Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

Ovipositional deterrents in Dacus oleae

The deterrent substances diverting D. oleae females from ovipositing on already attacked olives are contained, at least partly, in the juice which trickles from the oviposition wounds. Surprisingly, the water fraction of the olive juice had limited deterrent activity. The principal deterrent stimuli are present in the oil fraction. Acetophenone and benzaldehyde are likely to be involved.Some liposoluble volatile substances can be deterrent to the females as vapours. The deterrent power of olives containing D. oleae larvae is probably linked to these substances.Most deterrent hydro-soluble substances had two contiguous hydroxyls, characterized by a comparative acidity and located in the ortho position in diphenols and somewhat analogously in glycolic acid.Amongst orthodiphenols, Pyrocatechol-the simplest compound-was the most active substance.

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Résumé Les jus d'olive qui sourt des blessures dues à la ponte, contient des substances inhibitrices qui empêchent les attaques ultérieures de la mouche de l'olivier.Une telle activité était attribuée à des substances hydrosolubles et, en particuliet, au dihydroxyphenil ethanol.On a testé l'activité inhibitrice des fractions huileuse et acqueuse obtenues par centrifugation de la pulpe des olives, rendue homogène.De facon inattendue, la fraction huileuse s'estavérée posséder une activité inhibitrice environ 20 fois supérieure à celle de la fraction acqueuse.Les différences d'activités inhibitrices d'huile d'olives mûres ou non, ou d'huiles commerciales, permet de penser que l'activité inhibitrice principale est liée à des substances liposolubles présentes dans l'huile.En outre, on a constaté que la pulpe d'olives, broyée, présente une action inhibitrice en l'absence de contact direct avec les femelles. Des substances inhibitrices volatiles sont aussi présentes dans l'huile.Des inhibiteurs volatiles qui se libèrent des tissus lacérés par les larves, peuvent expliquer l'activité inhibitrice des olives attaquées durant la période de développement des larves. Les substances inhibitrices dont la femelle couvre la surface cireuse des fruits, sont, en effet, facilement éliminées par la pluie.Parmi les substances liposolubles dont on connaît la présence dans l'huile, l'aceto-phénone et le benzaldehyde, possèdent un pouvoir inhibiteur prononcé.Les substances hydrolsolubles jouent un rôle mineur dans l'ensemble des stimuli inhibiteurs. Le pouvoir inhibiteur relatif est lié à deux hydroxyles contigus à une fonction acide présente en position ortho dans les diphénols, ceci par analogie avec l'acide glycolique qui possède un hydroxyle en caractérisé par une certaine acidité.Le plus actif orthodiphénol s'est avéré être la pyrocatéchine. Aussi une part des inhibiteurs liposolubles sont des substances liées au biochimisme des phénols.

     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.     

Lead soaps formation and biodiversity in a XVIII Century wax seal coloured with minium

A multidisciplinary approach was carried out in order to study the biodeterioration and the associated microbiome of a XVIII Century wax seal coloured with minium. A small wax seal fragment was observed by scanning electron microscopy combined with energy dispersive spectroscopy in non-destructive mode. The same object was analysed by Raman and Fourier-transform infrared spectroscopy. The identification of the microbiota growing on the seal was performed with both a culture-dependent strategy, combined with hydrolytic assays, and high-throughput sequencing using the MinION platform. The whole bacterial 16S rRNA gene and the fungal markers ITS and 28S rRNA were targeted. It was observed that the carnauba wax coloured with lead tetroxide (minium) was covered by a biofilm consisting of a network of filaments and other structures of microbial origin. The culture-dependent and culture-independent investigations showed the presence of a complex microbiota composed mainly by fungal members, which demonstrated interesting properties related to lipids and lead processing. The formation of lead soaps and secondary biogenic minerals was also described.     

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.     

Purification and characterization of amine oxidase from pea seedlings

A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

The K(ATP)+ channel is involved in a low-amplitude permeability transition in plant mitochondria

Pea (Pisum sativum) stem mitochondria, energized by NADH, succinate or malate plus glutamate, underwent a spontaneous low-amplitude permeability transition (PT), which could be monitored by dissipation of the electrical potential (deltapsi) or swelling. The occurrence of the latter effects was dependent on O2 availability, because O2 shortage anticipated the manifestation of both deltapsi dissipation and swelling. Spontaneous deltapsi collapse was also monitored in sucrose-resuspended mitochondria and again O2 deprivation caused an anticipation of the phenomenon. However, in this case deltapsi dissipation was not accompanied by a parallel mitochondrial swelling. The latter effect was, indeed, evident only if mitochondria were resuspended in KCl (as osmoticum), or other cations with a molecular mass up to 100 Da (choline+). PT was also induced by protonophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or free fatty acids) or valinomycin (only in KCl). The FCCP-induced dissipation of deltapsi and swelling were inhibited by ATP and stimulated (anticipated) by cyclosporin A or O2 shortage. The FCCP-induced PT was accompanied by the release of pyridine nucleotides from the matrix and of cytochrome c from the intermembrane space of KCl-resuspended mitochondria. The spontaneous and FCCP-induced low-amplitude PT of plant mitochondria are interpreted as due to the activity of a recently identified K(ATP)+ channel whose open/closed state is dependent on polarization of the inner membrane and on the oxidoreductive state of some sulfhydryl groups.