plant dry weight, and the accumulation of proline and total soluble sugars in leaves were quantified after 2 weeks. All P. euphratica plantlets survived at all levels of mannitol and NaCl, while the mortality of P. alba cv. Pyramidalis × P. tomentosa increased both at the mannitol and the NaCl treatments. A significant increase in proline accumulation was observed in both young and mature P. euphratica leaves at 200 mM mannitol and above, and at 150 mM NaCl and above. The total soluble sugar content increased in young P. euphratica leaves at 250 mM NaCl; however, it decreased in the mature leaves. Similar increases of the total soluble sugar content were not seen in P. alba cv. Pyramidalis × P. tomentosa plants in response to either mannitol or NaCl treatment. Our results suggest that accumulated proline and sugars promote osmotic and salt tolerance. The effects of accumulated proline and total soluble sugars on leaves are discussed in relation to growth and osmotic adjustment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein

The cDNA clone (pNP24) coding for a protein induced by exogenous NaCl has been isolated from a tomato root cDNA library with the use of an inosine containing synthetic oligomer. The authenticity of the clone has been established by comparing the sequence of the clone to the NH₂-terminal sequence of the protein which has been purified to homogeneity by HPLC. The nucleotide sequence of pNP24 reveals a 5 signal sequence, an open reading frame of 718 nucleotides, a 3 AT rich untranslated region containing a probable polyadenylation signal sequence, and a poly A stretch. The mature polypeptide sequence as deduced from the nucleotide sequence reveals a protein with a molecular weight of 24226. This protein has been named NP24. It is slightly basic and has an unusually high number of cysteines (15). Northern blot analyses reveal that the abundance of mRNA for NP24 is at least 100-fold greater in tomato suspension cells in log phase grown in medium with NaCl than in cells grown in the control medium. The mRNA for NP24 is below the level of detection in roots of young control tomato plants until several weeks after germination but it is induced earlier and to higher levels in roots stressed by 0.171 M NaCl. Thus salt stress accelerates the accumulation of message in tomato roots. A comparison of the steady state levels of mRNA for NP24 to the accumulation of NP24 by immuno analyses indicates that the accumulation of this protein is determined by its mRNA level. The protein is not secreted and is localized within the cytoplasm or the soluble fraction of the nucleus, vacuole, or microbodies. NP24 has a high degree of homology (58%) with thaumatin, a protein which has considerable value as an artificial sweetener.