Chromotoxic effects of exogenous hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) in barley seeds exposed to salt stress

The effect of exogenous hydrogen peroxide (H₂O₂) on mitotic activity and chromosomal aberrations in root tip meristems of barley (Hordeum vulgare L. var. Tokak 157/37) germinated under salinity was analyzed. The inhibitory effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration (0.00 control, 0.35, 0.40, 0.45 M, molal NaCl). The frequency of chromosomal aberrations of seeds germinated in medium with 0.40 M NaCl after pretreatment with H₂O₂ (30 μM, micromolal) was significantly higher than the control group. The highest concentration of NaCl (0.45 M) together with H₂O₂ caused total inhibition of germination. In this study, the intention was to determine the performance of H₂O₂ in alleviating detrimental effect of salt stress on mitotic activity and chromosomal aberrations. However, H₂O₂ did not reduce the detrimental effect of NaCl on these parameters. Also, it caused higher chromotoxic effect compared to those of control groups.     

Role of some growth regulators on cytogenetic activity of barley under salt stress

The effects of gibberellic acid (GA₃), kinetin (KIN), benzyladenine and ethylene (E) on mitotic activity and chromosomal aberrations in root tips of barley seeds (Hordeum vulgare L. cv. “Bülbül 89”) germinated under salt stress were investigated. It was determined that all of these plant growth regulators (PGRs) decreased mitotic index in root tips of barley seeds germinated at 20 °C and in distilled water. Furthermore, some of the PGRs studied increased significantly the frequency of chromosomal aberrations. The frequency of chromosomal aberrations in seeds treated with E and KIN was considerably higher than in the seeds germinated under nonstress conditions. The inhibitory effect of salt stress on mitotic index increased with increasing salt concentration (0.30, 0.35, 0.40 and 0.45 molal, m). GA₃ and KIN pretreatments showed a successful performance in ameliorating the negative effects of increasing salinity on mitotic activity. The number of chromosomal aberrations also increased with increasing NaCl concentration. However, most of the PGR pretreatments studied alleviated the detrimental effects of increasing salinity on chromosomal aberrations. KIN pretreatment at 0.30 and 0.35 m salinity could not rescued the cytogenetic activity of salt stress on this parameter.     

Protective roles of exogenous polyamines on chromosomal aberrations in <Emphasis Type="Italic">Hordeum vulgare</Emphasis> exposed to salinity

The effects of exogenous polyamines (PAs): spermine (Spm), spermidine (Spd), cadaverine (Cad) and putrescine (Put) on mitotic activity and chromosomal aberrations in root meristem cells of Hordeum vulgare L. (barley) seeds exposed to salinity were analyzed. The PAs significantly inhibited cell division in distilled water. Furthermore, most of these PAs (except for Spd) caused a significant increase in the frequency of chromosomal aberrations as compared to control group. Seeds treated with Put caused the highest percentage of mitotic abnormalities in total. The negative effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration. PAs studied could not be successful in ameliorating of the negative effect of salinity on mitotic activity. Particularly, exposure to Cad and 0.40 M NaCl caused a complete block of cell division in total. However, most of the PA studied showed a perfectly performance in alleviating the detrimental effects of increasing salinity on chromosomal aberrations.     

Serial cultivation of Ehrlich ascites tumor cells in hypertonic media

Sublines of hyperdiploid Ehrlich ascites tumor cells (ED-0.15 cells) growing in Eagle basal medium (total salt concentration: 0.15 M) supplemented with 10 % fetal calf serum were adapted, by stepwise addition of NaCl to the medium, to growth in hypertonic media at salt concentrations of 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 M. Progressive increases in the NaCl concentration resulted in a progressive increase in the population doubling time of these high-salt-tolerant cultures. Cells growing in isotonic control medium were predominantly ‘epithelial-like’ in contrast to the flat, extended, ‘fibroblast-like’ morphology of cells growing in hypertonic media.     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

In vitro selection of salt tolerant variants following <Superscript>60</Superscript>Co gamma irradiation of long-term callus cultures of <Emphasis Type="Italic">Zoysia matrella</Emphasis> [L.] Merr.

