Search

The Southern North Sea Basin area, stretching from the UK to the Netherlands, has a rich hydrocarbon exploration and production history. The past, present and expected future hydrocarbon and geothermal exploration trends in this area are discussed for eight key lithostratigraphic intervals, ranging from the Lower Carboniferous to Cenozoic. In the period between 2007 and 2017, a total of 95 new hydrocarbon fields were discovered, particularly in Upper Carboniferous, Rotliegend and Triassic reservoirs. Nineteen geothermal systems were discovered in the Netherlands onshore, mainly targeting aquifers in the Rotliegend and Upper Jurassic/Lower Cretaceous formations. Although the Southern North Sea Basin area is mature in terms of hydrocarbon exploration, it is shown that with existing and new geological insights, additional energy resources are still being proven in new plays such as the basal Upper Rotliegend (Ruby discovery) for natural gas and a new Chalk play for oil. It is predicted that hydrocarbon exploration in the Southern North Sea Basin area will probably experience a slight growth in the coming decade before slowing down, as the energy transition further matures. Geothermal exploration is expected to continue growing in the Netherlands onshore as well as gain more momentum in the UK.

As geological information about the North Sea is increasingly made available to the public, there is great potential to build cross-border datasets to enable people to more rapidly filter relevant geological data and also to better understand and further study the subsurface. This paper discusses the construction of such a geological database and presents the associated map set. Public domain lithostratigraphic data from petroleum exploration and appraisal wells drilled in the Norwegian, Danish, German, Dutch and UK North Sea sectors were used to create a series of 13 well-penetration maps, ranging from Basement to Eocene age. In order to produce the maps, lithostratigraphic well tops had to be translated into a chronostratigraphic scheme, followed by building a database and subsequent mapping in a GIS. Three well categories were used for each of the maps: wells that (1) fully penetrate a chronostratigraphic interval, (2) prove it is absent or (3) reach total depth in the interval under consideration. The resulting maps, which will all be discussed and presented in this paper, form a starting point for multiple types of (petroleum) geological screening studies. The North Sea database is freely available in QGIS format and can be easily imported into any geospatial software platform.

The Devonian–Recent tectono-stratigraphic history of the Northern, Central and Southern North Sea is here reviewed at a regional scale and four novel cross-border pseudo-Wheeler diagrams are presented to summarize the stratigraphic evolution of the cycles of basin fill and uplift/erosion. In this scheme, six first-order megasequence boundaries have been defined, characterized by extensive and long-lasting erosional hiatuses and major coastal regressions: (1) Caledonian (or Base Devonian) Unconformity; (2) Variscan–Saalian (or Base Permian) Unconformity; (3) Mid Cimmerian (or Intra-Aalenian) Unconformity; (4) Late Cimmerian (or Base Cretaceous) Unconformity; (5) Atlantean (or Near-Base Tertiary) Unconformity; and (6) Eridanos (or Mid-Miocene) Unconformity. These surfaces have been linked to regional causal factors ranging from orogenesis-related compressional uplifts, in either active plate margin settings (1) or foreland basin settings (2), to intra-plate dynamically supported uplifts associated with the development of mantle plumes (3, 5 and 6) and the end-of-rifting and associated widespread erosion of tilted fault block crests (4). The aforementioned megasequence boundaries punctuate the geodynamic evolution of the North Sea area and facilitate the subdivision of the entire the North Sea sedimentary basin fill into six megasequences, named here A–F. All of the lithostratigraphic units of the North Sea (formations and members) have been described within the context of this first-order tectono-stratigraphic framework. The correlation powers of certain stratigraphic markers are also compared and contrasted, together with the potential cross-border equivalence of sedimentary units on different sides of the political median lines.

The North Sea has reached an ultra-mature state as a petroleum basin, entering a phase of infrastructure-led exploration in an attempt to extend the economic lives of the main fields and reduce the rate of production decline. At the same time, the transition to a future low-carbon use of the basin is also in progress. As the papers in this volume demonstrate, in order to find, appraise and develop the mostly smaller near-field opportunities as well as making sure to grasp the opportunities of the near-future energy transition, a regional understanding of the North Sea is still critical. Even more so, a cross-border approach is essential because: (1) some of the plays currently being targeted have a clear cross-border element; (2) it allows a comparison of stratigraphic names throughout the entire basin; and (3) it enables explorers to learn lessons from one part of the rift to be applied somewhere else.This volume offers an up-to-date ‘geology-without-borders’ view of the stratigraphy, sedimentology, tectonics and oil-and-gas exploration trends of the entire North Sea Basin. The challenges associated with data continuity and nomenclature differences across median lines are discussed and mitigated. Examples of under-exploited cross-border plays and discoveries are discussed.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinereato the bacteria Bacillus subtilisand Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinereasecretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinereato the bacteria Bacillus subtilisand Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinereasecretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized aß-defensins (Csaß-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

