Ultrastructural and protein changes in cell suspension cultures of peach associated with low temperature-induced cold acclimation and abscisic acid treatment

Cell suspension cultures were initiated from callus derived from xylem tissues of peach [Prunus persica (L.) Batsch]. Cold acclimation was induced (LT₅₀ of-13°C) in cell suspensions at 3°C in the dark for 10 days. Freezing tolerance returned to the level of nonacclimated cells (LT₅₀ of –4.5°C) when cold-acclimated cells were transferred to 24°C (in dark) for 3 days. Addition of 75 M abscisic acid (ABA) to the growth medium failed to induce cold acclimation after cells were cultured for 5 days at 24°C. Microvacuolation, cytoplasmic augmentation and disappearance of starch grains were observed in cells that were cold-acclimated by exposure to low temperature. Similar ultrastructural alterations were not observed in ABA-treated cells. Several qualitative and quantitative changes in proteins were noted during both cold acclimation and ABA treatment. Both the ultrastructural and protein changes observed during cold acclimation were reversed during deacclimation. The relationship of these changes to cold acclimation in peach cell-cultures is discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - Ms Murashige & Skoog - PMSF phenylmethylsulfonyl fluoride - LT₅₀ or Freezing Tolerance temperature that resulted in 50% decrease in TTC reduction - TTC 2,3,5-triphenyltetrazolium chloride     

Respiration deficient mutants of Schizosaccharomyces Pombe I

Summary By use of N-methyl-N-nitro-N-nitrosoguanidin (NG) respiration deficient (RD) mutants were induced. They could be selected by replica-plating on glycerol medium. RD mutants were also induced by UV irradiation, enriched by use of 2,3,5-triphenyltetrazolium chloride (TTC) and tested for their inability to grow on glycerol medium. The RD mutants were characterized enzymatically for their decrease or loss in cytochrome c oxidase activity and in succinate- cytochrome c reductase activity. These assays allowed the localization of the mutational blocks in complexes II, III and IV of the respiratory chain. Tetrad analysis and random spore analysis demonstrated that all mutants contained chromosomal defects.     

A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth

Different dilutions of chlorhexidine gluconate were tested by the Kelsey-Sykes procedure. The method was further modified on microtitration plates using 2,3,5-triphenyltetrazolium chloride (TTC) reduction as an indicator of bacterial growth. There was a good correlation with the results based on TTC reduction and the conventional method based on turbidity changes caused by bacterial growth. Furthermore, the modified method using TTC reduction is more rapid and can be read by the naked eye because of the red colour.     

A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth

Different dilutions of chlorhexidine gluconate were tested by the Kelsey-Sykes procedure. The method was further modified on microtitration plates using 2,3,5-triphenyltetrazolium chloride (TTC) reduction as an indicator of bacterial growth. There was a good correlation with the results based on TTC reduction and the conventional method based on turbidity changes caused by bacterial growth. Furthermore, the modified method using TTC reduction is more rapid and can be read by the naked eye because of the red colour.     

Secondary Metabolites of the Grapevine Pathogen <Emphasis Type="Italic">Eutypa lata</Emphasis> Inhibit Mitochondrial Respiration,Based on a Model Bioassay Using the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2-isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2-isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H₂O₂) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.     

NADH-dependent reduction of d-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase,d-proline reductase,and a third protein factor