To study growth in the presence of NaCl, in vitro plantlets regenerated from callus of manilagrass (Zoysia matrella [L.] Merr.) were cultured on regeneration medium supplemented with or without 0.3 M NaCl. The results indicated that growth was significantly inhibited by NaCl, with the leaves becoming relatively shorter and thicker. The differences of in vitro plantlets grown under NaCl stress provided specific criteria for the selection of salt tolerant variants. The 6-year maintained calli were treated with different doses (0, 5, 10, 20, 40, 80, 100, 150, 200, 250, and 300 Gy) of ₆₀Co γ rays. Regeneration rate and regeneration capacity of the calli were highest after treatment with 20 Gy ⁶⁰Co γ rays, 27.08 and 91.67% respectively. When the irradiation dose was increased to 100 Gy, 10.42% of the calli developed shoots, but at 150 Gy, both regeneration capacity and regeneration rate declined significantly, and no shoot was observed after 6 weeks of regeneration. Therefore, 100–150 Gy is the most appropriate irradiation span for inducing somaclonal variation. The irradiated calli were selected in vitro for NaCl tolerance. Five NaCl tolerant variant lines, Ze1, Fv1, Te1, Tw1, Fr1, were selected on subculture medium supplemented with 0.35 M NaCl, then transferred to regeneration medium containing 0.25 M NaCl, and grown in a greenhouse. The dark green colour index (DGCI) was used to identify the amount of injury caused by NaCl treatment. This was significantly higher in four of the lines, Ze1, Fv1, Te1, Fr1 (30.88, 31.17, 30.45 and 37.70%, respectively) compared to the control line (CK), which was regenerated from calli subcultured monthly (27.39%), indicating that watering with NaCl caused less injury in these four lines. These lines had lower proline contents than CK under salt stress. The superoxide dismutase (SOD) activity was higher in Ze1 under control condition and its peroxidase (POD) activity increased significantly under salt stress. With Fr1 catalase (CAT) activity was higher under salt stress. The higher activity of these antioxidant enzymes may contribute to the enhanced salt tolerance of the four plant lines.     

Ameliorative effect of brassinosteroid and ethylene on germination of cucumber seeds in the presence of sodium chloride

Brassinosteroids are a class of plant polyhydroxysteroids with a diverse of functions in plant growth and development, while ethylene is a gaseous hormone involved in regulation of numerous physiological processes. To evaluate the roles of BR and ethylene in seed germination under conditions of salt stress, effects of 24-Epibrassinolide (EBR) and 1-aminocyclopropane-1-carboxylic acid (ACC) on seed germination of cucumber (Cucumis sativus) seeds in the presence of 250 mM NaCl were investigated. Seed germination was significantly inhibited by the presence of NaCl in the incubation medium, and the inhibitory effect was significantly alleviated by addition of EBR and ACC to the incubation medium containing NaCl. There was an increase in ethylene evolution during seed germination and this increase was suppressed by salt stress. The reduction in ethylene evolution from imbibed seeds by salt stress was attenuated by EBR. Salt stress inhibited ACC oxidase (ACO) activity and EBR reversed the salt stress-induced decrease in ACO activity. Salt stress reduced expression of gene encoding ACO (CsACO2), and EBR reversed the salt stress-induced down-regulation of CsACO2. The alleviative effect of EBR on seed germination in the presence of NaCl was diminished by antagonist of ethylene synthesis, aminoethoxyvinylglycine. These results indicate that both ethylene and BR are likely to be associated with suppression of seed germination under salt stress and that the mitigating effect of BR on salt stress-induced inhibition of seed germination may occur through its interaction with ethylene synthesis.     

Pretreatment with 24-Epibrassinolide Synergistically Protects Root Structures and Chloroplastic Pigments and Upregulates Antioxidant Enzymes and Biomass in Na+-Stressed Tomato Plants

Salt stress reduces plant growth by negatively interfering with the division rate and cellular expansion, limiting the growth and development of the roots, stems, and leaves. 24-Epibrassinolide (EBR) is a molecule extracted from plant tissues and is a plant growth regulator with a high capacity to modulate tolerance to abiotic stresses. The objective of this study was to verify the possible improvements promoted by pretreatment with EBR in salt-stressed tomato plants, evaluating the variables related to root anatomy, photosynthetic pigments, antioxidant system, and biomass accumulation. The experiment comprised four treatments: two salt conditions (0 and 150 mM NaCl, described as Na⁺ (?) and Na⁺ (?+), respectively) and two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as EBR (?) and EBR (?+), respectively). EBR modulated the protection and vascularization of root structures, as demonstrated by the increases in epidermis thickness (12%) and metaxilem diameter (119%), respectively. This steroid relieved oxidative damage, which was clearly linked to elevated activities of superoxide ascorbate peroxidase (24%) and guaiacol peroxidase (31%). EBR also benefited photosynthetic pigments, reducing the degradation of chlorophylls. In addition, pretreatment with EBR favoured a higher biomass, which was due to positive effects on leaf and root tissues, including better performance of photosynthetic machinery.