AbstractIn this paper we develop renewal theorems for point processes with interarrival times ξ(Xn+1Xn…), where (Xn)n∈ℤis a stochastic process with finite state space Σ and ξ:ΣA→ℝ is a Hölder continuous function on a subset ΣA⊂Σℕ. The theorems developed here unify and generalise the key renewal theorem for discrete measures and Lalley's renewal theorem for counting measures in symbolic dynamics. Moreover, they capture aspects of Markov renewal theory. The new renewal theorems allow for direct applications to problems in fractal and hyperbolic geometry, for instance to the problem of Minkowski measurability of self-conformal sets.

Plant growth regulating properties of brevicompanines (Brvs), natural products of the fungus Penicillium brevicompactum, have been known for several years, but further investigations into the molecular mechanism of their bioactivity have not been performed. Following chemical synthesis of brevicompanine derivatives, we studied their activity in the model plant Arabidopsisby a combination of plant growth assays, transcriptional profiling, and numerous additional bioassays. These studies demonstrated that brevicompanines cause transcriptional misregulation of core components of the circadian clock, whereas other biological read-outs were not affected. Brevicompanines thus represent promising chemical tools for investigating the regulation of the plant circadian clock. In addition, our study also illustrates the potential of an unbiased -omics-based characterization of bioactive compounds for identifying the often cryptic modes of action of small molecules.

Jasmonates are lipid-derived signals that mediate plant stress responses and development processes. Enzymes participating in biosynthesis of jasmonic acid (JA) (1, 2) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants of Arabidopsisand tomato have helped to define the pathway for synthesis of jasmonoyl-isoleucine (JA-Ile), the active form of JA, and to identify the F-box protein COI1 as central regulatory unit. However, details of the molecular mechanism of JA signaling have only recently been unraveled by the discovery of JAZ proteins that function in transcriptional repression. The emerging picture of JA perception and signaling cascade implies the SCFCOI1complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S-proteasome pathway, thereby allowing the transcription factor MYC2 to activate gene expression. The fact that only one particular stereoisomer, (+)-7-iso-JA-l-Ile (4), shows high biological activity suggests that epimerization between active and inactive diastereomers could be a mechanism for turning JA signaling on or off. The recent demonstration that COI1 directly binds (+)-7-iso-JA-l-Ile (4) and thus functions as JA receptor revealed that formation of the ternary complex COI1-JA-Ile-JAZ is an ordered process. The pronounced differences in biological activity of JA stereoisomers also imply strict stereospecific control of product formation along the JA biosynthetic pathway. The pathway of JA biosynthesis has been unraveled, and most of the participating enzymes are well-characterized. For key enzymes of JA biosynthesis the crystal structures have been established, allowing insight into the mechanisms of catalysis and modes of substrate binding that lead to formation of stereospecific products.

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thalianaisoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative “substrate specificity motif,” only one residue, Met293, was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met293or Lys320, another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met293, by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met293by bulky aromatic amino acids increased the apparent affinity (Km) for caffeate up to 10-fold, whereas single substitutions of Val294did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.

A ubiquitous feature of plant/pathogen interactions is host cell death that is manifested as rapid collapse of tissue and is termed the hypersensitive response (HR). This response accompanies many but not all incompatible interactions and is considered one of the important mechanisms leading to resistance. The sites of HR the infection sites proper are invariably the focal points for transcriptional activation of a large variety of plant defence genes in neighbouring cells. The subsequent biosynthesis of protective secondary metabolites and inhibitory proteins around the infection sites are considered to be important for overall pathogen containment. In addition local HR is often associated with the onset of systemic acquired resistance (SAR) in distal plant tissues. This type of resistance is generally effective against a broad range of pathogens and it is associated with the transcriptional activation of whole set of marker genes many of which encode pathogenesis-related proteins such as chitinases and 1,3-β-glucanases. Cell death is also a feature of disease symptoms in many compatible interactions but in these cases it usually occurs rather late during the course of host colonisation by the pathogen. Necrotic lesions may develop but are not required for triggering SAR and systemic gene activation. Apparently different forms of cell death and mechanisms leading to HR exist and are executed in plant/pathogen interactions. Although the importance of small molecules such as reactive oxygen intermediates (ROI) for the establishment of HR cell death has been recognized a functional and causal link between ROI production initiation of HR cell death and induced local and systemic disease resistance remains to be unequivocally demonstrated.

