Plant resistance inductors and active forms of oxygen. I. The influence of salicylic acid on hydrogen peroxide production in common cultures of wheat callus and bunt pathogen

The influence of salicylic acid (SA) on cell resistance to bunt pathogen in wheat calluses has been studied. Cell staining by diaminobenzidin substratum (DAB-cells) was conditioned by hydrogen peroxide (H2O2) generation with the involvement of oxalate oxidase (OO). In the control group, DAB-staining was typical only of rhizoid cells (up to 50%). The infection caused no significant increase in the number of such cells; however, in the zone of fungus penetration parenchyma-like DAB-stained cells were observed (up to 24%). Under the influence of SA, the number of DAB-stained cells did not change, but increased in the zone of fungus penetration (up to 36%). Besides, SA increased OO activity and accelerated proembyogenic complex formation in the calluses, which, unlike rhizoids, were not sensitive to the phytopathogen. The infection caused an increase of OO activity in the cytoplasm and in an intercellular fraction, and an inhibition of an extracellular fraction of the enzyme.     

Plant resistance inductors and active forms of oxygen. II. The influence of chitooligosaccharides on the production of hydrogen peroxide with the involvement of oxalate oxidase in common cultures of wheat calluses and bunt pathogen

We have studied the influence of various concentrations of chitooligosaccharides (CO) on hydrogen peroxide (H2O2) generation, with the involvement of oxalate oxidase (OO), in rhizoids and in zones of hard brand pathogen penetration, as well as on oxalate oxidase activity in wheat calluses. In the control group, diaminobenzidin (DAB)-staining was typical of 30% of peripheral rhizoid cells, which provided their resistance during infection. In the zone of fungus penetration, the appearance of DAB-stained parenchyma-like cells was observed. Simultaneously, the activity OO cytoplasmic fraction increased, whereas OO activity in its ion-bound fraction was suppressed. Low concentrations of medication induced rhizoid formation, increased the number of DAB-stained cells in the pathogen penetration zone, and induced OO activity in cytoplasmic and ionically cell wall-bound fractions. On the contrary, a high concentration of CO (100 mg/ml) suppressed rhizoid formation, Oo activity and the fungus growth. The discovered correlation between enzymatic activation under CO influence, the high level of protective response during infection, and the intensity of rhizoid formation may suggest the community of protective and morphogenetic mechanisms of reactions in plant cells in respect to hydrogen peroxide production.     

The influence of some elicitors on growth and morphogenesis of Hypericum perforatum L. callus cultures

We studied the effects of elicitors, such as mannan, gβ-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.     

The influence of some elicitors on growth and morphogenesis of Hypericum perforatum callus cultures

We studied the effects of elicitors, such as mannan, beta-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.     

Influence of salicylic and succinic acids on formation of active oxygen forms in wheat coleoptiles

The comparative study of influence of exogenous salicylic (SaA) and succinic (SuA) acids on the production of reactive oxygen species by isolated wheat coleoptiles has been provided. Under the action of both acids the increase of generation of superoxide anion-radical (O2(.-)) was observed. This increase was partially suppressed by treatment of coleoptiles with inhibitors of peroxidase (salicylhydroxamic acid) and NADP H-oxidase (imidazole and alpha-naphthol). The increase of hydrogen peroxide content, activity of peroxidase and superoxide dismutase (SOD) was registered under the influence of SaA and SuA; catalase activity did not change essentially. The treatment of coleoptiles with the indicated acids resulted in the increase of their resistance to abiotic stress (damaging heating, 43 +/- 0,1 degrees C, 10 min). The conclusion is made, that the increase of O2(.-) generation in wheat coleoptiles under the action of SaA and SuA is related, probably, to the increase of apoplast peroxidase and NADP.H-oxidase activity, and the rise of H2O2 content is related to the growth of SOD activity. These enzymatic systems are involved in the induction of plant cells protective reactions to the hyperthermia.     

The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage

The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells.     

The influence of elicitation on the subcellular localization and content of sanguinarine in callus cells ofPapaver somniferum L.

The fungal elicitor prepared fromBotrytis cinerea affected sanguinarine alkaloid formation and accumulation in callus cells ofPapaver somniferum L. Ultrastructural changes have been observed in association with the accumulation and secretion of sanguinarine in elicitor-treated cells. Alkaloid content in elicited cells was showed as a electrondense material (osmiophilic aggregations), which occurred on the tonoplast and in freely floating bodies in the vacuole. A 30-times increasing of sanguinarine content was observed in elicitor-treated cultures.     

The influence of phenobarbital on cytochromes and reactive oxygen species in erythropoietin producing HepG2 cells

Light absorption photometry of HepG2 cells treated with phenobarbital for enhancing the content of cytochrome P-450 and the synthesis of erythropoietin revealed an influence on all cytochromes detectable in the wavelength range between 400 and 620 nm. No correlation was found between specific changes of cytochrome P-450 absorption and increased EPO synthesis as proposed earlier by Fandrey et al. (Life Sci. (1990) 47, 127–134). In the present study, however, the increased erythropoietin synthesis could be related to a decreased intracellular hydroxyl radical level described as crucial for the oxygen regulated gene expression (Kietzmann et al., Biochem. J. (1998) 335, 425–432; Porwol et al., Eur. J. Biochem. (1998) 256, 16–23).     

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.     

The influence of hepatic venous oxygen saturation on the liver's synthetic response to metabolic stress.

