Mauro Maccarrone

In vitro oxygenation of soybean biomembranes by lipoxygenase-2

The ability of soybean lipoxygenases-1 and -2 to oxygenate biomembranes isolated from soybean seedlings has been investigated. Constituents of the lipid bilayer were analyzed by means of reversed phase and chiral phase high performance liquid chromatography, gas chromatography/mass spectrometry, high performance thin layer chromatography and uv spectroscopy. Evidence is presented that soybean lipoxygenase-2, at variance with the type-1 enzyme, oxygenates the esterified unsaturated fatty acid moieties in biomembranes, whereas membrane-embedded free unsaturated fatty acid moieties were not a suitable substrate for either isoenzyme. The oxygenation products derived from the biomembranes were the 9- and 13-hydroperoxides of linoleic acid residues, in a molar ratio of 1.0 to 1.7, and the 9- and 13-hydroperoxides of alpha-linolenic acid residues, in a molar ratio of 1.0 to 0.1. The R/S ratios of 13-hydroperoxy-9Z,11E-octadecadienoic acid and 9-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid were found to be 0.5 and 25.0, respectively. These stereospecificity values were much higher than those of hydroperoxides isolated after incubation of lipoxygenase-2 with non-membraneous fatty acids or their methyl esters. The hydroperoxy fatty acids produced were distributed in neutral lipids and phospholipids isolated from soybean membranes, the former being oxidized to a larger extent. Furthermore, both intracellular and plasma membranes were substrates for the enzymic oxygenation, with a preference for those of chloroplasts followed by those of Golgi apparatus, endoplasmic reticulum, plasma membrane and mitochondria. These data point towards a different action of the two lipoxygenases in soybean cells. We suggest that the type-2 enzyme plays a role in the in vivo remodelling of biomembranes. The physiological relevance of these findings is discussed.

Thermal injury and ozone stress affect soybean lipoxygenases expression.

The effects of thermal injury (cold and heat shock) and ozone treatment on lipoxygenases 1 (LOX‐1) and 2 (LOX‐2) of soybean seedlings have been investigated. Cold stress led to a decrease of the specific activities of both isoenzymes, attributable at least in part to a down‐regulation of gene expression at the translational level. Both heat shock and ozone treatment enhanced lipoxygenases‐specific activities, acting at the level of transcription of the genes. It is proposed that LOX‐1 and LOX‐2 are involved in the thermotolerance of soybeans and in the precocious aging induced by ozone.

Modulation of soybean lipoxygenase expression and membrane oxidation by water deficit

The modulation of the activity and expression of soybean lipoxygenases 1 (LOX‐1) [Vliegenthart, J.F.G. and Veldink, G.A. (1982) in: Free Radicals in Biology (Pryor, W.A., Ed.) pp. 29–64, Academic Press, New York] and 2 (LOX‐2) by water deficit (osmotic stress) has been investigated, by following gene expression at the transcriptional and translational levels. Osmotic stress enhanced the transcription of the genes of both isoenzymes, leading to increased levels of the corresponding mRNAs, protein contents and specific activities. Abscisic acid (ABA) did not mediate enhancement of LOX expression, but caused a decrease of LOX‐2 activity and was ineffective on LOX‐1. Water deficit also caused oxidative modification of soybean membrane pool lipids [Schmidt, W.E. and Ebel, J. (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121], attributable to the increase of conjugated hydroperoxides in the esterified fatty acids of the lipid bilayer.

Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules

Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.

Soybean lipoxygenase‐1 is not a quinoprotein

Soybean lipoxygenase‐1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase‐1 contains pyrroloquinoline quinone as the organic cofactor [1]. Because spectroscopie data were found to be inconsistent [2] with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase‐1 is not a quinoprotein.

Phytochrome control and anoxia effect on the activity and expression of soybean seedling lipoxygenases 1 and 2

The effects of different light irradiations and anoxia treatment on the activity and expression of the lipoxygenases 1 (LOX‐1) and 2 (LOX‐2) of soybean seedlings have been investigated. A phytochrome‐mediated decrease of the specific activities of both isoenzymes was found which was due to the regulation of the gene expression at the transcriptional level. The anoxia treatment of the seedlings led to a decrease of both lipoxygenase specific activities as well, but this effect was attributable to the reduction of the amount of the two isoenzymes, without changes in the steady‐state levels of specific mRNAs. Therefore, light and anoxia are both regulators of soybean lipoxygenases, but their mechanism of action at the molecular level is different.