Terence M. Murphy

Plasma Membrane Redox Enzyme Is Involved in the Synthesis of O2- and H2O2 by Phytophthora Elicitor-Stimulated Rose Cells.

An elicitor prepared from the autoclaved cell walls of Phytophthora sp. induced O2- generation and H2O2 accumulation by cultured cells of Rosa damascena Mill. cv Gloire de Guilan. N,N-Diethyldithiocarbamate, a superoxide dismutase inhibitior, blocked H2O2 accumulation and caused a dramatic accumulation of O2- by elicitor-treated rose cells. In the absence of N,N-diethyldithiocarbamate no detectable O2- was accumulated. Diphenyleneiodonium, quinacrine, pyridine, and imidazole, inhibitors of the mammalian neutrophil NADPH oxidase responsible for the generation of O2- during phagocytosis, inhibited O2- generation by elicitor-treated rose cells. Diphenyleneiodonium also inhibited NADH-dependent O2- production by plasma membranes isolated from rose cells. None of the four compounds inhibited the peroxidase activity in the cell-suspension medium. These results demonstrate that elicitor-stimulated accumulation of H2O2 comes only from superoxide dismutase-catalyzed dismutation of O2-. The data are inconsistent with the hypothesis that the synthesis of O2- is catalyzed by extracellular peroxidase and suggest that the enzyme responsible for the synthesis of O2- by elicitor-treated rose cells might be similar to the mammalian neutrophil NADPH oxidase.

The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells

Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells.

Solar ultraviolet-B radiation effects on growth and pigment composition of the intertidal alga Ulva expansa (Setch.) S. & G. (Chlorophyta)

Abstract Segments of Ulva expansa (Setch.) S. & G. (Chlorophyta) were grown in outdoor tanks under UV-B-transparent or UV-B-opaque screens. Growth rates of the segments were measured by changes in surface area. Amounts of chlorophyll a , carotenoids, and UV-absorbing compounds per segment did not vary systematically with treatments, but chlorophyll a and carotenoids per unit weight were higher after growth under UV-B-transparent screens than under UV-B-opaque screens. These results indicate that chlorophyll a and carotenoid synthesis was not inhibited by UV-B. Nitrogen supplementation (10 μM NaNO 3 ) enhanced the growth rates of both UV-B-exposed and -unexposed segments; however this supplementation did not affect the sensitivity of the algal segments to UV-B. Growth rates of Ulva segments grown attached to frames on the mudflats were similar to those measured in the outdoor tanks, supporting the assumption that the effects of UV-B measured in outdoor tanks used in this and other studies are relevant to natural populations.

INHIBITION OF GROWTH OF ULVA EXPANSA (CHLOROPHYTA) BY ULTRAVIOLET‐B RADIATION1

We examined the effect of ultraviolet‐B radiation (UV‐B, 290–320 nm) on the growth rate of the intertidal marine alga Ulva expansa (Setch.) S. & G. (Chlorophyta). Segments of thallus collected from a natural population were grown in outdoor seawater tanks. Combinations of UV‐B‐opaque screens, UV‐B‐transparent screens, and UV‐B lamps were used to investigate the effects of solar UV‐B and solar plus supplemental UV‐B on the growth of these segments. Growth was measured by changes in segment surface area, damp weight, and dry weight. Growth rates of segments were inhibited under both solar UV‐B and solar plus supplemental UV‐B treatments. Growth rates were also inhibited by high levels of photosynthetically active radiation, independent of UV‐B fluence. These results indicate that increases in UV‐B resulting from further ozone depletion will have a negative impact on the growth of this alga.

A comparison of the effects of a fungal elicitor and ultraviolet radiation on ion transport and hydrogen peroxide synthesis by rose cells

Abstract An elicitor preparation from a Phytophthora sp. induced an efflux of K+ and a production of H2O2 by cell suspensions of Rosa damascena. The kinetics and amounts of K+ efflux and of H2O2 production and degradation were similar to those observed when the cells were treated with u.v. radiation (254 nm). The K+ efflux from both elicitor-treated and u.v.-treated rose cells was inhibited at 0 °K+ efflux from elicitor-treated cells was stimulated by a pretreatment of the cells at 40°C; efflux from u.v.-treated cells was inhibited by the same pretreatment. Washed cells lost sensitivity to elicitor (but not u.v.) over a period of several hours. Like u.v.-treated cells, elicitor-treated cells lost sensitivity to a second treatment with u.v. given 15–18 hr later. The results are interpreted in terms of a common ion transport process stimulated by elicitor and u.v. through different receptors.

