Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response. Recently, we have demonstrated that treatment of tobacco ( Nicotiana tabacum ) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li⁺, Na⁺, K⁺), alkali earth metals (Mg²⁺, Ca²⁺) or lanthanides (La³⁺, Gd³⁺). In this study, we tested the effect of extracellular supplementation of Zn²⁺ and Mn²⁺ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent. Extracellular supplementation of Zn²⁺ and Mn²⁺ inhibited the generation of superoxide in response to addition of salts. Although both Zn²⁺ and Mn²⁺ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn²⁺ simply inhibited total production of superoxide while Zn²⁺ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn²⁺ and Zn²⁺ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed.     

Salinity adaptation in the snakehead, Ophiocephalus maculatus Lacépède: changes in oxygen consumption, branchial Na+-K+-ATPase and body composition

Snakeheads were adapted to fresh water (1 mOsm kg⁻¹), 25% sea water (230 mOsm kg⁻¹), 33% sea water (320 mOsm kg⁻¹) and 40% sea water (380 mOsm kg⁻¹) for 20 days. Exposure to salt water resulted in tissue dehydration, elevations of plasma osmolality, Na⁺, Mg²⁺, Cl⁻and protein concentrations and stimulation of branchial Na⁺-K⁺-ATPase activity. These changes were accompanied by concomitant decline of the hepatosomatic index and liver glycogen concentration. The routine rate of oxygen consumption was increased in snake-heads adapted to 33% sea water. These data were taken to indicate a stressful effect of salinity to the snakehead despite documentation of its ability to penetrate into brackish waters.     

Relationship between polar basipetal auxin transport and acropetal Ca2+ transport into tomato fruits

The dependence of acropetal Ca²⁺ transport on polar basipetal indoleacetic acid (IAA) transport was investigated in excised tomato fruits ( Lycopersicon esculentum L. Mill.) using an in vitro fruit system. Auxin transport inhibitors like triiodobenzoic acid (TIBA), chlorofluorenolmethyl ester (CME) and naphthylphthalamic acid (NPA) were used in order to investigate the effect of restricted polar basipetal auxin transport on the acropetal transport of ⁴⁵Ca²⁺, ⁸⁶Rb⁺ and ⁹⁸Sr²⁺ into the same fruits. TIBA and CME inhibited basipetal transport of IAA. particularly in 10- to 12-day-old tomato fruits, and simultaneously restricted the acropetal transport of ⁴⁵Ca²⁺. The auxin transport inhibitors failed to significantly reduce the upward transport of ⁸⁶Rb⁺ and the transport of ⁹⁶Sr²⁺ was less inhibited than that of ⁴⁵Ca²⁺. TIBA and CME did not significantly affect the acropetal transport of labelled water into the fruit, nor the cation-exchange capacity or K⁺ and Mg²⁺ concentrations in the tomato fruit. These results support the view that a part of the Ca²⁺-specific acropetal transport into tomato fruits is associated with the polar basipetal IAA transport. This Ca²⁺ transport is independent of the transpiration stream into the fruit and the cation exchange capacity of the fruit tissue.     

Osmotic stress- and indole-3-butyric acid-induced NO generation are partially distinct processes in root growth and development in Pisum sativum

In this work, the effects of osmotic stress and exogenous auxin (indole-3-butyric acid, IBA) on root morphology and nitric oxide (NO) generation in roots were compared in pea plants. Five-day-old plants were treated with 0, 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹  M IBA or with PEG 6000 at concentrations that determined 0, 50, 100, 200 or 400 mOsm in the medium, during 5 days. NO generation was examined by in situ and in vivo fluorescence method. Increasing concentrations of PEG as well as IBA resulted in shortening of primary root (PR), enhancement of lateral root (LR) number and significant increase of NO generation. Time-dependent investigations revealed that in the case of IBA treatments, the LR number increased in parallel with an intensified NO generation, while elongation of PR was not followed by changes in NO levels. Under osmotic stress, the time curve of NO development was distinct compared with that of IBA-treated roots, because significantly, the appearance of lateral initials was preceded by a transient burst of NO. This early phase of NO generation under osmotic stress was clearly distinguishable from that which accompanied LR initiation. It is concluded that osmotic stress and the presence of exogenous auxin resulted in partly similar root architecture but different time courses of NO synthesis. We suppose that the early phase of NO generation may fulfill a role in the osmotic stress-induced signalization process leading to the modification of root morphology.     

