Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.     

Salt-induced Inhibition of Phosphate Transport and Release of Membrane Proteins from Barley Roots

Osmotic shock with sequential 30-minute treatments in ice-cold saline solutions and H₂O released proteins from excised barley roots and inhibited the subsequent uptake of orthophosphate (Pi). The amount of protein released increased sharply at NaCl concentrations above 0.05 molar, approximately the threshold concentration above which Pi uptake was increasingly suppressed. About 60% of the nearly 100 micrograms of protein per gram fresh weight of roots that was eluted in 0.16 molar NaCl treatments apparently had no function in Pi transport, since it was eluted at NaCl concentrations (≤0.05 molar) that did not affect Pi uptake. Although 0.16 molar NaCl completely inhibited Pi uptake, active uptake resumed at about 60% of control rates within 1 to 2 hours. The presence of either puromycin or cycloheximide greatly reduced the recovery of Pi uptake activity after the NaCl treatment. Mannitol and various monovalent and divalent salts at concentrations isosmotic with NaCl also inhibited Pi uptake, but CaCl₂ was consistently the least inhibitory. The correlation between the concentration of the osmotic treatments and the simultaneous loss of protein and Pi uptake activity, together with the evidence that uptake recovery requires protein synthesis, support the hypothesis that the proteins eluted are required for active Pi transport.     

A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts

Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.Abbreviations CPW Cell and Protoplast Wash Solution - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - MS Murashige and Skoog medium (1962) - UM Uchimiya and Murashige medium (1976)     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

Isolation of sodium chloride-tolerant callus line of Cicer arietinum L. cv. BG-203

A NaCl-tolerant callus line of Cicer arietinum has been isolated, as a spontaneous variant, on agar-solidified MS-medium supplemented with 100 mM NaCl. The growth of this line, in the presence of 100 mM NaCl, was comparable to that of the NaCl-sensitive callus line growing in the absence of NaCl. Regarding relative tolerance of the two callus lines towards NaCl (0 to 200 mM), the tolerant line performed poorly in the absence of NaCl and exhibited optimal growth at 50 mM NaCl. The tolerance persisted even after three passages of 4-wk each, tested so far, away from the selective agent.     

Plantlet regeneration from a NaCl-selected salt-tolerant callus culture of Shamouti orange (Citrus sinensis L. Osbeck)

Plantlets were regenerated from a selected salt-tolerant cell line of Shamouti orange (Citrus sinensis L. Osbeck). Embryogenesis was carried out both in the presence and absence of NaCl, yielding green and white globular embryos, respectively. Greening could be induced subsequently and normal heart shape embryo development was obtained. Plantlet formation required exposure to kinetin prior to the introduction of the root-inducing hormone naphthalene acetic acid. This system differs from the designed protocol for plant regeneration from the salt-sensitive, i.e., unselected callus. It is concluded that NaCl interferes with the regeneration process, with embryogenesis and/or embryo development into plantlets. Its presence during callus growth probably changes the balance of the phytohormones which is later manifested in plant regeneration. Citrus salt-tolerant callus yields salt-tolerant embryos. Salt-tolerant calli derived from regenerated plantlets indicate acquisition of salt tolerance on the whole plant level.     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.     

Marker proteins for embryogenic differentiation patterns in pea callus

Polypeptide pattern alterations during somatic embryogenesis were investigated using callus cultures of two Pisum sativum genotypes. Both genotypes show the formation of two different callus lines from the same explant after six to eight weeks in culture: a nodular yellowish callus line, which forms somatic embryoids in suspension cultures (e⁺) and a white compact callus line with no regenerative capacity (e^–). The cytosol proteins of the two different callus lines were separated in a semi-preparative two-dimensional system and the polypeptide patterns were compared. Two protein bands were found (P1: M_r=45000 D, pI=7.0–7.1; P2: M_r=7000 D, pI=<4.5), which were characteristic of the putatively embryogenic (e⁺) callus line in all tissues investigated (two genotypes × two explant sources). These proteins found in nodular (e⁺) pea cultures are very similar to two proteins found in Daucus carota suspension cultures preceding the formation of somatic embryos.Abbreviations BA 6-benzyl-aminopurine - Bistris 2-(bis(2-hydroxyethyl)imino)-2-(hydroxymethyl)-1.3-propanediol - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane     