UV-ACTION SPECTRUM (254-405 nm) FOR INHIBITION OF A K+ -STIMULATED ADENOSINE TRIPHOSPHATASE FROM THE PLASMA MEMBRANE OF ROSA DAMASCENA

A K +‐stimulated ATPase from suspension‐cultured rose cells was isolated and subjected to UV radiation. The characteristics of the ATPase resembled those of a plasma‐membrane associated enzyme and not those of the mitochondrial enzyme. The ATPase required Mg2+ and was further stimulated up to 100% by K+. K+ stimulation was specific for ATP. The order of stimulation by monovalent cations was K+ > Na+ > Li+. The enzyme had a pH optimum of 6.5 in the presence of 50 mM K+. It was almost completely inhibited by diethylstilbestrol and partially inhibited by vanadate. but was not affected by azide or oligomycin. The inhibition of ATPase activity by various fluences of UV indicated that one fraction of the K+‐stimulated activity was very sensitive to radiation, while another fraction was relatively insensitive. It is possible that UV distinguished between two enzymes. The action spectra for inhibition of both fractions showed maxima at 290 nm and significant but much lower action throughout the near‐UV region, resembling spectra in the literature for the inhibition of transport processes in bacteria.

Requirement for Abasic Endonuclease Gene Homologues in Arabidopsis Seed Development

Arabidopsis thaliana has three genes, Ape1L, Ape2, and Arp, that show homology to abasic (apurinic/apyrimidinic) endonuclease genes of bacterial, yeast, or animal cells. In bacteria, yeast, and animals, abasic endonucleases function in base excision repair of oxidized and other modified DNA bases. Here we report that plants with knock-out mutations in any one of Ape1L, Ape2, or Arp show no apparent differences from wild type in growth rate, growth habit, and fertility. However, coincident knock-out mutations in Ape1L and Ape2 are lethal and lead to abortion of developing embryos. Mutations of Arp are not deleterious, even in combination with one of the other two mutations. The results are consistent with the interpretation that the process of base excision repair, involving at least one intact copy of Ape1L or Ape2, is required in the process of embryogenesis.

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane‐associated K+‐stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m2). ATPase activity measured in the absence of K+ and K+‐stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m2.

Alternative Forms of Formamidopyrimidine-DNA Glycosylase from Arabidopsis thaliana¶

Abstract Formamidopyrimidine-DNA glycosylase (FPG) catalyzes the initial steps in the repair of DNA containing oxidized purines. Two complementary DNA clones encoding homologs of bacterial FPG, designated Atfpg-1 and Atfpg-2, have been isolated from Arabidopsis thaliana. They are products of alternative splicing of the transcript of a single gene. Proteins encoded by both clones, AtFPG-1 and AtFPG-2, engineered to contain oligohistidine sequences on their C-terminal ends, were expressed in Escherichia coli and purified, and their activities were assayed. Both proteins cleaved DNA that contained apurinic sites, indicating that they have abasic lyase activity. AtFPG-1, but not AtFPG-2, showed significant cleavage of a double-stranded oligonucleotide that contained 8-oxo-guanine, indicating that the structural differences between the two proteins influence their enzymatic activities. However, both proteins were able to cleave the same sites in DNA that was treated with visible light in the presence of methylene blue.

Novel Expression Pattern of Cytosolic Gln Synthetase in Nitrogen-Fixing Root Nodules of the Actinorhizal Host, Datisca glomerata

Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform, DgGS1-1, are abundant in uninfected nodule cortical tissue, but expression was not detectable in the infected zone or in the nodule meristem. Similarly, the GS holoprotein is immunolocalized exclusively to the uninfected nodule tissue. Phylogenetic analysis of the full-length cDNA of DgGS1-1 indicates affinities with cytosolic GS genes from legumes, the actinorhizal species Alnus glutinosa, and nonnodulating species, Vitis vinifera and Hevea brasilensis. The D. glomerata nodule GS expression pattern is a new variant among reported root nodule symbioses and may reflect an unusual nitrogen transfer pathway from the Frankia nodule microsymbiont to the plant infected tissue, coupled to a distinctive nitrogen cycle in the uninfected cortical tissue. Arg, Gln, and Glu are the major amino acids present in D. glomerata nodules, but Arg was not detected at high levels in leaves or roots. Arg as a major nodule nitrogen storage form is not found in other root nodule types except in the phylogenetically related Coriaria. Catabolism of Arg through the urea cycle could generate free ammonium in the uninfected tissue where GS is expressed.