AbstractTwo distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojaeresulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.

4‐Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thalianaAt4CL2. Determination of specific activities and Kmvalues for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild‐type Kmvalues; enzymes with drastically altered Kmvalues; and enzymes with almost wild‐type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate‐forming enzymes such as luciferases, peptide synthetases, and fatty acyl‐CoA synthetases.

We have isolated cDNA clones encoding class I chitinase (ChtC) from potato leaves which share a high degree of nucleotide and amino acid sequence similarity to other, previously described basic (class I) chitinases (ChtB) from potato. Despite this similarity, characteristic features distinguish ChtC from ChtB, including an extended proline-rich linker region between the hevein and catalytic domains and presence of a potential glycosylation site (NDT) in the deduced protein. These differences are in accordance with the properties of purified chitinase C which is glycosylated and hence has a higher molecular mass in comparison to chitinase B. In contrast to the coding sequences, the 3′-untranslated regions of ChtC and ChtB exhibited a low degree of similarity, which allowed us to generate gene-specific probes to study the genomic organization and expression of both types of gene. Genomic DNA blots suggest that ChtC and ChtB are each encoded by one or two genes per haploid genome. RNA blot analysis showed that in healthy potato plants ChtC mRNA is most abundant in young leaves, the organs which also contain high levels of chitinase C. By contrast, ChtB mRNA abundance is highest in old leaves, which accumulate chitinase B. By in situ RNA hybridization with gene-specific probes we could demonstrate that ChtC mRNA in leaves is restricted to epidermal cells, whereas ChtB mRNA showed no distinct pattern of cell-type-specific localization. Infection of potato leaves with Phytophthora infestans, or treatment with fungal elicitor, ethylene, or wounding resulted in accumulation of both ChtC and ChtB mRNAs; however, for ChtC, in contrast to ChtB, no corresponding accumulation of the encoded protein could be detected, suggesting a post-transcriptional mechanism of regulation. Salicylic acid treatment did not induce accumulation of either mRNA. The possible functional implications of these findings for pathogen defence and developmental processes are discussed.

The distribution of enzymes interconverting fructose 6-phosphate and fructose 1,6-bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PP i:fructose 6-phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6-bisphosphatase. PP i:fructose 6-phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3-ß-glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3-ß-glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3-ß-glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3-ß-glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3-ß-glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3-ß-glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3-ß-glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3-ß-glucanase are encoded by gene families of considerable complexity.

Zusammenfassung: Seien B1, B2,..., Bk kleinere ähnliche Bestände versicherter Personen, von denen gewisse Wahrscheinlichkeiten geschätzt werden sollen. Die Beobachtungen in den Beständen Bj, j ≠ i, können jedoch durchaus nützliche Zusatzinformationen zu den Beobachtungen aus Bi sein. Es wird eine Schätzung Si (B{im1},..., Bi,..., Bk) für Bi konstruiert, die die in Bj, j ≠ i, bezüglich Bi enthaltenen Zusatzinformationen einbezieht und im Rahmen des Modells besser als Si (Bi) ist.

Chitinase {poly[1,4-(N-acetyl-β-D-glucosaminide)]glycanohydrolase, EC 3.2.1.14} and 1,3-β-glucanase (1,3-β-D-glucan 3-glucanohydrolase, EC 3.2.1.6) activities increased rapidly in potato (Solanum tuberosum) leaves inoculated with the pathogenic fungus Phytophthora infestansor treated with fungal elicitor. The enzyme activities were resolved into a total of two distinct 1,3-β-glucanases and six proteins with chitinase activity. By several criteria, all of these proteins are classified as “pathogenesis-related” proteins whose biochemical functions have so far been unknown. Some of them constitute a major portion of the proteins accumulating in the intercellular space of infected potato leaves and are assumed to play an important role in pathogen defense.

Abstract: The levels of ribulose 1,5-bisphosphate carboxylase small-subunit (SSU) mRNA and protein decreased considerably in potato (Solanum tuberosum L.) leaves upon infection with the pathogenic fungus,Phytophthora infestans, or upon treatment with an elicitor preparation from the fungal culture fluid. This effect occurred systemically throughout the affected leaf, regardless of whether the interaction withP. infestans was compatible or incompatible. Using the comparatively drastic and synchronous response to fungal elicitor, we demonstrated that the repression of SSU synthesis was caused by rapid gene inactivation. The timing of repression was similar to that observed previously for the transcriptional activation of various pathogen defense reactions. This supports the hypothesis that induction of the extensive, multi-component defense response requires repression of other cellular functions to ensure metabolic balance.