The enzyme system from Clostridium sticklandii catalyzing the NADH-dependent reduction of d-proline was co-purified by chromatography on DEAE-cellulose at pH 8.2 and ammonium sulfate fractionation, and resolved into fractions containing three different protein components, NADH dehydrogenase, d-proline reductase and a third protein factor, by chromatography on DEAE-cellulose at pH 7.0. Upon recombination of the fractions containing the three different protein components, the NADH-dependent reduction of d-proline was successfully reconstituted. The NADH dehydrogenase fractions oxidized NADH in the presence of artificial electron acceptors, and were inhibited by p-hydroxymercuriphenylsulfonate (50% at 80 nM). They contained 3–4 different enzyme bands as revealed by polyacrylamide-gel electropherograms stained with the NADH-dependent reduction of 2,3,5-triphenyltetrazolium chloride. d-Proline reduction was also coupled to a leuco-methylene blue-generating system containing d-glucose and glucose-oxidase (EC 1.1.3.4). Circumstantial evidence indicated that, among the clostridial proteins, only d-proline reductase and the third protein factor were needed for this reaction.Non-standard abbreviations TTC 2,3,5-triphenyltetrazolium chloride     

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.     

Phenotypic characteristics of thermotolerantCampylobacter from human and animal sources

Five reference strains and 314 field strains ofCampylobacter growing at 42°C, but not at 25°C, were characterized by tests of hippurate hydrolysis and sensitivity to nalidixic acid, to metronidazole, and to 2,3,5-triphenyltetrazolium chloride (TTC). All strains but seven were TTC-resistant by the disc test used. Of 168 human isolates, 87% hydrolyzed hippurate; three of these strains were nalidixic acid-resistant. Also hippurate-positive were all 23 strains from ovine abortion, 79% of 43 avian strains, two of six bovine isolates, and one of two strains of equine origin. Seventy-two porcine strains were all hippurate-negative. Metronidazole sensitivity was found to be a variable property in hippurate-positive strains, although present in all but four hippurate-negative strains. A group of eight nalidixic acid-resistant, hippurate-negative isolates of porcine and bovine origin probably represent a new group ofCampylobacter.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements.A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species.By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (−) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography.From a pool of 2.0 × 10⁸ CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.     

2,3,5-triphenyltetrazolium chloride as a viability assay for immobilized plant cells

Grape (V. vinifera L. cv Gamay Fréaux) cells were entrapped in calcium alginate beads. The effect of bead diameter and gel concentration on the viability of the cells was checked. The reduction of 2,3,5-triphenyltetrazolium chloride as well as O₂ consumption were used as viability test, and their results compared. The existence of diffusional limitations for O₂ introduces an unaccuracy as high as 25% for the O₂ consumption method. The reduction assay is more simple, precise and reliable.     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO₄. However, the activity of dehydrogenase was inhibited by the addition of CdSO₄ to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO₄.High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Development of cyanide-resistant respiration in mitochondria from potato tubers treated with ethanol,acetaldehyde, and acetic acid

Treatment of intact potato (Solanum tuberosum L.) tubers with acetaldehyde, ethanol or acetic-acid vapors led to a respiratory upsurge which was further increased when the volatiles were applied in 100% O₂. Mitochondria from tubers held in 100% O₂ (O₂ control) displayed a substrate state, state 3, and state 4 in respiration, whereas in mitochondria from the volatile-treated tubers the respiratory rate of the different states was virtually indistinguishable. This respiratory pattern was companied by the development of a cyanide-resistant respiration since these mitochondria exhibited resistance to CN and sensitivity to CN+salicylhydroxamic acid. Acetaldehyde-treated potatoes showed a time-course development (up to 36 h) of cyanide resistance and concomitant sensitivity to salicylhydroxamic acid, indicating the onset of synthetic processes leading to the observed changes in mitochondrial respiration.Abbreviations V total respiration rate - V_cyt velocity of O₂ uptake attributable to cytochrome oxidase - V_alt velocity of O₂ uptake attributable to the alternate oxidase - RCR respiratory control ratio - SHAM salicylhydroxamic acid Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, N.J., USA     

