Paul Finch

A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

Complexes of copper(II), calcium, and other metal ions with carbohydrates: Thin-layer ligand-exchange chromatography and determination of relative stabilities of complexes

Abstract Thin-layer ligand-exchange chromatography with sodium, magnesium, aluminium, calcium, chromium(III), iron(III), nickel, copper(II), zinc, strontium, cadmium, and barium as the central atoms has been investigated. With copper(II) as the central atom, the method is a simple, inexpensive, and speedy means of resolution of mixtures of carbohydrates not easily achieved by other methods. The molar ratios of complexed to uncomplexed polyhydroxy compounds, which give an indication of the relative stabilities of the complexes, are calculated from the chromatographic migration rates. For a particular compound, this ratio is, in general, greatest for the complex with the copper(II) ion.

Comparison of modern and fossil plant cuticles by selective chemical extraction monitored by flash pyrolysis-gas chromatography-mass spectrometry and electron microscopy

Abstract In order to investigate the preservation processes influencing the occurrence of plant cuticles (and hence leaves etc.) in the fossil record we have undertaken a comparative study of modern and fossil Ginkgo cuticles by chemical and microscopical methods. Cuticles are stripped or released from modern leaf tissue with hydrogen peroxide in aqueous acetic acid. The polysaccharide component and lignin can be selectively removed by acetyl bromide in acetic acid, and the cutin (polyester) by saponification. These treatments reveal the presence of a non-saponifiable residue of a resistant biomacromolecule with a characteristic dominantly aliphatic pyrolysis pattern as demonstrated and named cutan in the prototypical Agave americana cuticle. However, the same chemical treatments of recent Ginkgo biloba, the untreated cuticle of which shows an aliphatic signature upon pyrolysis, results in complete solubilisation of the sample with no resistant residue. The pyrolysis patterns can be clearly related to electron microscopic observations of the cuticles at different stages of chemical treatment. In particular the initial presence and extent of extracuticular cellular material, and its subsequent removal by the acetylation treatment can be visualised and explained. The saponifiable cutin polyester has a structural function even when associated with a resistant biomacromolecule in the Agave americana cuticle because electron microscopy shows that the resistant residue consists only of cuticle fragments. Fossil cuticles of Ginkgo huttonii were examined by Py-GC-MS and electron microscopy. These consist of extensive cuticle sheets on which SEM reveals gross morphology closely comparable to that in the modern cuticle whilst TEM shows that extra cuticular cellular material is lacking but that the outermost amorphous cuticle zone is preserved. An aliphatic pattern of alkene/alkane doublets has been identified in all of the samples. In addition, phenolic compounds have been found in the modern and fossil Ginkgo cuticle. The presence of a series of alkene/alkane doublets and some phenolic compounds in the fossil sample, combined with preservation of outer cuticle morphology and ultrastructure suggests that a less resistant, saponifiable polymer like that in modern Ginkgo cuticle is one possible source of the morphologically-preserved organic matter in the fossil. A highly resistant macromolecule is not responsible for the preservation of fossil Ginkgo cuticles.

Determination of the structures of cystocarpic carrageenans from Gigartina skottsbergii by methylation analysis and NMR spectroscopy

The combined use of methylation analysis and high-field 1H and 13C NMR spectroscopy allows the determination of the fine structure of the carrageenans produced by the cystocarpic stage of Gigartina skottsbergii.

Development of non‐invasive monitoring methods for larvae and adults of the stag beetle, Lucanus cervus

Abstract.  1. The stag beetle, Lucanus cervus is Nationally Scarce in the UK, yet no methods exist for monitoring the abundance of adults or presence of the subterranean larvae.

Geochemical evidence for combustion of hydrocarbons during the K-T impact event.