     

Evaluating cadmium toxicity in the root meristem of <Emphasis Type="Italic">Pisum</Emphasis><Emphasis Type="Italic">sativum</Emphasis> L.

The effect of cadmium (Cd) was studied on root tips of Pisum sativum L. Seeds of P. sativum were treated with a series of concentrations ranging from 0.125, 0.250, 0.500 and 1.000 mM for 6 h. The effect of Cd was analyzed by studying the percentage seed germination, radicle length (RL), mitotic index (MI) and chromosomal aberrations (CAs) in root tip. The results revealed that Cd had significant impeding effect on the root meristem activity of P. sativum at 0.500 and 1.000 mM as noticed by reduction in seed germination percentage and RL compared to control. Furthermore, it also reduced MI in dose-related manner compared to control. Additionally, the variation in the percentage of mitotic abnormalities was observed. The overall percentage of aberrations generally increased with increasing concentrations of Cd. Among these abnormalities laggards, bridges, stickiness, precocious separation and fragments were most common. The obtained results demonstrated that the Cd treatment leads to a significant reduction in MI and increase in CAs. Overall results allow us to suggest that the Cd has clastogenic effect on the crop.     

Alleviation of Oxidative Stress Induced by 24-Epibrassinolide in Soybean Plants Exposed to Different Manganese Supplies: UpRegulation of Antioxidant Enzymes and Maintenance of Photosynthetic Pigments

Adverse effects promoted by inadequate manganese (Mn) supply (deficiency or toxicity) causes inefficiency of the antioxidant system and degradation of chlorophylls. However, 24-epibrassinolide (EBR) is a natural steroid that exhibits beneficial effects on antioxidant metabolism, chlorophyll levels and stress indicators. Therefore, this research aims to evaluate whether EBR application via spray can alleviate oxidative stress in soybean plants exposed to different Mn concentrations and to determine possible contributions of the antioxidant enzymes and photosynthetic pigments. Experiment followed a completely randomized factorial design with two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as − EBR and + EBR, respectively) and three Mn doses (0.25, 25 and 2500 µM Mn, described as low, control and high supply of Mn, respectively). Plants treated with low and high concentrations of Mn + EBR exhibit significant increases in all enzymes evaluated (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase). To superoxide dismutase (SOD), EBR spray promoted increments of 77%, 38% and 76% under low, control and high Mn supplementation, respectively, compared to same treatment in absence of EBR. Clearly intense activity is linked to SOD contributed by dismutation of superoxide into hydrogen peroxide, being subsequently decomposed by other enzymes (catalase, ascorbate peroxidase and peroxidase). Concomitantly, plants with Mn deficiency and toxicity sprayed with 100 nM EBR presented maintenance of chlorophylls and carotenoids due to reduction of superoxide and hydrogen peroxide and consequently reduced chloroplast membrane damages as indicated by malondialdehyde levels and electrolyte leakage.

     

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.     

Effects of Fusicoccin on the Early Stages of Sorghum Growth at High NaCl Concentrations

The effects of fusicoccin (FC) on the early growth processes in sorghum (Sorghum vulgare M.) seeds germinated in water and in 0.1 M or 0.2 M NaCl solutions were investigated. We studied the rate of seed imbibition, the onset of radicle protrusion, the occurrence of the first mitoses, the mitotic index, the distribution of cells according to the phases of the cell cycle, as well as the length and weight of roots. Seed imbibition was considerably accelerated by treating them with 5 × 10^–6 M FC for 1 h. In these FC-treated seeds placed on NaCl solutions, FC stimulated water influx into seeds, radicle protrusion, and occurrence of the first mitoses. FC pretreatment did not affect substantially the distribution of meristematic cells according to the periods of the cell cycle after 72 h of seed germination on water or 0.1 M NaCl. Root growth was inhibited by 0.1 M NaCl, but it was partially recovered in the presence of FC. 0.2 M NaCl caused a decrease in the mitotic index and in the number of cells in the S phase, an accumulation of cells in the G₂ period and in the prophase, as well as a considerable inhibition of root growth. FC pretreatment of seeds subsequently germinated on 0.2 M NaCl resulted in an increase in the number of cells in the G₁ period, in the mitotic index, and in the root-growth rate. FC virtually did not affect the growth of sorghum in the absence of salt. Pretreatment of seeds with FC followed by salinization resulted in an increase in the water content in seeds. It is suggested that a FC-induced increase in the water content of seeds accelerated metabolic processes in seeds germinating on NaCl solutions, thus regulating ionic homeostasis and thereby stimulating the initial growth processes.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 378–383.Original Russian Text Copyright © 2005 by Lutsenko, Marushko, Kononenko, Leonova.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

