The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila

The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45 degrees C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis sigmaA-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.     

Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula

The impacts of salt stress (75 mM NaCl) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners’ variability for symbiotic traits and of the symbiosis integrity as indicated by: (a) the specificity of the partners’ interactions-the nonadditive inputs of their genotypes into the variation of symbiotic parameters and (b) the correlative links between these parameters. The structure of the nodD1 locus and the plasmid content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N₂-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass: height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.     

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K⁺/Na⁺ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.     

Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation

The application of safe and cheap inducers is important in the field of fermentation technology, which persuades employing new expression systems. In this study, a Bacillus subtilis expression system was induced by applying starvation and environmental stresses to produce xylanase. The expression plasmid harbors SigB-dependent ohrB promoter. The target gene was expressed by inoculating the recombinant strain into glucose-limited synthetic medium resulting in a sharp increase of xylanase activity at the end of logarithmic growth phase. The recombinant strain was able to express the xylanase enzyme 14-fold higher than that of the control one. The induction was also performed by exposing the recombinant strain to NaCl and ethanol stresses, and heat shock; the strain growing in LB showed 5-, 15- and 6-fold increases in xylanase activity, respectively. The best induction using environmental stresses was achieved by applying the salt stress in the synthetic medium. The maximum expression for NaCl and ethanol stresses occurred after 40 min of induction. All observed inductions were related to activation of SigB protein causing expression of the SigB-dependent xylanase gene. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers.     

Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity

Changes in biomass yield rates, cell membrane stability (CMS), malondialdehyde (MDA) content and in the levels of physiological stress markers such as proline and glycine betaine in two high yielding genotypes (S1 and ATP, salt tolerant and salt sensitive, respectively) of mulberry under NaCl salinity were studied. Biomass yield rates and CMS were significantly decreased in both the genotypes under stress conditions. Per cent of decrease in biomass yield rate and CMS was relatively less in S1 than in ATP. Salt stress results a significant increase in the accumulation of proline, by 6-fold in S1 and 4-fold in ATP. Glycine betaine content was also increased significantly in stressed plants. However, the per cent increase was more in S1 than in ATP. The level of lipid peroxidation as indicated by MDA formation was greater in ATP than in S1. These results clearly support the better salt tolerant nature of S1 compared to ATP genotype.     

Preferential accumulation of d-pinitol in Acrostichum aureum gametophytes in response to salt stress

Growth and metabolic responses to salt stress were studied in gametophytes of Acrostichum aureum L. (a mangrove fern). Gametophytes were cultured in 0–170 m M NaCl solutions. The growth of gametophytes was best in low NaCl concentrations (35 and 85 m M ), and was retarded at higher NaCl concentrations. Photosynthetic rate of gametophytes was also significantly reduced when grown in high NaCl concentrations. Severe salt stress (>120 m M NaCl) led to a preferential accumulation of d -pinitol in gametophytes, whereas the sporophyte accumulated d -1- O -methyl- muco -inositol. The content of d -pinitol reached up to 50% of the soluble carbohydrate pool of gametophytes under 155 and 170 m M NaCl. The accumulation of d -pinitol and other cyclitols in gametophytes was correlated with the retention of photochemical efficiency of photosystem II and the survival of gametophytes after transfer to solutions containing 340 and 600 m M NaCl, respectively.     

Physiological mechanisms of enhancing salt tolerance of oilseed rape plants with brassinosteroids

The ability of brassinosteroids, such as 24-epibrassinolide (EBL) to increase the resistance of oilseed rape plants (Brassica napus L.) to salt stress (175 mM NaCl) was investigated along with the possible mechanisms of their protective action. Seedlings were grown for three weeks on the Hoagland-Snyder medium under controlled conditions. The experimental plants were treated with either (1) 175 mM NaCl, or (2) 10^?10 M EBL, or (3) 175 mM NaCl plus 10^?10 M EBL by adding the corresponding components to the growth medium. The exposure was 7 and 14 days. As compared to the control, salinization inhibited plant height by 33–35%, reduced leaf area by 2.0–2.5 times, reduced 2.5- and 2-fold plant fresh and dry weight, respectively, reduced water content of plant tissues by 26–31% and, twofold, the content of chlorophylls a and b. Plants responded to NaCl by developing oxidative stress conditions, lowering the osmotic potential of the cell contents down to ?2 MPa, accumulating proline (by 43–52 times) and low-molecular-weight phenolics (by 1.9–2.7 times). Oilseed rape plants were shown to respond to salinization with an increase of endogenous content of steroid hormones: 24-epibrassinosteroids (24-epibrassinolide and 24-epicastasterone), 24S-methyl-brassinosteroids (brassinolide and castasterone), and 28-homobrassinosteroids (28-homobrassinolide and 28-homocastasterone); such evidence indirectly confirms the involvement of brassinosteroids in the development of salt tolerance. Adding EBL to the nutrient medium under optimal growth conditions did not significantly affect the indices under study. Under salt stress, EBL showed a pronounced protective effect: stem growth was fully restored, plant assimilation area increased by as much as 67–76% as compared to the control index, fresh and dry weight largely recovered (up to 85–92% of the control values), and the inhibitory effect of NaCl on photosynthetic pigments was diminished. Exogenous EBL impeded the development of NaCl-dependent lipid peroxidation and increased the osmotic potential of the leaf cell contents. The protective effect of EBL under salt stress was probably associated with EBL antioxidant effect, rather than the hormone-induced accumulation of proline and of low-molecula-weight phenolics, as well as with the ability to regulate water status by maintaining intracellular ion homeostasis.     

谷子SiRLK35基因克隆及功能分析

为探讨谷子(Setaria italica L.)耐旱抗逆机制,解析类受体蛋白激酶(receptor like protein kinase, RLKs)基因功能,进而为培育谷子抗逆新品种提供依据,本文以干旱处理的谷子“豫谷1号”为材料,通过iTRAQ技术筛选到1个干旱响应的类受体蛋白激酶基因,命名为SiRLK35。以谷子RNA反转录的单链cDNA为模板,经PCR扩增获取SiRLK35基因全长序列。应用qRT-PCR方法,对SiRLK35在NaCl、PEG、ABA、GA、MeJA等不同处理下的表达模式进行分析。进一步构建基因原核表达载体pET28a-SiRLK35,结合斑点法对SiRLK35的抗盐能力进行初步评价。同时构建过表达载体pCAMBIA1301P-SiRLK35转化水稻,并对转基因植株抗盐能力进行检测。结果显示：胁迫及激素处理均可不同程度诱导SiRLK35基因的表达;斑点法研究结果显示,在相同NaCl浓度的LB平板上,含有SiRLK35基因的原核表达载体的大肠杆菌菌株生长状态较阴性对照好,SiRLK35具有一定的抗盐能力;获得的转SiRLK35基因水稻植株对盐胁迫的耐受性高于对照。SiRLK35基因对不同胁迫均可以产生响应,但对盐胁迫的响应较为明显,推测该基因可能在谷子的抗盐及抗逆过程中发挥作用。     

Response of mycorrhizal and nonmycorrhizal Arachis hypogaea to NaCl and acid stress

 The response of peanut to salt (NaCl) and acid (HCl) stress was studied in association with Glomus caledonium, an arbuscular mycorrhizal (AM) fungus. The plants were exposed to salt stress by irrigation on alternate days with 1% or 5% NaCl solutions, or with 0.1 N HCl to induce acid stress. Plant yield almost tripled in mycorrhizal plants compared with nonmycorrhizal control plants. AM inoculation significantly increased plant yield and biomass at 1% NaCl, while at 5% NaCl AM was less effective in alleviating salt stress. Percentage AM colonization was also lowest at 5% NaCl. AM inoculation was found to promote the establishment of peanut plants under acid stress conditions. Accepted: 2 October 1995     

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.     

Growth, zearalenone production and some metabolic activities of Fusarium moniliforme under salt stress

With a increasing salinity, a decrease in the growth rate of Fusarium moniliforme was observed. The percentage of germinated conidia decreased with increasing salinity (% germination ranged from 80.3% at 0.0% NaCl to 0.0% at 15% NaCl). Concentration of 12.5% NaCl produced the highest chlamydospore-like structures. Concentration of 5% NaCl increased zearalenone production which decreased with increasing salt stress. Escherichia coli was more tolerant to the toxin than Bacillus subtilis. The total amount of lipids produced by F. moniliforme changed with increasing the concentration of NaCl in the growth medium. The genomic DNA of the control and treated samples showed a common band of more than 20 kilobases. Similar RAPD-PCR patterns were also produced.     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

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.     

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.     

Seed germination of the halophyteSuaeda japonica under salt stress

Suaeda japonica Makino belonging to the family Chenopodiaceae, is a halophyte and grows at the shore of Ariake sea in Japan. This plant presumably possesses high salt resistant nature, thus, we examined the mechanisms of seed germination under salt stress. The seeds maintained 80% germination rates on the medium containing 0.7 M NaCl. Germination rates varied depending on salt type; the germination rates under NaCl or KCI exhibited relatively lower values than ones under sodium gluconate or potassium gluconate. This different responses for salts seemed to be as a result of the presence of Cl⁻ ions. Although very high levels of betaine (compatible solute), were kept in the seedlings grown under no salt stress, the contents gradually increased as concentration of NaCl increased. Betaine is a factor present in plants that works to alleviate the effects of excessive soil salts. It is synthesized in leaves from betaine aldehyde, and this process is catabolized by betaine aldehyde dehydrogenase (BADH). When the seedlings were cultivated on the medium without NaCl, relatively high level of BADH activity was found. The activity increased 5-fold in the seedlings grown under 0.5 M NaCl stress. Increases in betaine content and BADH activity were found during seed germination. InS. japonica, the salt stress promoted BADH activity, subsequently endogenous betaine contents were increased, and increased betaine seemed to secure seed germination under salt stress.