Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

Ultrastructural changes in gametophytes of Acrostichum aureum L. cultured in different sodium chloride concentrations

Gametophytes cultured in solutions containing 0.0 to 0.7 % NaCl exhibited no change in ultra structural organization of chloroplasts. In 1.0% NaCl-grown gametophytes, there were thinner granal stacks, relatively larger spaces between granal thylakoidal membranes and larger plastoglobuli in the chloroplasts. These changes were accompanied by a decrease in photosynthesis. Cup shape, horseshoe shape, ring shape, and amoeboid mitochondria were observed in gametophytes grown in 0.0 to 0.7% NaCl. Only round mitochondria were observed in the gametophytes grown in 1.0 % NaCl. Mitochondria seemed to be more resistant to salt stress compared to chloroplasts. There was no direct relationship between changes in respiration rate and changes in mitochondrial shape among gametophytes grown in different NaCl concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth, gas exchange and ion content in Olea europaea plants during salinity stress and subsequent relief

Olive ( Olea europaea L. cv. Frantoio) plants grown hydroponically in a glasshouse were supplied with half-strength Hoagland solutions containing 0, 50, 100, and 200 m M NaCl for 4 weeks and subsequently supplied with the standard solution without NaCl to relieve salinity stress. Two complete stress-relief cycles were repeated on the same plant material during one growing season. Growth was inhibited at all salt levels, but most growth parameters of plants treated with 50 or 100 m M NaCl returned to control levels after 4 weeks of relief. More severely stressed plants (200 m M NaCl) recovered to only 60% of the growth of the controls after 4 weeks. During relief, plants treated with 50 and 100 m M NaCl had net photosynthetic rates and stomatal conductances higher than the controls. Increasing the NaCl concentration of the external solution from 0 to 200 m M decreased both leaf pre-dawn water potential (from -0.3 to -1.0 MPa) and osmotic potential (from -2.1 to -2.7 MPa). The sodium concentration in the leaves of plants treated with 200 m M NaCl reached maximum levels of 211 and 388 m M (expressed on a tissue water basis) at the end of the first salinity and relief periods, respectively. Leaf chloride concentrations were 359 and 223 m M at the same sampling dates. These data indicate that the inhibitory effects of salinization on growth and gas exchange of the salt-tolerant olive cv. Frantoio can be readily reversed when salinity is relieved, despite the marked accumulation of potentially toxic ions (Na⁺. Cl) in the leaf.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

The influence of NaCl on Δ1-pyrroline-5-carboxylate reductase in proline-accumulating cell suspension cultures of Mesembryanthemum nodiflorum and other halophytes

Pyrroline-5-carboxylate (P-5-C) reductase (EC 1.5.1.2) was extracted from cell suspension cultures, which proved to be very suitable for investigation of proline accumulation. Proline accumulation in cell suspensions of M. nodiflorum L., as well as P-5-C reductase activity and substrate affinity, increased with progressive adaptation to NaCl stress. In vitro NaCl treatment inhibited enzyme activity and decreased substrate affinity, independent of pretreatment of the cells. NaCl concentrations below 100 m M did not inhibit enzyme activity of adapted cells. High substrate concentrations counteracted in vitro NaCl inhibition (up to 200 m M ). Cycloheximide inhibited the increase of P-5-C reductase activity, as well as proline accumulation, after NaCl treatment, indicating stress-induced de novo synthesis of the enzyme. The different reactions of P-5-C reductase upon salt treatment are discussed with respect to its possible role in the regulation of proline accumulation.     

Responses of photosynthesis to NaCl in gametophytes of Acrostichum aureum

Gametophytes of Acrostichum aureum were cultured in 0.0 to 1.0% NaCl solutions or in NaCl‐free solution and then transferred to 1.0% NaCl solution. Photosynthetic light‐response curves, efficiency of the primary photochemical reaction, relative electron transport rate, and photochemical and non‐photochemical quenching at steady state were determined by photosynthetic O₂ evolution and in vivo chlorophyll fluorescence. Results obtained showed that the chlorophyll fluorescence parameters, F_v/F_m and F'_v/F'_m and αO₂ (the initial linear slope of the photosynthetic light‐response curve) increased in gametophytes grown in NaCl. Linear electron transport rate was stimulated by NaCl. Based on the chlorophyll content, light‐saturated photosynthesis in gametophytes grown in 0.2 to 0.7% NaCl increased slightly; it decreased in gametophytes grown in 1.0% NaCl. Photochemical quenching decreased in NaCl‐grown gametophytes at all photosynthetic photon flux density (PPFD) levels measured, but there was no increase in non‐photochemical quenching. The chlorophyll a/b ratio increased with increasing NaCl concentration in culture solutions. These results indicated that NaCl enhanced photochemical efficiency of photosystem II (PSII) and photosynthetic linear electron transport, thus resulting in the development of an excitation pressure in PSII. Such excitation pressure might act as a signal for photosynthetic acclimation to salt stress, thus allowing the gametophytes to grow in their natural habitats.     