Osmostress-induced changes in yeast gene expression

When Saccharomyces cerevisiae cells are exposed to high concentration of NaCl, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCl) by a previous treatment with 0.7 M NaCl, but not by a previous heat shock. Two-dimensional analysis of [3H]-leucine-labelled proteins from salt-shocked cells (0.7 M NaCl) revealed the elevated rate of synthesis of nine proteins, among which were the heat-shock proteins hsp12 and hsp26. Northern analysis using gene-specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol-3-phosphate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso-osmolar NaCl concentration (0.7 M).     

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.     

Regulation of protein synthesis in root, shoot and embryonic tissues of germinating barley during salinity stress

Abstract Protein synthesis during seed germination, a stage vulnerable to salinity stress, was investigated. The responses of barley genotypes, CM72 (California Mariout 72) and Prato, toward salinity were different during seed germination. Germination of CM72 was unaffected up to 0.34 kmol m^?3 (2%) NaCl, but that of Prato was reduced 30% by 0.17 kmol m ³ NaCl and 75% by 0.34 kmol m^?3 NaCl. Therefore, the former genotype is relatively more salt-tolerant than the latter. Protein synthesis in roots, shoots, and embryos was investigated in these two genotypes before and after salinity stress. The uptake of S-methionine and its incorporation into protein were significantly reduced by salinity in both genotypes. The inhibition of global protein synthesis was significant in roots and shoots. Proteins from different tissues were resolved by single and two dimensional gels. The steady-state protein levels were maintained remarkably well during salinity stress in roots and shoots. Likewise, proteins in germinating embryos were stable except for a 42-kilodalton protein unique to the salt tolerant genotype which was apparently degraded during salinity stress. Salinity, around 0.34 kmol m^?3 NaCl, induced both quantitative and qualitative changes in the expression of some proteins labelled in vivo. The quantitative changes included repression or enhancement of synthesis of selected groups of proteins. Around 8% of the nearly 400 resolved proteins in a tissue was affected this way. Some of the proteins in this category were specific to each genotype. About 1 % of the total showed qualitative changes; these proteins were expressed only during salinity stress. In roots, two proteins (28, 41.7 kilodaltons) were detected in CM72 and five (28, 45, 60.5, 76.5, 82.5 kilodaltons) in Prato; only the 28-kilodalton protein was common to both genotypes. In shoots, four proteins (45, 60.5, 76.5, 82.5 kilodaltons) were found only in Prato and these were similar to those induced in roots. The four new proteins (32, 37.5, 89, 92 kilodaltons) in germinating embryos were apparently induced only in CM72; these were distinctly different from those detected in developed roots and shoots. The unique protein changes induced by salinity stress during germination (this study) and seedling growth studies reported earlier (Ramagopal, 1987b) are apparently different. The findings demonstrate that ontogeny plays an important role in the expression of tissue-specific proteins during salinity stress in the salt tolerant and sensitive barley genotypes.     

Transformation of Medicago arborea L. with an Agrobacterium rhizogenes binary vector carrying the hygromycin resistance gene

Plants of Medicago arborea have been infected with Agrobacterium rhizogenes strain LBA9402 harbouring the plasmids Ri 1855 and AGS125 carrying a gene conferring resistance to the antibiotic hygromycin. About 7056 of the hairy roots showed callus formation on hygromycin-supplemented medium. Regeneration took place on antibiotic free medium only. Plantlets suitable for transfer to soil were obtained after the manual removal of most of the leaves. Plant morphology showed the usual alterations induced by the Ri plasmid; moreover, two years after soil-transfer, transformants have not flowered. Molecular analysis indicates the presence of T-DNA from both pAGS 125 and p1855. The expression of the hygromycin phosphotransferase gene allowed callus and protoplasts of transformed plants to grow on media supplemented with the antibiotic. This trait will be utilized as a marker in protoplast fusion between Medicago arborea and Medicago sativa (alfalfa).Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - kin kinetin - GA3 Gibberellic acid - IAA Indole-3-acetic acid - HPT hygromycin phosphotransferase - NOS nopaline synthase - MS Murashige and Skoog (1962) - B5 Gamborg et al. (1968) - B5hy B5 supplemented with 20 mg 1-1 of hygromycin - YMB yeast mannitol broth     