Hepatic oxygen consumption (HVO2) and hepatic venous oxygen saturation (ShvO2) were assessed in the isolated perfused rat liver under conditions that mimic critical illness in an effort to assess their utility in predicting the functional status of the liver. Flow rates were adjusted over the physiologic range of oxygen transport as indicated by the hepatic venous O2 saturation range of 10%-75%. HVO2 was found to be transport (HDO2) dependent only when perfusate conditions contained an increased counterregulatory hormone (glucagon, epinephrine, dexamethasone) stimulus or a high lactate concentration. In the absence of a metabolic load, (substrate and hormone-free perfusate), HVO2 was transport independent even at an ShvO2 as low as 10%. Although transport dependency of HVO2 is frequently used to infer tissue ischemia, hepatic oxygen consumption was poorly correlated with synthetic function under all conditions. In contrast, hepatic albumin production was directly related to ShvO2 at all levels of HDO2 and under all perfusion conditions indicating that this metabolic process is particularly sensitive to reductions in oxygen availability, which is more reliably predicted by venous saturation measurements. However, glucose and urea synthesis were almost independent of ShvO2. These findings indicate that various hepatic processes are affected differentially by stress conditions and flow alterations that may exist during critical illness, and protein synthesis is particularly sensitive to oxygen deprivation. Additionally, hepatic venous oxygen saturation measurement, but not HVO2, serves as a useful surrogate marker for hepatic albumin production suggesting that regional venous oximetry may play an important role in the detection of hepatic functional impairment.     

The effect of specific chromosome and cytoplasm substitutions on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of four lines of hexaploid wheat (Triticum aestivum L. em. Thell), the euplasmic nuclear donor Chinese Spring, Chinese Spring in which both 4B chromosomes were substituted by those of the variety Cappelle-Desprez and two alloplasmic lines in which these nuclei were substituted into the cytoplasm of Aegilops ovata. The calli were found to differ in their initia growth rates and their ability to organise shoot primordia and regenerate shoots. The Cappelle 4B chromosomes had a very significant effect on all these characters. The potential for modelling genotypes for improved tissue culture characteristics is discussed.     

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.     

A time course assessment of changes in reactive oxygen species generation and antioxidant defense in hydroponically grown wheat in response to lead ions (Pb2+)

We examined the effect of Pb(2+) (8 and 40?mg?l(-1)) on reactive oxygen species generation and alterations in antioxidant enzymes in hydroponically grown wheat at 24, 72, and 120?h after exposure. Pb(2+) toxicity was more pronounced on root growth, and it correlated with the greater Pb accumulation in roots. Pb exposure (40?mg?l(-1)) enhanced superoxide anion, H(2)O(2), and MDA content in wheat roots by 1.9- to 2.2-folds, 56-255%, and 41-90%, respectively, over the control. Pb-induced loss of membrane integrity was confirmed by the enhanced electrolyte leakage and in vivo histochemical localization. Activities of scavenging enzymes, superoxide dismutases and catalases, enhanced in Pb-treated wheat roots by 1.4- to 5.7-folds over that in the control. In contrast, the activities of ascorbate and guaiacol peroxidases and glutathione reductases decreased significantly, suggesting their non-involvement in detoxification process. The study concludes that Pb(2+)-induced oxidative damage in wheat roots involve greater H(2)O(2) accumulation and the deactivation of the related scavenging enzymes.     

Genetic resistance to helminths. The influence of breed and haemoglobin type on the response of sheep to re-infection with Haemonchus contortus.

The influence of genetic factors on acquired resistance to Haemonchus contortus infections in sheep was investigated. Animals whose primary infections were terminated with an anthelmintic failed to develop any immunity against subsequent challenge as judged by worm numbers. Nevertheless, all were better able to retard the development and reduce the fecundity and haematophagic activities of their parasite populations than animals undergoing primary infections. High levels of resistance, as judged by all these parameters, were observed in most animals when the challenge larvae were superimposed on existing worm populations. The patterns of worm establishment and disease indicated that genetic factors operated in determining resistance, since fewer worms became established and less severe clinical and pathophysiological changes were observed in Scottish Blackface than in Finn Dorset sheep with the same haemoglobin type. Similar advantages were displayed by animals with haemoglobin AA and to a lesser extent those with haemoglobin AB over haemoglobin BB types. The importance of breed was further indicated by the occurrence of 'self-cure' in the majority of the Scottish Blackfaces but in only one Finn Dorset. There was no evidence that this reaction was associated with haemoglobin type.     

The influence of protonophore high concentration on the structure and functions of wheat root cells

Protonophore induced structural and functional changes in cells of excised roots of wheat seedlings have been investigated. The vector transfer of H+ inside the cells was accompanied by a decrease in energy supply of these cells (suppression of oxygen consumption and heat release), an output of K+ ions to the incubation medium, and by an increase in its pH value. The initial increase in heat release by roots (1 h) apparently reflects the process of dissipation of deltamicro H+ in plasma membrane. Within the first 5-10 min of exposure of 50 microM CCCP, changes in cell ultrastructure were observed that involved activation of Golgi apparatus, secretion of vesicle contents to the vacuole, and swelling of endoplasmic reticulum canals. Following a 2 h treatment with CCCP, structural and functional changes acquired a destructive character, and after 5-6 h of treatment with protonophore a complete desintegration of cell structure occurred demonstrating formations of myelin-like bodies, fragmentation of plasma membrane, and destruction of the nucleus. Thus, the protonophore induced proton excessive transport inside cells is fast and may cause an irreversible cell de-energization followed by serious disruption of ultrastructural organization of cells leading eventually to their death.     

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.