Biomedical subjects

Axel Mithöfer

Publications and source records attributed to Axel Mithöfer.

13 recordsLinked to original sources

PubMed2006

Purification and characterization of proline/hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola.

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components, which are also involved in response to pathogen attack. In pearl millet, deposition and cross-linking of HRGPs in plant cell walls was shown to contribute to the formation of resistance barriers against the phytopathogenic oomycete Sclerospora graminicola. In the present study, the purification and characterization of HRGPs that accumulated in coleoptiles of pearl millet seedlings in response to S. graminicola inoculation has been carried out. Periodic acid Schiff's staining revealed that the purified protein was a glycoprotein. The protein to carbohydrate ratio was determined to be 95.5%:4.5% (w/w). Proline amounted for 20 mol% of the total amino acids as indicated by amino acid composition analysis. The isolated protein had a pI of 9.8 and was shown to be composed of subunits of 27, 17, and 14 kDa. Cross reactivity with the monoclonal antibody MAC 265 and the presence of the signature amino acid sequence, PVYK, strongly suggested to classify the purified glycoprotein as a member of the P/HRGPs class. In the presence of horseradish peroxidase and H2O2 the purified glycoprotein served as a substrate for oxidative cross-linking processes.

Chromatography, Ion Exchange↗

PubMed2006

Activation of members of a MAPK module in beta-glucan elicitor-mediated non-host resistance of soybean.

Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a beta-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identification of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of specific antisera enabled the identification of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the beta-glucan signal transduction pathway. An upstream GmMKK1 was identified based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating beta-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants.

Cells, Cultured↗

PubMed2006

Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide.

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Aequorin↗

PubMed2005

Flavones and flavone synthases.

Within the secondary metabolite class of flavonoids which consist of more than 9000 known structures, flavones define one of the largest subgroups. Their natural distribution is demonstrated for almost all plant tissues. Various flavone aglyca and their O- or C-glycosides have been described in the literature. The diverse functions of flavones in plants as well as their various roles in the interaction with other organisms offer many potential applications, not only in plant breeding but also in ecology, agriculture and human nutrition and pharmacology. In this context, the antioxidative activity of flavones, their use in cancer prevention and treatment as well as the prevention of coronary heart disease should be emphasized. The therapeutic potential of flavones makes these compounds valuable targets for drug design, including recombinant DNA approaches. The biosynthesis of flavones in plants was found to be catalyzed by two completely different flavone synthase proteins (FNS), a unique feature within the flavonoids. The first, FNS I, a soluble dioxygenase, was only described for members of the Apiaceae family so far. The second, FNS II, a membrane bound cytochrome P450 enzyme, has been found in all other flavone accumulating tissues. This phenomenon is particularly of interest from the evolutionary point of view concerning the flavone biosynthesis and functions in plants. Recently, FNS I and FNS II genes have been cloned from a number of plant species. This now enables detailed biochemical and molecular characterizations and also the development of direct metabolic engineering strategies for modifications of flavone synthesis in plants to improve their nutritional and/or biopharmaceutical value.

Antineoplastic Agents↗

PubMed2005

Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission.

Herbivore feeding elicits defense responses in infested plants, including the emission of volatile organic compounds that can serve as indirect defense signals. Until now, the contribution of plant tissue wounding during the feeding process in the elicitation of defense responses has not been clear. For example, in lima bean (Phaseolus lunatus), the composition of the volatiles induced by both the insect caterpillar Spodoptera littoralis and the snail Cepaea hortensis is very similar. Thus, a mechanical caterpillar, MecWorm, has been designed and used in this study, which very closely resembles the herbivore-caused tissue damage in terms of similar physical appearance and long-lasting wounding period on defined leaf areas. This mode of treatment was sufficient to induce the emission of a volatile organic compound blend qualitatively similar to that as known from real herbivore feeding, although there were significant quantitative differences for a number of compounds. Moreover, both the duration and the area that has been mechanically damaged contribute to the induction of the whole volatile response. Based on those two parameters, time and area, which can replace each other to some extent, a damage level can be defined. That damage level exhibits a close linear relationship with the accumulation of fatty acid-derived volatiles and monoterpenes, while other terpenoid volatiles and methyl salicylate respond in a nonlinear manner. The results strongly suggest that the impact of mechanical wounding on the induction of defense responses during herbivore feeding was until now underestimated. Controlled and reproducible mechanical damage that strongly resembles the insect's feeding process represents a valuable tool for analyzing the role of the various signals involved in the induction of plant defense reactions against herbivory.

Animals↗

PubMed2005

Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots.

Inducible and specific ion fluxes on plasma membranes represent very early events during elicitation of plant cells. The hierarchy of such ion fluxes involved is still unknown. The effect of Phytophthora sojae-derived beta-glucan elicitors on the plasma membrane potential as well as on surface K+, Ca2+, and H+ fluxes has been investigated on soybean roots using ion-selective microelectrodes. Beta-Glucans with different degrees of polymerization transiently depolarized the plasma membrane. The elicitor concentration necessary for half-maximal depolarization closely resembled the corresponding binding affinities of soybean root membranes toward the respective beta-glucans. Upon repeated elicitor treatment, the root cells responded partially refractory, suggesting a complex responsiveness of the system. Within the root hair space, characteristic decreasing K(+)- and Ca(2+)-free concentrations were induced by the elicitors, probably causing depolarization through the influx of positive charges. Whereas K+ fluxes were inverted after passing the K+ equilibrium (Nernst-) potential, Ca2+ influx continued. No anion fluxes sufficient to account for charge compensation were observed under the same experimental conditions. K+ and Ca2+ fluxes as well as depolarization were inhibited by 100 microM or less of the Ca2+ antagonist La3+. Contrasting other systems, in soybean the main cause for elicitor-induced plasma membrane depolarization is the activation of cation instead of anion fluxes.

Cations↗

PubMed2005

Direct and indirect defences induced by piercing-sucking and chewing herbivores in Medicago truncatula.

Direct and indirect defences against feeding induced by chewing (Spodoptera littoralis) and piercing-sucking (Tetranychus urticae) herbivores, as well as components of signal transduction, were investigated in the model legume Medicago truncatula. Emitted volatiles, representing a mechanism of indirect defence, were measured and identified by gas chromatography/mass spectrometry (GC-MS). As elements of direct defence, the accumulation of phenolic compounds and of reactive oxygen species (ROS) was assessed using microscopic techniques. Jasmonic acid (JA) and salicylic acid (SA) concentrations were assessed as putative components of signal transduction. Volatile profiles revealed a sizeable number of different substances emitted, particularly sesquiterpenoids. The qualitative composition clearly differed depending on the type of herbivory. The same held true for JA and SA concentrations. Also, deposition of phenolic compounds and the production of ROS around the wounding sites could be detected. Conspicuous differences were found in indirect defence and signalling for different types of herbivory. In contrast, no divergence in direct defences was observed; furthermore, the traits investigated exhibited striking similarities to reactions known to occur upon pathogen attack.

Animals↗

PubMed2004

Biotic and heavy metal stress response in plants: evidence for common signals.

In higher plants, biotic stress (e.g., herbivore or pathogen attack) as well as abiotic stress (in particular heavy metals) often induce the synthesis and accumulation of the same defense-related secondary metabolites. This well-known finding still awaits an explanation regarding the common features of both stress types. In this study, a mechanism is proposed that links reactive oxygen species (ROS) generation with lipid oxidation processes, ultimately resulting in the formation of similar, highly active signalling compounds. The generation of ROS is a common event in both heavy metal treatment and biotic stress although it can depend on quite different, enzymatic and non-enzymatic reactions. Regardless, ROS are involved in the oxidation of unsaturated fatty acids which initiate the formation of oxylipins, a highly variable class of lipid-derived compounds in plants. Oxylipins represent new endogenous signals involved in biotic- and abiotic-induced stress responses.

Animals↗

PubMed2004

Coronalon: a powerful tool in plant stress physiology.

Coronalon, a synthetic 6-ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12-oxo-phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense-related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress-related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.

Cells, Cultured↗

PubMed2004

Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components.