Effect of NaCl and mannitol on plasma membrane proteins in corn roots

Summary The short-term effects of NaCl and mannitol stress on plasma membrane (PM) polypeptides from corn roots (Zea mays L.) were determined using two-dimensional gel electrophoresis following radiolabeled amino acid incorporation. After 2.5 hours, both stress treatments altered synthesis of several polypeptides. Changes included up-regulation of some polypeptides with concomitant down-regulation of others. Some changes were unique to the stress treatment while others were common to both NaCl and mannitol. No new polypeptides appeared in either case. Pulse-chase experiments following 0.5-hours and 2.5-hours incubation periods with radiolabeled amino acids did not reveal differences in turnover of PM polypeptides. The results support the contention that altered synthesis of PM proteins under stress may contribute to the alteration of membrane function.Abbreviations ER endoplasmic reticulum: GA Golgi - PM plasma membrane - PVPP polyvinylpolypyrrolidone     

Proline is not the primary determinant of chilling tolerance induced by mannitol or abscisic Acid in regenerable maize callus cultures

Chilling sensitive regenerable maize (Zea mays L.) callus cultures can be induced to survive prolonged exposure to 4°C by treatments with mannitol, abscisic acid (ABA), and/or high levels of proline. Maize callus with a free proline content of about 122 micromoles/grain fresh weight survived longer exposures to 4°C than did callus with a free proline content of about 68 micromoles/grain fresh weight. The addition of 0.53 molar mannitol or 0.1 millimolar ABA to culture medium produced a free proline content in maize callus of about 136 and 145 micromoles/grain fresh weight, respectively, if the medium contained 12 millimolar proline or about 36 and 1 micromoles/grain fresh weight, respectively, if no proline was in the medium. Although these mannitol and ABA treatments produced drastically different free proline levels in maize callus, callus grown on these media survived longer exposures to 4°C than did maize callus grown on any proline treatment alone. Thus, the internal free proline level of treated callus is not the primary factor conferring chilling tolerance on these tissues.     

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.     

Correlation of glyoxalase I activity with cell proliferation inDatura callus culture

Glyoxalase-I activity in growingDatura callus showed 184% increase with the age of the culture. Spermidine increased the enzyme activity together with DNA and protein synthesis. With the addition of mitotic inhibitors, vinblastine and methylglyoxal in the growth medium, the enzyme activity was inhibited by 92 percent and 50 percent respectively, at the most effective concentration and the callus growth was also reduced. Similar results were obtained with specific glyoxalase I inhibitors, iso-ascorbate and squaric acid.     

Influence of formaldehyde in control of bacterial and fungal contaminants in plant cell cultures: Its effect on growth and secondary metabolite production

Summary Influence of formaldehyde on growth and secondary metabolite production inin vitro grown tissues of pyrethrum, capsicum and carrot were studied. Formaldehyde concentration above 0.025% completely inhibited the growth of addedBacillus andAspergillus in the culture medium. Formaldehyde up to 0.025% level caused slight inhibition in the growth of pyrethrum callus. Callus subjected to 0.05, 0.1 and 0.2% formaldehyde enhanced the level of pyrethrin production in pyrethrum callus, whereas production of phenolics was lower in all the treatments. Growth of carrot callus and anthocyanin production was not inhibited up to a formaldehyde concentration of 0.04%. Similarly, the production of capsaicin in immobilised cell cultures ofCapsicum frutescens was not inhibited up to 0.04% level of formaldeyde. The results demonstrate the usefulness of formaldehyde to control contamination in plant tissue culture.     

Heat shock protects cultured neurons from glutamate toxicity.

Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions.