A.G. Sanz

PIOX, a New Pathogen-Induced Oxygenase with Homology to Animal Cyclooxygenase

Changes in gene expression induced in tobacco leaves by the harpin HrpN protein elicitor were examined, and a new cDNA, piox (for pathogen-induced oxygenase), with homology to genes encoding cyclooxygenase or prostaglandin endoperoxide synthase (PGHS), was identified. In addition to the amino acid identity determined, the protein encoded by piox is predicted to have a structural core similar to that of ovine PGHS-1. Moreover, studies of protein functionality demonstrate that the PIOX recombinant protein possesses at least one of the two enzymatic activities of PGHSs, that of catalyzing the oxygenation of polyunsaturated fatty acids. piox transcripts accumulated after protein elicitor treatment or inoculation with bacteria. Expression of piox was induced in tissues responding to inoculation with both incompatible and compatible bacteria, but RNA and protein accumulation differed for both types of interactions. We show that expression of piox is rapidly induced in response to various cellular signals mediating plant responses to pathogen infection and that activation of piox expression is most likely related to the oxidative burst that takes place during the cell death processes examined. Cyclooxygenase catalyzes the first committed step in the formation of prostaglandins and thromboxanes, which are lipid-derived signal molecules that mediate many cellular processes, including the immune response in vertebrates. The finding of tobacco PIOX suggests that more similarities than hitherto expected will be found between the lipid-based responses for plant and animal systems.

ALPHA -OXIDATION OF FATTY ACIDS IN HIGHER PLANTS: IDENTIFICATION OF A PATHOGEN-INDUCIBLE OXYGENASE (PIOX) AS AN ALPHA -DIOXYGENASE AND BIOSYNTHESIS OF 2-HYDROPEROXYLINOLENIC ACID

A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsiswere recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523–1537). Linolenic acid incubated at 23 °C with preparations containing the recombinant enzymes underwent α-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z),14(Z)-heptadecatrienal (83%), an α-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 °C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid in α-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same α-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 °C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 °C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for α-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic α-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into α-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both α-dioxygenases, and that members of this new class of enzymes catalyze the first step of α-oxidation in plant tissue.

Activation of the Fatty Acid α-Dioxygenase Pathway during Bacterial Infection of Tobacco Leaves FORMATION OF OXYLIPINS PROTECTING AGAINST CELL DEATH

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid α-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of α-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing α-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. α-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.

Activation of defence-related genes during senescence: a correlation between gene expression and cellular damage.

The correlation between activation of defence-related gene expression and plant senescence was investigated by evaluating the presence of specific transcripts in various leaves of tobacco senescing plants. Expression of most genes examined was found to be induced shortly after flowering; however, each gene had its own characteristic timing of expression and level of RNA accumulation. Studies of the symptoms developed in senescing leaves responding to bacterial inoculation suggest that the accumulation of defence-related transcripts in these tissues might be related with the mechanism of senescence rather than with protection of the plant against pathogen infection. We observed that the high level of GUS expression directed by the β-1,3-glucanase gn1 promoter, in senescing leaves of transgenic tobacco plants, decreased after bacterial inoculation, in correlation with the formation of symptoms. Reduction of gene expression was likely to be the reflection of the additional damage caused by the bacteria in the senescent tissues inoculated.

Fatty acid α-dioxygenases

Abstract This paper will briefly review the biochemistry, molecular biology and functionality of α-dioxygenases from tobacco, Arabidopsis , cucumber, pea, rice and algae. α-Dioxygenase treated in this review has emerged as a third group of fatty acid dioxygenases along with lipoxygenases and endoperoxide synthases.

Electron scattering cross section calculations for polar molecules over a broad energy range

We report computational integral and differential cross sections for electron scattering by two different polar molecules, HCN and pyrimidine, over a broad energy range. We employ, for low energies, either the single-centre expansion (ePOLYSCAT) or the R-matrix method, while for the higher energies we select a corrected form of the independent-atom representation (IAM-SCAR). We provide complete sets of integral electron scattering cross sections from low energies up to 10,000 eV. Our present calculated data agree well with prior experimental results.

Current prospects on Low Energy Particle Track Simulation for biomedical applications

The Low Energy Particle Track Simulation code is a radiation interaction simulation tool specifically designed to describe electron and positron interactions below 10 keV at a molecular level. Relying on carefully selected, preferentially experimental input parameters that account for all expected scattering processes, it provides detailed results about all collisional events undergone by an incident radiation particle during its slowdown until thermalisation. Here, we give an up-to-date description of its input data sources and selection procedure and summarise the current contents of the resulting database.

Thymidine Decomposition Induced by Low-Energy Electrons and Soft X Rays under N2 and O2 Atmospheres

A novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0–30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N2 or O2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2′-deoxyuridine (5-HMdUrd), 5-formyl-2′-deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N2 and O2 environment, we report a dramatic effect from 6 O2: an approximately threefold increase in the yield of products, attributed to the reaction of O2 with initial carbon-centered thymidine radicals generated in the film during irradiation.

Electron interactions with tetrahydrofuran

In this paper, we summarize our recent experimental and theoretical results on electron scattering from gaseous tetrahydrofuran (THF). Electron-impact ionization and total scattering cross sections were determined experimentally for energies between 50-5000 eV. Electron energy loss spectra were measured in the keV range using a transmission beam technique and for smaller energies (15-50 eV) with a crossed-beam apparatus. Using an optical potential method assuming the screening-corrected additivity rule, total, elastic and inelastic cross sections including dipole interactions were calculated (leV - lOkeV) in order to complement the experimental data. Elastic differential cross sections were also obtained. An empirical approximation to the inelastic angular distributions based on differential cross sections is proposed. The available integral and differential cross sections and energy loss distributions in the range 1 eV - 10 keV are combined into a table of recommended electron interaction cross sections with THF.

α-Dioxygenase, a new enzyme in fatty acid metabolism

Abstract Infection of tobacco leaves with bacteria resulted in the accumulation of a 75-kDa oxygenase. This enzyme and a homologous one from Arabidopsis were expressed in insect cells and their catalytic properties were studied. Linolenic acid incubated with preparations containing the recombinant enzymes was converted into 2(R)-hydroperoxylinolenic acid, an unstable oxylipin which underwent spontaneous degradation into a mixture of 8(Z), 11(Z), 14(Z)-heptadecatrienal, 2(R)-hydroxylinolenic acid, and 8(Z), 11(Z), 14(Z)-heptadecatrienoic acid. Studies with stereospecifically deuterated linolenic acid demonstrated that the enzymatic α-dioxygenation proceeded with retention of absolute configuration. The α-dioxygenases from tobacco and Arabidopsis showed homology to mammalian prostaglandin endoperoxide synthases-1 and -2 and to linoleate diol synthase from certain fungi. His-388 and His-207 (heme-binding), Gln-203 (charge-stabilizing), and Tyr-385 (hydrogen-abstracting) of ovine endoperoxide synthase-1 were conserved in the α-dioxygenases, and mutation studies of the Arabidopsis enzyme showed that their replacement with other amino acids resulted in loss of catalytic activity. The α-dioxygenase activity in tobacco leaves increased from three- to fourfold upon mechanical wounding, about fourfold in response to infection by Pseudomonas syringae pv syringae (incompatible strain), and from six- to eightfold in response to P. syringae pv tabaci (compatible strain).