The electron-microscopic structure of nucleoprotein fibrils from metaphase chromosomes treated with salt

The diameter of nucleoprotein fibres of metaphase chromosomes is sensitive to salt concentrations. Treatment of human lymphocyte cells in metaphase with a hypotonic medium and spreading them on a water surface causes swelling of the chromosome fibres from 150–180 Å to 230–250 Å. Treatment of the chromosomes with 0.15–0.45 M NaCl, a concentration at which histones are not yet removed from the nucleoprotein complexes, apparently does not affect the chromosome structure. Treatment with NaCl solutions between 0.6 M and 2.0 M NaCl leads to a progressive extraction of the chromosomal proteins and decreases the diameter of the chromosome fibres to 80–100 Å and less.Dedicated to Prof. Dr. A. Butenandt on the occasion of his 70th birthday.     

Evaluation of cytological effects of Zn2+ in relation to germination and root growth of Nigella sativa L. and Triticum aestivum L

The effects of different treatments with zinc sulfate (Zn(2+)) on the cytology and growth of Nigella sativa and Triticum aestivum were investigated. Five concentrations of zinc sulfate ranging from 5 to 25mg/l were applied for 6, 12, 18, and 24h. The treatments reduced the germination percentages of N. sativa seeds and T. aestivum grains and inhibited the root growth of both plants. Concentrations higher than 25mg/l of Zn(2+) applied for 24h were toxic for both plants. The non-lethal concentrations of Zn(2+) showed an inhibitory effect on cell division in root tips of both plants and caused a decrease in their mitotic index values. The reduction in MI in root tips of T. aestivum was more evident than that of N. sativa. All treatments changed the frequency of mitotic phases as compared with the control values. The total percentage of abnormalities in N. saliva was more than that in T. aestivum. Zn(2+) treatments produced a number of mitotic abnormalities in dividing cells in root tips of both plants resulting from its action on the spindle apparatus such as C-metaphases, lagging chromosomes and multipolar anaphases and telophases. Also, Zn(2+) induced vacuolated nuclei and irregular prophases. The induction of chromosomal stickiness and chromosomal aberrations such as bridges and breaks indicates its action on the chromosome. These abnormalities (chromosome breaks and chromosomal bridges at ana-telophases) indicate true clastogenic potential of the ions tested.     

Cryo-electron microscopy of an extremely halophilic microbe: technical aspects

Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.

     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

Regulation of Root Growth in Lactuca sativa L. Seedlings by the Ent-Kaurane Diterpenoid Epinodosin

Epinodosin, an ent-kaurane diterpenoid isolated from Isodon japonica var. galaucocalyx, had a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in Lactuca sativa L. seedlings. Lower levels of epinodosin (25–100 μM) significantly promoted root growth, but higher concentrations (150–200 μM), by contrast, had inhibitory effects. In addition, all of the tested concentrations (20–80 μM) inhibited root hair development of lettuce seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of epinodosin (25–100 μM) resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in lettuce seedling root tips. On the other hand, epinodosin at higher concentrations inhibited root growth by strongly affecting both the cell length in the mature region and the division of meristematic cells. Comet assay analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced by higher levels of epinodosin.     

Osmotic Shock Augments Ethanol Stress in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 2918

Yeast cells sense and respond to hypertonicity. Saccharomyces cerevisiae MTCC 2918 was tested for its metabolic status in 1 M NaCl by cell viability analysis, intracellular glycerol content and total antioxidant capacity. Yeast cell viability was maximum in 1 M NaCl and 24 h addition of 1 M NaCl was effective in induction of hyperosmolarity. Increased glycerol contents in cells treated with salt indicated adaptation to osmotic stress with a maximum of 240.87 ± 0.38 mg/g dry weight (DW) at 72 h. The total antioxidant status with 1 M NaCl was 9.29 ± 0.39 mM/g DW at 96 h reflecting free radical quenching to overcome stress with increasing growth period. Considering that pre-adaptation to one type of stress evoked a protective response to other stress factors, we have attempted the cross adaptation of osmotic shock to high ethanol concentrations. In effect, we observed that osmotic shock lowered the cell survival by augmentation of cell toxicity by ethanol due to stress induction during exponential phase. Glycerol accumulation to an order of 470.27 ± 0.53 mg/g DW at 48 h in 1 M NaCl and 12% ethanol indicated that both stresses culminated in membrane disruption further leading to cell burst and contributed to the stress overload.