It has been proposed that extensive wildfires occurred after the Cretaceous–Tertiary (K-T) impact event. An abundance of soot and pyrosynthetic polycyclic aromatic hydrocarbons (pPAHs) in marine K-T boundary impact rocks (BIRs) have been considered support for this hypothesis. However, nonmarine K-T BIRs, from across North America, contain only rare occurrences of charcoal yet abundant noncharred plant remains. pPAHs and soot can be formed from a variety of sources, including partial combustion of vegetation and hydrocarbons whereby modern pPAH signatures are traceable to their source. We present results from multiple nonmarine K-T boundary sites from North America and reveal that the K-T BIRs have a pPAH signature consistent with the combustion of hydrocarbons and not living plant biomass, providing further evidence against K-T wildfires and compelling evidence that a significant volume of hydrocarbons was combusted during the K-T impact event.

The effects of organic solvents on the synthesis of galactose disaccharides using β-galactosidases

Abstract The use of β-galactosidases from five different sources in syntheses through transglycosylation leads to 3- O -methyl allo lactose, N -acetyllactosamine, N -acetyl allo lactosamine and Gal(β1–6)Gal. The yield and distribution of products depends on the source of the enzyme and on the reaction conditions, viz. the nature of added organic co-solvent. Yields of 3- O -methyl allo lactose (47%), Gal(β1–6)Gal (6%), N -acetyl allo lactosamine (30%) were obtained in aqueous buffer with β-galactosidase from E . coli . The same reactions occurred at much lower rates in the presence of water-miscible organic solvents. With β-galactosidases from K. fragilis and A. oryzae , however, the synthesis of the above disaccharides occurred only in the presence of organic solvent (60% v/v triethyl phosphate, trimethyl phosphate or tetraglyme) but not in aqueous buffer solution. β-galactosidases from D. pneumoniae and B. circulans in systems incorporating organic solvent produced 3- O -methyl allo lactose and N -acetyllactosamine in yields of 30–40%. The direct separation of oligosaccharides on a preparative scale can be achieved by Ca 2+ -ligand exchange chromatography. Ultrafiltration was also used for the efficient recycling of enzymes.

The reverse anomeric effect: further observations on N-glycosylimidazoles

Abstract The conformational analysis of a number of N-hexopyranosylimidazoles and their tetra-O-acetyl derivatives has been carried out using 1H-n.m.r. data obtained after computer simulation of spectra. Evidence is presented that, for some compounds, mixtures of 4C1 and 1C4 conformers are present in solution, and the possible contributions of steric effects and polar, reverse anomeric effects are discussed. It is concluded that the results can in large part be accounted for by steric factors, but that the operation of additional polar factors is likely. Present rationalisations of the reverse anomeric effect are discussed and a stereoelectronic interpretation is presented. The conformations of the exocyclic hydroxymethyl groups are analysed and shown to give additional information about the presence of alternative chair conformations.

Simulated diagenesis of plant cuticles — implications for organic fossilisation

Abstract The possible origin of the characteristic compositions of pyrolysates of organic fossil leaf cuticles has been investigated by heat treatment of cuticles of recent Kalanchoe grandiflora followed by flash pyrolysis–gas chromatography. Dewaxed cuticles, whether heat-treated or not, yield mainly cyclic compounds from adventitious cell-wall material together with cutin monomers. The pyrolysate of waxy cuticle includes the same compounds but is dominated by wax components which are simply desorbed unchanged under the conditions of pyrolysis. However prior heat treatment of waxy cuticle transforms the pyrolysate so that its chromatogram shows a prominent series of alkene–alkane peaks from C 8 to C 34 , typical of fossil leaf cuticles. It is concluded that leaf waxes, instead of or in addition to cutan, are a principal source of the aliphatic constituents of organic fossil leaf cuticles.

Laboratory stimulation of oil-spill effects on marine phytoplankton

Continuous culture conditions designed to achieve a dynamic equilibrium between phytoplankton growth and nutrient input were established for Phaeodactylum tricornutum, Isochrysis galbana and Chlorella salina. The technique was used to determine the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for algae after spiking with diesel oil. P. tricornutum (NOEC=0.25mg/l, LOEC=0.3mg/l) was more sensitive than I. galbana (NOEC=2.5mg/l, LOEC=2.6mg/l), while C. salina (NOEC=16.0mg/l, LOEC=17.0mg/l) was the most tolerant. Continuous renewal of medium ensured that experimental conditions remained stable throughout the test period and is a more environmentally relevant method for assessing the effects of many contaminants.