Tolerance of gametophytes of Acrostichum aureum (L.) to salinity and water stress

Tolerance of gametophytes of Acrostichum aureum to NaCl and dehydration was investigated under controlled conditions following the changes in chlorophyll fluorescence parameters (Fv/Fm, qP, qN). Salt tolerance was increased by growing gametophytes in low concentrations of NaCl. However, such treatment could not increase the tolerance of gametophytes to dehydration. Under water stress, a decrease in photochemical quenching (qP) was accompanied by an increase in non-photochemical quenching (qN). Under salt stress, qP also decreased, but qN did not change significantly in salt-hardened gametophytes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tolerance of gametophytes of Acrostichum aureum (L.) to salinity and water stress

Tolerance of gametophytes of Acrostichum aureum to NaCl and dehydration was investigated under controlled conditions following the changes in chlorophyll fluorescence parameters (Fv/Fm, qP, qN). Salt tolerance was increased by growing gametophytes in low concentrations of NaCl. However, such treatment could not increase the tolerance of gametophytes to dehydration. Under water stress, a decrease in photochemical quenching (qP) was accompanied by an increase in non-photochemical quenching (qN). Under salt stress, qP also decreased, but qN did not change significantly in salt-hardened gametophytes.     

Is salinity tolerance related to Na accumulation in Upland cotton (Gossypium hirsutum) seedlings?

Physiological responses to salt stress were studied in two cotton cultivars previously selected on the basis of growth under salinity. Plants were grown in nutrient solutions under controlled conditions. In the first experiment, the genotypes were grown at different salt concentrations (0, 100 and 200 mt M NaCl) and growth rates, water contents and ion accumulation were determined. In a second experiment, both genotypes were grown at the same salt concentration (200 mt M NaCl). Dry matter partitioning in individual leaves, stem and roots, water contents, specific leaf area (SLA), ion accumulation (K⁺, Na⁺, Cl^–) and leaf water potentials were measured. Finally, an experiment with low salt levels (2.7 and 27 mt M NaCl) was run to compare K and Na⁺ uptake and distribution.There were no differences in growth between the cultivars in the absence of salt stress, whereas under stress genotype Z407 had higher leaf area and dry matter accumulation than P792. Leaf water potential and leaf water content were lower in cv P792 than in cv Z407. There were no significant differences in the levels of Cl^– accumulation between genotypes. The main feature of the tolerant genotype (Z407) was a higher accumulation of Na⁺ in leaves and an apparent capacity for K⁺ redistribution to younger leaves.We postulate that the higher tolerance in Z407 is the result of several traits such as a higher Na⁺ uptake and water content. Adaptation through adequate, but tightly controlled ion uptake, typical of some halophytes, matched with efficient ion compartmentation and redistribution, would result in an improved water uptake capacity under salt stress and lead to maintenance of higher growth rates.     

Polyamines in various rice (Oryza sativa) genotypes with respect to sodium chloride salinity

Physiological responses of various rice genotypes were studied in relation to salt (NaCl) stress. Cultivars CSR-1 and Dular germinated well in different NaCl regimes compared to cvs Rupsail, Assam Getu and M-1–48. At 100 m M NaCl, the lowest germination was observed in cv. M-1–48. Cvs CSR-1 and Dular were relatively effective in maintaining high concentrations of polyamines as well as arginine decarboxylase (EC 4.1.1.19) activity in coleoptiles and roots in a non-stressed condition. The activities of two biodegradative enzymes, diamine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.4.3.4), were lowest in cv. CSR-1. The polyamine content was not significantly altered when seedlings of cv. CSR-1 were exposed to 100 m M NaCl. However, in cv. M-1–48 enhancement of arginine decarboxylase activity with concomitant accumulation of polyamines was observed. Leakage of metabolites and changes in the levels of Na⁺ and Cl⁻ were prominent in cv. M-1–48 under saline conditions. The results suggest a correlation between polyamine and salt stress-induced responses in rice genotypes.     

Accumulation of a 22-kDa protein and its mRNA in the leaves of Raphanus sativus in response to salt stress or water deficit

The response of 10-day-old seedlings of Raphanus sativus L. cv. Fakir to salt stress (100 m M to 200 m M NaCl) was investigated. Three weeks after initiation of salt treatment, the fresh weight of the shoots of salt-treated plants was half that of untreated plants. The salt stress resulted in the accumulation of Na⁺, preferably in the old leaves. The K⁺ level was reduced by as much as 50% in the old leaves of NaCl-treated plants, whereas this reduction was only 20–25% in the young leaves. Free proline accumulated in all aerial organs, and the highest levels were found in the young leaves. Patterns of total proteins extracted from the leaves of control or salt-treated plants were compared. The most obvious change concerned a 22-kDa, pl 7.5 polypeptide, which accumulated after exposure of the plants to NaCl. The appearance of this polypeptide was also mediated by a rapid drought stress, and sequencing indicated that it is related to the Künitz protease inhibitor family. A cDNA clone corresponding to the radish 22-kDa polypeptide was obtained and sequenced. Northern blot analysis showed that salt stress induces a large accumulation of this mRNA in the leaves of radish.     

Enhancement of salt tolerance in soybean with NaCl pretreatment

Acclimation response to salt stress in soybean ( Glycine max [L.] Merr. cv. Lee) was found in this study. Soybean seedlings were exposed to 0, 34 and 68 m M NaCl for 23 days (pretreatment), thereafter plants were exposed to 0, 68 and 137 m M NaCl until maturity (main treatment). There was no effect of pretreatment on growth, but at 137 m M NaCl, Na⁺ concentration in leaves of the plants pretreated with 34 m M NaCl was lower than that of plants pretreated with 0 and 68 m M NaCl. Furthermore, the survival rate under 137 m M NaCl improved with the 34 m M NaCl pretreatment. Therefore, it is possible that soybean can acclimate to salt stress by its increased survival rate, without showing any improvement in growth. The regulation of Na⁺ or Cl⁻ concentration in leaves could be one of the possible factors involved in salt acclimation of soybean.     

Higher accumulation of gamma-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots.

Polyamines (PAs) are assumed to perform their functions through their oxidative product such as gamma-aminobutyric acid (GABA) formation. However, there is only limited information on the interrelation between PA degradation and GABA accumulation under salt stress. In order to reveal a quantitative correlation between PA oxidation and GABA accumulation, the effects of treatments with different NaCl concentrations, along with aminoguanidine (AG, a specific inhibitor of diamine oxidases (DAO; EC: 1.4.3.6)) and a recovery test from salt stress on endogenous free PAs, gamma-aminobutyric acid (GABA) accumulation and DAO activity were determined in roots of soybean [Glycine max (L.) Merr.] cultivar Suxie-1. The results showed that the levels of putrescine (Put), cadaverine (Cad), and spermidine (Spd) decreased significantly with increasing salt concentrations. This occurred because salt stress strongly promoted DAO activity to stimulate PA degradation. GABA accumulation increased with growing NaCl concentrations, about an 11- to 17-fold increase as compared with the control plants. AG treatment increased the accumulation of endogenous free PAs as a result of a strong retardation of DAO activity, but decreased GABA accumulation. The recovery for 6 days in 1/2 Hoagland solution from 100mM NaCl stress resulted in a decrease in DAO activity, a rebound of PA levels and a simultaneous reduction of GABA content. A close correlation was observed between the changes in DAO activity and GABA accumulation. The results indicated that higher GABA accumulation (about 39%) induced by salt stress could come from PA degradation, suggesting that PAs might perform their functions through GABA formation under salt stress.     

Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots

Salt-stress-induced ABA accumulation in maize root tissues was compared with that in leaf tissues. While salt stress with NaCl resulted in a significant ABA accumulation in root tissues (up to 10-fold), the same stress only led to a small ABA accumulation in leaf tissues (about 1-fold). Pretreatment with ethylene glycol (EG), a permeable and inert monomer of PEG, could prevent the shrinkage of cell volume and completely block the ABA accumulation in leaf tissues under salt stress, but substantial salt-induced ABA accumulation was still observed in root tissues following such pretreatment. Hypotonic salt solutions, i.e. below 100 mM NaCl, still induced a significant ABA accumulation (more than 3-fold) in roots, but showed no effect on that in leaf tissues. Results suggest that the salt-stress-induced ABA accumulation in roots may also be triggered by an osmosensing mechanism, which is in addition to the perception of the changes in reduced cellular volume or plasmalemma tension that leads to ABA accumulation in leaves. When leaf and root tissues were immersed into salt solutions, salt entered into the cells as a function of time and salt concentrations. Such entrance apparently led to a loss of sensitivity of leaf tissues to accumulate ABA under the salt stress, and also prevented the leaf tissues from responding to further air-drying in terms of ABA accumulation. Roots showed no such responses. Results suggest that the entrance of salt into leaf cells brought about some toxic effect that might have reduced the capability of leaf cells to produce ABA under dehydration.     