Heat shock and the protection against metal toxicity in wheat leaves1

Abstract. Wheat (Triticum aestivum L. var. TAM-W101) leaf segments exhibited an acquired protection against metal toxicity following exposure of the seedlings to heat shock temperatures in the dark. The acquired protection of the leaf segments to cadmium was 400-fold greater than leaf segments from seedlings kept at 25°C and exhibited the greatest change in protection of the five metals tested. Increased protection against aluminium and iron toxicity was also detected in the leaf segments from heat shocked seedlings. The concentration of aluminium at which a 50% loss of 2,3,5-triphenyltetrazolium chloride reduction occurred was 5.5 mol m^?3 in control leaf segments and 20 mol m^?3 in the leaf segments from heat shocked seedlings. The 50% loss of 2,3,5-triphenyltetrazolium chloride reduction in the leaf segments from heal shocked seedlings was four-fold higher for iron. A small, yet reproducible change in the 2,3,5-triphenyltetrazolium chloride reduction was observed for copper and no change in reduction was observed for zinc treatments in the leaf segments from heat shocked seedlings. These data indicate that exposure of wheat seedlings to heat shock temperatures results in the acquisition of protection against metal toxicity to otherwise lethal concentrations of aluminium, cadmium, and iron.     

In Vitro Reduction of Tetrazolium Indicators by Sulfhydryl Compounds

There was considerable variation between the sulfhydryl induced in vitro reduction of the oxidation-reduction indicators (2,3,5-triphenyltetrazolium chloride, 2,3-diphenyl 5-methyl tetrazolium chloride, neotetrazolium chloride, neotetrazolium phosphate-2B, blue tetrazolium, tetrazolium violet, potassium tellurite, methylene blue, and resazurin). The neotetrazolium salts and potassium tellurite showed the greatest reducing activity. The reduction of the indicators by oxidized sulfhydryl compounds in the presence of potassium cyanide closely paralleled the reduction of the sarrte indicators by reduced sulfhydryl compounds. Iodoacetamide was the most effective sulfhydryl inhibitor as demonstrated by indicator reduction.     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

Agar Medium for the Selective Enumeration of Coagulase-positive Staphylococcus from the Rat Alimentary Tract

A selective agar medium (medium J(1)) is proposed for the quantitative enumeration of egg yolk-positive (EYP) and egg yolk-negative (EYN) Staphylococcus pyogenes from the digestive tract and feces of the rat. This medium, buffered at pH 5.0, is composed of acid casein hydrolysate and yeast extract with 7.5% sodium chloride, 1.6% sodium pyruvate, 0.0008% 2,3,5-triphenyltetrazolium chloride (TTC), and 6% egg yolk emulsion. Inoculation is by the pour-plate method and incubation is at 38 C in a water-jacketed incubator for 36 hr. Colonies of S. pyogenes reduce TTC; EYP strains are surrounded by a halo of opacity; and EYN strains may be surrounded by a red halo, but no opacity. Small, white colonies of S. epidermidis may develop, but Micrococcus, and all other groups of Staphylococcus recognized in the rat intestinal flora, are inhibited. Other bacterial genera, notably Bacillus, Corynebacterium, Proteus, and Streptococcus, are also inhibited.     

Quantitative viability of seaweed tissues assessed with 2,3,5-triphenyltetrazolium chloride

A spectrophotometric quantification method was optimized to evaluate its utility in seaweed tissue viability tests using the enzymatic reduction of colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a colored triphenylformazan (TPF). To allow accurate determination of TPF in the seaweed Porphyra thallus and conchocelis, 0.2 g of tissues are incubated with 4 mL of 0.8% TTC reagent in the dark at 20°C for 1 h under a mineral oil layer. The TPF formed in tissues was extracted for 15 min at 60°C with 2 mL of 0.2 N KOH in 25% ethanol. Then TPF is partitioned away by prompt addition of hexane and vortexing. By this procedure, we have observed nearly complete separation of TPF, and observed good spectrophotometric discrimination between TPF and other hexane-soluble pigments at 545 nm. This procedure has proved applicable to a wide range of seaweed taxa; 1 species of Chlorophyta, 4 species of Phaeophyta and 7 species of Rhodophyta tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship