Early Responses of Tobacco Suspension Cells to Rhizobacterial Elicitors of Induced Systemic Resistance

International audienceColonization of roots by selected strains of fluorescent Pseudomonas spp. can trigger induced systemic resistance (ISR) against foliar pathogens in a plant species-specific manner. It has been suggested that early responses in cell suspension cultures in response to rhizobacterial elicitors, such as generation of active oxygen species (AOS) and extracellular medium alkalinization (MA), are linked to the development of ISR in whole plants. Perception of flagellin was demonstrated to elicit ISR in Arabidopsis, and bacterial lipopolysaccharides (LPS) have been shown to elicit several defense responses and to act as bacterial determinants of ISR in various plant species. In the present study, the LPS-containing cell walls, the pyoverdine siderophores, and the flagella of Pseudomonas putida WCS358, P. fluorescens WCS374, and P. fluorescens WCS417, which are all known to act as elicitors of ISR in selected plant species, were tested for their effects on the production of AOS, MA, elevation of cytoplasmic Ca2+ ([Ca2+]cyt), and defense-related gene expression in tobacco suspension cells. The LPS of all three strains, the siderophore of WCS374, and the flagella of WCS358 induced a single, transient, early burst of AOS, whereas the siderophores of WCS358 and WCS417 and the flagella of WCS374 and WCS417 did not. None of the compounds caused cell death. Once stimulated by the active compounds, the cells became refractory to further stimulation by any of the active elicitors, but not to the elicitor cryptogein from the oomycete Phytophthora cryptogea, indicating that signaling upon perception of the different rhizobacterial compounds rapidly converges into a common response pathway. Of all compounds tested, only the siderophores of WCS358 and WCS417 did not induce MA; the flagella of WCS374 and WCS417, although not active as elicitors of AOS, did induce MA. These results were corroborated by using preparations from relevant bacterial mutants. The active rhizobacterial elicitors led to a rapid increase in [Ca2+]cyt, peaking at 6 min, whereas the inactive siderophores of WCS358 and WCS417 elicited a single spike at 1 min. Elicitation of the cells by cell-wall LPS of WCS358 or the siderophore of WCS374 induced a weak, transient expression of several defense-related genes, including PAL and GST. The spectrum of early responses of the suspension cells was not matched by the expression of ISR in whole tobacco plants against Erwinia carotovora pv. carotovora. Of the live bacterial strains, only WCS358 elicited significant ISR, but application of the LPS or the siderophore of all three strains also elicited ISR. Notably, the absence of elicitation of AOS and MA in suspension-cultured cells but induction of ISR in whole plants by the siderophore of WCS358, which was lost upon treatment with the siderophore-minus mutant of WCS358, indicates that the early responses in suspension cells are not predictive of the ability to induce ISR in whole plants. Possible explanations for these discrepancies are discussed

Heavy metals and acid volatile sulphide in lake Ketelmeer: vertical and horizontal distribution

In het Ketelmeer zijn op vier locaties, die verschillen in sedimentatiesnelheid, sedimentkernen gestoken. Hieruit zijn 4 profielen bemonsterd over een diepte varierend van 7 tot maximaal 15 cm. Bovendien zijn van 2 kernen per locatie mengmonsters gemaakt van de bovenste 7 tot 15 cm van het sediment. De profielen en mengmonsters zijn gekarakteriseerd met betrekking tot gehalten aan Acid Volatile Sulphide (AVS), Simultaan Geextraheerd Metaal (SEM), totaal metaal, droge stof, totaal koolstof, totaal zwavel, carbonaat en de deeltjesgrootteverdeling. Het effect van beluchting van het sediment op de distributie van een aantal zware metalen is bestudeerd aan de hand van een sequentiele extractie gebaseerd op de methode van Tessier. Het AVS gehalte blijkt voornamelijk to te nemen met de diepte, het SEM gehalte vertoont geen eenduidig patroon. In alle kernen neemt de SEM/AVS ratio in de bovenste 2-4 cm snel af, maar blijft overal gelijk aan of groter dan 1. Het droge stof gehalte neemt toe met de diepte. Het carbonaatgehalte is vrij constant in de diepte en er is weinig verschil in gehalten tussen de vier locaties (11-19 % carbonaat). Ook de geconstateerde deeltjesgrootteverdeling met de diepte is in overeenstemming met de andere analytische data en de verwachte sedimentatiesnelheden. De variatie van gemeten parameters in de mengmonsters op de verschillende locaties is gering. waaruit geconcludeerd wordt dat de samenstelling van het sediment horizontaal in de ruimte gezien homogeen is. Uit de sequentiele extractie blijkt dat voor alle gemeten zware metalen het grootste deel teruggevonden wordt in de fractie gebonden aan organisch materiaal en sulfides. Dit beeld blijft gelijk na beluchten, waarbij er voor Zn en Cd wel een significante verschuiving optreedt in de verdeling over de andere fracties, te weten naar water oplosbaar, uitwisselbaar, gebonden aan carbonaten en het residu.Sediment cores were taken in Ketelmeer at 4 locations, differing in sedimentation rate. Slicing the cores under anaerobic conditions (0.5 - 1 cm slices), 4 profiles were subsampled over a depth varying from 7 to a minimum of 15 cm of the sediment. For each location, two cores were used to prepare mixed samples of the top 7 to 15 cm of the sediment. The profiles and mixed samples were characterised with respect to Acid Volatile Sulphide (AVS), Simultaneously Extracted Metals (SEM), total metal dry weight, total carbon, total sulphur, and carbonate and particle size distribution. Using a sequential extraction method based on the method of Tessier, the effect of aeration of the sediment on the distribution of a number of heavy metals (Cu, Pb, Ni, Zn and Cd) was studied. AVS concentrations mainly increase with depth, for SEM there was more variation in the four profiles. For all profiles in the top 2-4 cm, the SEM/AVS ratio decreased sharply, but is higher than or equal to 1 at all depths. Dry weight content increases with depth. Carbonate content is reasonably constant in depth without much variation between the four locations (11-19% carbonate). The particle size distribution is also in accordance with the other analytical data and expected sedimentation rates. The mixed samples of the different locations contained little variation in the measured parameters. Horizontally, the sediment is rather homogeneous. The results of the sequential extraction show the largest part of the metals to be found in the fraction bound to organic material and sulphides. After aeration, this situation is similar, although for Zn and Cd a significant shift i.e. towards water soluble, exchangeable, bound to carbonated and residual, occurred in the distribution over the other fractions.DGM/SV

Acid Volatile Sulfide (AVS) as instrument in the risk assessment of sediments

Een analyse van de huidige en de in de toekomst te verwachten kwaliteit van Nederlandse sedimenten laat zien dat vooral Zn, Cu and Ni de stoffen zullen zijn die in belangrijke mate de verontreinigingsgraad zullen gaan bepalen. De mobiliteit van deze elementen wordt sterk gereduceerd door de aanwezigheid van een overmaat aan sulfide (aangeduid als AVS). Daarnaast wijzen ecotoxicologische gegevens uit dat ook de toxiciteit van sedimenten die een overmaat aan sulfide bevatten, aanzienlijk lager kan zijn dan metaaltoxiciteit in sulfide-arme sedimenten. Dit is de basis van het zogenaamde AVS-concept. In dit rapport worden de mogelijkheden nagegaan om, in aanvulling op de huidige risicobeoordeling van sedimenten op basis van totaalgehaltes, rekening te houden met de aanwezigheid van metaalbindend sulfide. Dit in relatie tot het geochemische gedrag van zware metalen in sulfiderijke sedimenten en ecotoxicologische data. Geconcludeerd wordt dat het AVS-concept niet goed geschikt is voor het inschatten van daadwerkelijke ecotoxicologische risico's in het veld. Daarentegen draagt de aanwezigheid van AVS w5l bij aan het verminderen van de verspreiding van metalen uit sedimenten, zodat het AVS-concept een effectief instrument kan zijn bij het prioriteren van te saneren sedimenten.Risk assessment of sediments is primarily based on the determination of total content of polluted components. Analysis of sediment composition in the Netherlands, both at present and expected in the future, shows that heavy metals, especially Zn, Cu and Ni, appear to be the main compounds determining the pollution status of contaminated sediments. The mobility of these elements is strongly reduced in the presence of an excess of sulfide (AVS). The geochemical behaviour of heavy metals in sulfide-rich sediments and the relation with ecotoxicological data, provide the basis for the AVS concept presented in this report. Although there are some further limitations attached to its exploration, the AVS concept would seem, at present, to be an effective instrument for determining the cleaning order of polluted sediments, especially in view of the fairly simple procedures to be implemented.DGM-SA

Simultaneous release of sulphide with Fe, Mn, Ni and Zn in marine harbour sediment measured using a combined metal/sulphide DGT probe.

The technique of DGT (Diffusive Gradients in Thin Films) was further developed to allow simultaneous measurement of sulfide and trace metals at the same location in sediment. The new combined DGT probe consisted of a layer of gel impregnated with AgI, overlain by (1) a layer of gel containing Chelex, (2) a layer of gel and (3) a filter membrane. Diffusion of sulfide was controlled by layers (1) to (3), while diffusion of metals was controlled by layers (2) and (3). The Chelex gel trapped metals that were measured after elution with acid. The AgI gel trapped sulfide through the formation of Ag2S. This was then measured densitometrically as the colour changed from pale yellow to grey. Experiments demonstrated that concentrations of metal or sulfide measured by the combined device were no different to the concentrations measured by more conventional devices. The presence of Chelex in the gel did not impede the diffusion of sulfide. Deployment of a combined probe in marine sediment revealed simultaneous remobilisation of metals and sulfide at the same location. Solubility calculations indicated that some precipitation of amorphous FeS was probably occurring at the maxima in sulfide concentrations. There was general undersaturation with respect to NiS, but ZnS was supersaturated at all locations. There appeared to be localised active zones of organic matter decomposition, where reduction of manganese oxides, iron oxides and sulfate occurred simultaneously. Mass balance calculations indicated that Ni could not be supplied by release from decomposing organic matter. Manganese oxides were the most likely source, but supply from reductive dissolution of iron oxides could not be entirely discounted. Supply from either Fe or Mn oxides could account for the Zn maxima. Application of the newly developed combined probe provides new information that helps understanding of the complex nature of trace metal and sulfur chemistry in sediments

X-ray absorption spectroscopy of soybean lipoxygenase-1 : Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 23 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X-ray absorption fine structure shows clear differences in active-site structure as a result of this conversion. Curve-fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active-site structure proposed earlier (6 ± 1 N/O ligands at 0.2050.209 nm with a maximum variance of 0.009nm, including 4 ± 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al-Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 7076], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 ± 1 N/O ligands at 0.2000.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data

EXAFS of soybean lipoxygenase-1: influence of lipid hydroperoxide activation and lyophilization on the structure of the soybean lipoxygenase non-heme iron active site

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 23 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X-ray absorption fine structure shows clear differences in active-site structure as a result of this conversion. Curve-fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active-site structure proposed earlier (6 ± 1 N/O ligands at 0.2050.209 nm with a maximum variance of 0.009nm, including 4 ± 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al-Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 7076], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 ± 1 N/O ligands at 0.2000.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data