Novel role of Wag31 in protection of mycobacteria under oxidative stress

Wag31 of Mycobacterium tuberculosis belongs to the DivIVA family of proteins known to regulate cell morphology in Gram-positive bacteria. Here we demonstrate an unrecognized, novel role of Wag31 in oxidatively stressed mycobacteria. We report the cleavage of penicillin-binding protein 3 (PBP3) by the intramembrane metalloprotease Rv2869c (MSMEG_2579) in oxidatively stressed cells. Amino acids ¹⁰²A and ¹⁰³A of PBP3 are required for Rv2869c-mediated cleavage. Wag31_MTB, by virtue of its interaction with PBP3 through amino acid residues ⁴⁶NSD⁴⁸, protects it from oxidative stress-induced cleavage. PBP3 undergoes cleavage in Mycobacterium smegmatis (strain PM2) harbouring wag31 (Δ⁴⁶NSD⁴⁸) instead of the wild type, with concomitant reduction in ability to withstand oxidative stress. Overexpression of Wag31(Δ⁴⁶NSD⁴⁸) attenuates the survival of M. tuberculosis in macrophages with concomitant cleavage of PBP3, and renders the organism more susceptible towards hydrogen peroxide as well as drugs which generate reactive oxygen species, namely isoniazid and ofloxacin. We propose that targeting Wag31 could enhance the activity of mycobactericidal drugs which are known to generate reactive oxygen species.     

Herbicidal activity of 4-chloroindoleacetic acid and other auxins on pea, barley and mustard

The natural auxins, 4-chloroindoleacetic acid and its methyl ester have strong herbicidal effects on pea, Pisum sativum , a plant in which they occur naturally. The standard herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) is only 5 times more effective than 4-chloroindoleacetic acid. The I₅₀, the dose inhibiting yield by 50%, for 4-chloroindoleacetic acid and its methyl ester is 0.5 kg ha⁻¹ or 15 mg kg⁻¹ fresh weight, close to the concentration of 4-chloroindoleacetic acid methyl ester in maturing pea seeds. Naphthaleneacetic acid and indoleacetic acid are also inhibitory, but at much higher concentrations. In its inhibiting effect on white mustard, Sinapis alba , 4-chloroindoleacetic acid approximates that of 2,4-D, whereas in barley, Hordeum vulgare , it is a stronger herbicide than 2,4-D. All auxins tested killed white mustard at low doses, but none killed barley. Both 4-chloroindoleacetic acid and 2,4-D killed pea. The chloroindole auxins of pea may be the hypothetic death hormones or senescence factors that are secreted from the developing seeds into the parent plant which is strongly inhibited or killed and from which the nutrients are mobilized and translocated to the seeds. The action mechanism of auxin type herbicides may be to simulate the action of endogenous herbicides.     

Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

Patch-clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER-located auxin binding protein from maize (Zm-ERabp1), was investigated using the patch-clamp technique (whole-cell configuration). Following a lag-phase of 30–40 seconds, indole-3-acetic acid and 1-naphthylacetic acid induced an outwardly directed current of positive charge in a concentration-dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg²⁺-ATP in the patch-pipette and was abolished after addition of erythrosin B, an inhibitor of H⁺-ATPase, to the protoplasts indicating that the plasma membrane H⁺-ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm-ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm-ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm-ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H⁺-ATPase is activated, and (ii) that the activation of the H⁺-ATPase by such proteins is initiated from outside the plasma membrane.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Chilling-induced leaf abscission of Ixora coccinea plants. III. Enhancement by high light via increased oxidative processes

The role of increased oxidation induced by successive stresses of chilling and high light in the induction of leaf abscission was studied in Ixora coccinea plants in relation to auxin metabolism and oxidative processes. Exposure of plants following dark chilling (7°C for 3 days) to high light (500–700 μmol m⁻² s⁻¹ photosynthetically active radiation) for 5 h at 20–25°C enhanced chilling-induced leaf abscission. This abscission was inhibited by pretreatment with the antioxidant butylated hydroxyanisole, α -naphthaleneacetic acid or the ethylene action inhibitor, 1-methylcyclopropene. The oxidative processes initiated during the low light period following the dark chilling period, such as indoleacetic acid (IAA) decarboxylation and lipid peroxidation, were further enhanced by subsequent exposure to high light. Photoinhibition, expressed by the reduction of the chlorophyll fluorescence parameter Fv/Fm, was evident following exposure to high light, irrespective of the temperature of the pretreatment, but this reduction persisted only in chilled plants. This suggests that oxidative processes generated during and after the chilling period might have inhibited the recovery from photoinhibition. The chilling stress under darkness induced a 60% reduction in superoxide dismutase (SOD) activity and significant increases (130–600%) in the activities of several other antioxidative enzymes. These data suggest that the chilling-induced reduction in SOD activity may well be responsible for the increase in the oxidative stress induced by the subsequent light treatment, as expressed by the increased enzymatic activities. Taken together, this study provides further support for the involvement of oxidative processes in the events occurring in tissues exposed to sequential chilling and light stresses, leading to reduction in free IAA content in the abscission zone and to leaf abscission.     

Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Female control of the embryonic environment in a terrestrial amphipod, Mysticotalitrus cryptus (Crustacea)

1. In amphipod crustaceans the ventral chamber plays an integral role in a number of physiological processes and in the female forms the marsupium in which eggs are brooded. The ventral chamber can be viewed as a pre-adaptation to the colonization of land by the family Talitridae. The hypothesis that the female of the terrestrial species, Mysticotalitrus cryptus , can control the osmotic concentration of the marsupial fluid ([MF]) bathing the eggs, thereby buffering the brood from potential physiological stresses presented by the terrestrial environment, is examined.
2. [MF] was maintained significantly higher than the concentration of the external medium ([Medium]) on both dechlorinated tap-water and 400 mOsm kg^–1 media. In each case, [MF] was intermediate to [Medium] and the concentration of the female haemolymph ([Haem]): when [Medium] = 40 mOsm kg^–1, [MF] = 277 mOsm kg^–1 and [Haem] = 590 mOsm kg^–1, respectively, and when [Medium] = 413 mOsm kg^–1, [MF] = 516 mOsm kg^–1 and [Haem] = 722 mOsm kg^–1, respectively.
3. Evidence is provided that females produce urine that is isosmotic with the haemolymph and that the urine is directed, by capillarity, into the marsupium via cuticular channels. It is suggested that this urine plays a role in controlling [MF] in combination with other behavioural mechanisms.
4. Some preliminary observations are presented on the ontogeny of embryonic osmoregulation in M. cryptus which suggest that osmoregulatory ability improves with developmental stage. There is also limited evidence for the ability of the late embryonic stages to hypo-osmoregulate on concentrated media, even though adults lack this capacity.
5. The results are discussed in relation to the colonization of the terrestrial environment by the Talitridae.     

Phosphate Induces Rapid H2O2 Generation in Soybean Suspension Cells

Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H₂O₂ generation induced by the addition of phosphate in soybean ( Glycine max L.) cell suspension cultures. This H₂O₂ generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg -induced H₂O₂ generation, phosphate-induced H₂O₂ generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H₂O₂ accumulation, which could be involved in sensing of phosphate availability in the environment.     

Role of ethylene and auxin in regenerative differentiation and orientation of tracheids in Pinus pinea seedlings

A low auxin concentration (0.1% naphthalene acetic acid) induced tracheids with longitudinal polarity parallel to the hypocotyl axis in young Pinus pinea seedlings. Application of 0.1% ethrel laterally and 0.1% naphthalene acetic acid apically disturbed axial tracheid polarity and promoted the differentiation of tracheids with a lateral orientation. Ethrel by itself, with no auxin background, did not affect tracheid differentiation. Apical application of 1% gibberellic acid with the low auxin, reversed the polarity disorder induced by ethrel. Disturbance of axial tracheid polarity was observed under a high auxin concentration (0.5% naphthalene acetic acid) which was similar to the combined effect of ethrel and auxin. The high auxin concentration increased tracheid number significantly. This effect was curtailed following treatment with inhibitors of ethylene formation (Co²⁺; 1-aminoethoxy- vinylglycine) and action (Ag²⁺). The role of ethylene in controlling the differentiation of radial tracheids, which characterize the vascular rays of pines, is discussed.     

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (P_i) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca²⁺]. Cultured fetal rat cortical neurons constitutively import P_i, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular P_i is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular P_i dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. P_i similarly increased neuronal survival after exposure to kainic acid or H₂O₂. P_i-exposed neurons had higher basal intracellular [P_i], [ATP], and [GSH], and slightly lower cytosolic free [Ca²⁺], compared with P_i-deprived neurons. P_i-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H₂O₂, compared with P_i-deprived neurons. These findings demonstrate that changes in extracellular and intracellular P_i can affect neuronal survival after excitotoxic or oxidative insults.     

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA²⁻) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o- phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA²⁻ in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ' ex vivo' showed TA²⁻ to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe²⁺ + H₂O₂) indicate a mechanism whereby TA²⁻ forms a primary complex with Fe²⁺ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.     

Effects of nitrogen source, calcium concentration and osmotic stress on protoplasts and protoplast-derived cell cultures of Pinus Pinaster cotyledons

The experiments described emphasize the effects of several factors crucial to the maintenance of cell divisions leading to increased cell numbers in suspension and colony formation from cotyledon protoplasts of Pinus Pinaster Ait. Osmotic potential of the incubation and culture media are critical. Reducing the osmolality from 680 mOsm kg H₂O⁻¹ during protoplast isolation to 610 mOsm kg H₂O⁻¹ during washing and culture was essential to achieve a high frequency of cell division. Survival of the cells beyond 3 weeks of culture occurs only if the calcium concentration is decreased from 5.6 m M to 1.5 m M . Glutamine as sole source of nitrogen shortens the lag phase of response of the protoplasts and increases their plating efficiency. After 6 weeks of culture, a combination of low osmolality (225 mOsm kg H₂O⁻¹) and high level of glutamine (40 m M ) is a prerequisite for obtaining actively growing cell suspensions.