Membrane potentials (V(m)) and intracellular calcium variations were studied in Lima bean (Phaseolus lunatus) leaves when the Mediterranean climbing cutworm (Spodoptera littoralis) was attacking the plants. In addition to the effect of the feeding insect the impact of several N-acyl Glns (volicitin, N-palmitoyl-Gln, N-linolenoyl-Gln) from the larval oral secretion was studied. The results showed that the early events upon herbivore attack were: a) a strong V(m) depolarization at the bite zone and an isotropic wave of V(m) depolarization spreading throughout the entire attacked leaf; b) a V(m) depolarization observed for the regurgitant but not with volicitin [N-(17-hydroxy-linolenoyl)-Gln] alone; c) an enhanced influx of Ca(2+) at the very edge of the bite, which is halved, if the Ca(2+) channel blocker Verapamil is used. Furthermore, the dose-dependence effects of N-acyl Gln conjugates-triggered influx of Ca(2+) studied in transgenic aequorin-expressing soybean (Glycine max) cells, showed: a) a concentration-dependent influx of Ca(2+); b) a configuration-independent effect concerning the stereochemistry of the amino acid moiety; c) a slightly reduced influx of Ca(2+) after modification of the fatty acid backbone by functionalization with oxygen and; d) a comparable effect with the detergent SDS. Finally, the herbivore wounding causes a response in the plant cells that cannot be mimicked by mechanical wounding. The involvement of Ca(2+) in signaling after herbivore wounding is discussed.

Animals↗

PubMed2003

The role of octadecanoids and functional mimics in soybean defense responses.

Oxylipins of the jasmonate pathway and synthetic functional analogs have been analyzed for their elicitor-like activities in an assay based on the induced accumulation of glyceollins, the phytoalexins of soybean (Glycine max L.), in cell suspension cultures of this plant. Jasmonic acid (JA) and its methyl ester showed weak phytoalexin-inducing activity when compared to an early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), as well as to the bacterial phytotoxin coronatine and certain 6-substituted indanoyl-L-isoleucine methyl esters, which all were highly active. Interestingly, different octadecanoids and indanoyl conjugates induced the accumulation of transcripts of various defense-related genes to different degrees, indicating distinct induction competencies. Therefore, these signaling compounds and mimics were further analyzed for their effects on signal transduction elements, such as the transient enhancement of the cytosolic Ca2+ concentration and MAP kinase activation, which are known to be initiated by a soybean pathogen-derived beta-glucan elicitor. In contrast to the beta-glucan elicitor, none of the other compounds tested triggered these early signaling elements. Moreover, endogenous levels of OPDA and JA in soybean cells were shown to be unaffected after treatment with beta-glucans. Thus, OPDA and JA, which are functionally mimicked by coronatine and a variety of 6-substituted derivatives of indanoyl-L-isoleucine methyl ester, represent highly efficient signaling compounds of a lipid-based pathway not deployed in the beta-glucan elicitor-initiated signal transduction.

Acetates↗

PubMed2002

Aequorin-based measurements of intracellular Ca2+-signatures in plant cells.

Due to the involvement of calcium as a main second messenger in the plant signaling pathway, increasing interest has been focused on the calcium signatures supposed to be involved in the patterning of the specific response associated to a given stimulus. In order to follow these signatures we described here the practical approach to use the non-invasive method based on the aequorin technology. Besides reviewing the advantages and disadvantages of this method we report on results showing the usefulness of aequorin to study the calcium response to biotic (elicitors) and abiotic stimuli (osmotic shocks) in various compartments of plant cells such as cytosol and nucleus.

Journal Article↗

PubMed2002

Suppression of plant defence in rhizobia-legume symbiosis.

The symbiosis between rhizobia and legumes is characterized by the formation of dinitrogen-fixing root nodules. Although rhizobia colonize roots in a way that is reminiscent of pathogenic microorganisms, no host plant defence reactions are triggered during successful symbioses. Nevertheless, the plants obviously control the invading bacteria; failure in effective nodule formation or infections with rhizobia defective in surface polysaccharides often result in pathogenic responses. This article focuses on whether and how defence responses in effective symbiosis might be suppressed. Recent results suggest a central role for rhizobial polysaccharides acting as antagonists in the negative regulation of defence induction.

Carbohydrate Sequence↗