Role of sodium in the ABA‐mediated long‐term growth response of bean to salt stress

Long‐term salt effects on plant growth have often been related to direct ion toxicity due to the accumulation of high ion concentrations in plant tissue. This work examines the relative importance of endogenous ABA, as well as Na⁺ and Cl⁻ toxicity, in the inhibition of leaf growth and photosynthesis, in bean plants grown at 1, 25, 50 and 75 m M NaCl until the fruit‐bearing stage. All salt‐treated plants showed very high leaf Cl⁻ concentrations, with little difference between plants exposed to 50 or 75 m M NaCl. The 25 and 50 mM salt‐treated plants were able to successfully exclude Na⁺ from their leaves, and only suffered an initial decline in the rate of leaf growth. Plants exposed to 75 m M NaCl showed an increase in Na⁺ leaf concentrations with an accompanying decrease in growth and photosynthesis as salt exposure progressed. A high correlation was found between leaf Na⁺ and leaf growth. Leaf ABA significantly increased with salt supply, and was highly correlated with both leaf Na⁺ and leaf growth. Our results suggest that in bean plants under long‐term salt stress, leaf ABA may participate in the regulation of leaf growth, and leaf Na⁺ would be at least partly responsible for increased ABA levels.     

The role of arbuscular mycorrhizal fungi in alleviating salt stress in Medicago sativa L. var. icon

Medicago sativa L. is the most important forage crop in arid and semi-arid areas, where increased salinity is a major factor limiting plant growth and crop productivity. The role of arbuscular mycorrhizal (AM) fungus Glomus viscosum H.T. Nicolson strain A6 in protecting alfalfa plants from salt stress, induced by sodium chloride (NaCl), was studied in two ways. Firstly, the root systems of 3-month old M. sativa plants, both mycorrhizal (AM+) and non-mycorrhizal (non-AM) (M. sativa L. var. icon), were placed in solutions of increasing salt concentrations (0, 50, 100, 150, 200 mM NaCl) to study the wilting response. G. viscosum improved the tolerance to salinity stress and the benefit was expressed in terms of the time required to reach the T4 stage in the wilting experiment. Secondly, to evaluate the ability of the Glomus-alfalfa symbiosis to tolerate salt, a pot experiment was set up in a glasshouse in which 3-month old alfalfa plants (M. sativa var. icon) were grown in a peat substratum at three salinity levels (0, 100, 150 mM NaCl). The AM symbiosis stimulated plant height, leaf area, root density, fresh and dry plant weight under saline conditions. Furthermore, proline accumulation was higher in mycorrhizal M. sativa plants than in non-mycorrhizal plants under conditions of salt stress. These and other results indicated that the micropropagated selected clone of M. sativa var. icon, when in symbiosis with G. viscosum H.T. Nicolson strain A6, exhibited better growth and physiological activities under saline conditions than non-AM plants. The AM+ plants also had lower sodium and chloride concentrations in tissues than non-AM plants.     

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.     

Differences in salt tolerance of three sugar beet genotypes

The effect of increasing NaCl concentrations (up to 150 m M ) on growth and mineral composition of three genotypes of sugar beet ( Beta vulgaris L., MONOHILL, ADA and FIA) has been studied. Growth was stimulated or little affected in water culture by 50 m M NaCl in all 3 genotypes. Further increase in NaCl concentration depressed growth in ADA more than in MONOHILL, whereas in FIA growth did not significantly differ from the untreated control. In all 3 genotypes, particularly in FIA, increasing NaCl concentrations decreased potassium content in the shoots more than in the fibrous and storage roots. Simultaneously, the accumulation of sodium and chloride in the shoots was considerably higher in FIA than in ADA, where in contrast larger proportions of these ions were retained in the roots. The results demonstrate considerable genotypic differences in salt tolerance of sugar beet and indicate a positive correlation between salt tolerance and accumulation of sodium and chloride in the shoots. FIA but not ADA may be suited for a breeding programme of sugar beet for improved salt tolerance.     

盐分和水分胁迫对芦荟幼苗渗透调节和渗调物质积累的影响

用不同浓度NaCl和等渗聚乙二醇(PEG 6000)处理芦荟(Aloe vera L.)幼苗,10 d后测定叶片相对生长速率和厚度、叶片中主要有机溶质、无机离子含量及渗透调节能力.结果表明,-0.44、-0.88 MPa NaCl和PEG处理使芦荟叶片的相对生长速率和叶片厚度明显下降,且盐胁迫对幼苗生长的抑制和叶片含水量降低的效应明显高于等渗的水分胁迫,其叶片渗透调节能力随处理渗透势的降低而增加, -0.88 MPa PEG胁迫的芦荟幼苗的渗透调节能力高于等渗盐分胁迫.在主要渗透调节物质可溶性糖、有机酸、K 、Ca2 和Cl-中,-0.88 MPa PEG处理下含量比相同渗透势的NaCl处理下显著增加的是有机溶质,因此推断有机溶质含量高是PEG胁迫下渗透调节能力较强的主要因素.