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of a-glucan phosphorylase were visible. Using specific a-glucans incorporated into the gel before electrophoresis we have tentatively correlated a-amylase and ß-amylase with both hydrolytic activities. The activities of a-glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of a-glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and a-glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.

The distribution of enzymes interconverting fructose 6‐phosphate and fructose 1,6‐bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PPi:fructose 6‐phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6‐bisphosphatase. PPi:fructose 6‐phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3-ß-glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and ‘pathogenesis-related’ proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.

A cDNA library from cultured parsley (Petroselinum crispum) cells was differentially screened using labeled run-off transcripts derived from nucleic of elicitor-treated and untreated cells. This resulted in the isolation of 18 independent cDNA families representing putative defense-related genes. All genes are rapidly and transiently activated after elicitor application, but the time courses of transcriptional activity exhibit considerable variations, indicating differences in the mechanisms of gene regulation.

The extent of induction of some metabolic activities in cultured parsley cells (Petroselinum crispum) by an elicitor preparation from Phytophthora megasperma f. sp. glycinea varied with the growth stage of the cell culture. On the basis of cell fresh weight, the induction of phytoalexin accumulation was high until cell mass reached a maximum, and then declined to a low level which was indistinguishable from a level caused by an endogenous mechanism operating at this late growth stage. The induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities by the elicitor showed a high degree of coordination and a sharp maximum preceding the stage of maximal cell mass. 1,3-ß-Glucanase activity was induced to about the same level throughout all growth stages, with a large contribution by an endogenous mechanism at late stages.

Infection of potato (Solanum tuberosum) leaves by the late blight fungus Phytophthora infestans or treatment with fungal elicitor leads to a strong increase in chitinase activity. We isolated cDNAs encoding acidic (class II) chitinases (ChtA) from potato leaves and determined their structures and expression patterns in healthy and stressed plants. From the total number of cDNAs and the complexity of genomic DNA blots we conclude that acidic chitinase in potato is encoded by a gene family which is considerably smaller than that encoding basic (class I) chitinase (ChtB). The deduced amino acid sequences show 78 to 96% identity to class II chitinases from related plant species (tomato, tobacco) whereas the identity to basic chitinases of potato is in the range of 60%. RNA blot analysis revealed that both acidic and basic chitinases were strongly induced by infection or elicitor treatment and that the induction occurred both locally at the site of infection and systemically in upper uninfected leaves. In contrast, a differential response to other types of stress was observed. Acidic chitinase mRNA was strongly induced by salicylic acid, whereas basic chitinase mRNA was induced by ethylene or wounding. In healthy, untreated plants, acidic chitinase mRNA accumulated also in an organ-, cell-type- and development-specific manner as revealed by RNA blot analysis and in situ RNA hybridization. Relatively high transcript levels were observed in old leaves and young internodes as well as in vascular tissue and cells constituting the stomatal complex in leaves and petioles. Lower, but appreciable mRNA levels were also detectable in roots and various flower organs, particularly in sepals and stamens. The possible implications of these findings in pathogen defense, development and growth processes are discussed.

No relevant conflicts of interest to declare.

Abstract 3188

The conversion of soluble fibrinogen to an insoluble fibrin matrix following thrombin cleavage of the Aα and Bβ chains is critical to hemostasis. However, even soluble fibrinogen holds potential biological significance apart from fibrin polymer formation; for example, fibrinogen contributes significantly to overall blood rheology and can engage a number of integrins (e.g., αIIbβ3), enzymes (e.g., fXIII) and matrix proteins (e.g. fibronectin). To provide a system to study mice carrying fibrinogen but no capacity for fibrin formation, we employed a gene-targeting strategy to eliminate the Aα chain thrombin cleavage site and replace it with a sequence recognized by the highly-selective alternate protease, enterokinase (P6-P1: ADDDDK). Based on the prevailing view that thrombin cleavage of the Aα chain is essential to polymer formation, we hypothesized that this mutant fibrinogen, termed FibEK, would be locked in the soluble form in vivo, but would be readily clotted in vitro by exogenous enterokinase. The FibEK allele was transmitted through the germline and supported the expected level of mRNA expression and plasma protein production. Like previously established fibrinogen-null mice, FibEK mice uniformly developed to term. However, survival beyond the perinatal period was vastly superior in mice carrying fibrinogen-EK relative to fibrinogen-null animals in the same (C57Bl/6) genetic background; ~90% of FibEK mice survive to adulthood, whereas less than 30% of fibrinogen-null mice survive the perinatal period. Consistent with our initial hypothesis, FibEK plasma was found to be unclottable following the addition of excess exogenous murine thrombin at 37°C and complementary fibrinopeptide release assays by HPLC revealed that murine thrombin is completely incapable of releasing fibrinopeptide A (FpA) from fibrinogen-EK. Murine thrombin did support quantitative FpB release from fibrinogen-EK, albeit at a slower rate than wild-type fibrinogen. No thrombin-induced fibrin polymer formation could be appreciated by standard turbidity assays in incubation mixtures containing FibEK plasma at 37°C, even after 24 hrs. In contrast, wild-type plasma supported rapid polymer formation, with turbidity peaking within minutes of thrombin addition. Interestingly, despite the complete absence of FpA release, both plasma and purified fibrinogen-EK supported the formation of small and irregular clots (characterized by thin fibrils in scanning EM) following long incubations at reduced temperatures (i.e., 24 hr at 22 °C). Unlike fibrinogen-null mice, platelet-rich plasma prepared from FibEK mice supported robust ADP-induced platelet aggregation. However, FibEK animals essentially phenocopy fibrinogen-null mice in their inability to efficiently clear the microbial pathogen S. aureus. Following intraperitoneal infection with 109 CFU of S. aureus, control mice successfully cleared >99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of both FibEK and FibAα−/− mice remained similar to the original input CFU. In summary, FibEK mice provide a unique opportunity to further explore the biochemistry of thrombin-mediated clot formation in vitro and in vivo. These animals will also be a vital tool in defining the specific contribution of fibrin matrices to broad array of physiological and pathological processes in vivo.

The conversion of soluble fibrinogen to an insoluble fibrin matrix following thrombin cleavage of the Aα and Bβ chains is critical to hemostasis. However, even soluble fibrinogen holds potential biological significance apart from fibrin polymer formation; for example, fibrinogen contributes significantly to overall blood rheology and can engage a number of integrins (e.g., αIIbβ3), enzymes (e.g., fXIII) and matrix proteins (e.g. fibronectin). To provide a system to study mice carrying fibrinogen but no capacity for fibrin formation, we employed a gene-targeting strategy to eliminate the Aα chain thrombin cleavage site and replace it with a sequence recognized by the highly-selective alternate protease, enterokinase (P6-P1: ADDDDK). Based on the prevailing view that thrombin cleavage of the Aα chain is essential to polymer formation, we hypothesized that this mutant fibrinogen, termed FibEK, would be locked in the soluble form in vivo, but would be readily clotted in vitro by exogenous enterokinase. The FibEK allele was transmitted through the germline and supported the expected level of mRNA expression and plasma protein production. Like previously established fibrinogen-null mice, FibEK mice uniformly developed to term. However, survival beyond the perinatal period was vastly superior in mice carrying fibrinogen-EK relative to fibrinogen-null animals in the same (C57Bl/6) genetic background; ~90% of FibEK mice survive to adulthood, whereas less than 30% of fibrinogen-null mice survive the perinatal period. Consistent with our initial hypothesis, FibEK plasma was found to be unclottable following the addition of excess exogenous murine thrombin at 37°C and complementary fibrinopeptide release assays by HPLC revealed that murine thrombin is completely incapable of releasing fibrinopeptide A (FpA) from fibrinogen-EK. Murine thrombin did support quantitative FpB release from fibrinogen-EK, albeit at a slower rate than wild-type fibrinogen. No thrombin-induced fibrin polymer formation could be appreciated by standard turbidity assays in incubation mixtures containing FibEK plasma at 37°C, even after 24 hrs. In contrast, wild-type plasma supported rapid polymer formation, with turbidity peaking within minutes of thrombin addition. Interestingly, despite the complete absence of FpA release, both plasma and purified fibrinogen-EK supported the formation of small and irregular clots (characterized by thin fibrils in scanning EM) following long incubations at reduced temperatures (i.e., 24 hr at 22 °C). Unlike fibrinogen-null mice, platelet-rich plasma prepared from FibEK mice supported robust ADP-induced platelet aggregation. However, FibEK animals essentially phenocopy fibrinogen-null mice in their inability to efficiently clear the microbial pathogen S. aureus. Following intraperitoneal infection with 109CFU of S. aureus, control mice successfully cleared >99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of both FibEK and FibAα−/− mice remained similar to the original input CFU. In summary, FibEK mice provide a unique opportunity to further explore the biochemistry of thrombin-mediated clot formation in vitro and in vivo. These animals will also be a vital tool in defining the specific contribution of fibrin matrices to broad array of physiological and pathological processes in vivo.