PHYSIOLOGIA PLANTARUM 111: 165 – 171. 2001 Copyright © Physiologia Plantarum 2001 ISSN 0031-9317 Printed in Ireland —all rights resered Effects of moderately enhanced levels of ozone on the acyl lipid composition and dynamical properties of plasma membranes isolated from garden pea (Pisum satium ) Lars I. Hellgren 1, , Gun Sellde ´n and Anna Stina Sandelius Department of Plant Physiology, Botanical Institute, Go ¨teborg Uniersity, P.O. Box 461, SE-405 30 Go ¨teborg, Sweden 1 Present address: Dept. Biochemistry and Nutrition, Technical Uniersity of Denmark, Building 224, Søltoft Plads, DK-2800 Lyngby, Denmark *Corresponding author, e -mail: hellgren@mimer.be.dtu.dk Received 15 May 2000; revised 25 August 2000 percentage of campesterol increased, concomitant with a de- Plasma membranes were isolated from leaves of 16-day-old garden pea, Pisum satium L., that had been grown in the crease in stigmasterol. The dynamical properties of the iso- lated plasma membranes were assessed using Laurdan absence or presence of 65 nl l -1 ozone for 4 days prior to membrane isolation. Plasma membranes from ozone-fumi- fluorescence spectroscopy, which monitors water penetration and mobility at the hydrophilic-hydrophobic interface of the gated plants contained significantly more acyl lipids per protein than those from leaves of plants grown in filtered air membrane. At 0°C, the molecular mobility was slightly lower in plasma membranes from ozone-fumigated plants than in on a molar/weight ratio. The ratio between the major acyl lipids, phosphatidylethanolamine (PE) and phosphatidyl- plasma membranes from control plants, possibly reflecting the increased PE/PC, campesterol/stigmasterol and lipid/protein choline (PC), also increased due to the ozone fumigation, ratios, and suggesting that ozone-fumigated pea plants may be while the fatty acid unsaturation level was unaltered in total plasma membrane acyl lipids, as well as in PC and PE. The more susceptible to freezing injuries. amount of free sterols per protein was unaltered, but the The plasma membrane will be the first symplastic compo- nent to be affected by ozone (Heath 1986). It has been shown that plasma membrane vesicles isolated from ozone- exposed plants have a reduced Ca 2 + transport capacity but increased passive Ca 2 + flux over the membrane (Castillo and Heath 1990). Because ozone readily oxidizes fatty acid double bonds (Mudd et al. 1971), alterations in plasma membrane acyl lipids could be an early event in the effects of ozone exposure. When garden pea plants were fumigated with 65 nl l -1 ozone, the amounts per leaf area of phos- phatidylethanolamine (PE) and phosphatidylcholine (PC) increased, while the level of fatty acid unsaturation de- creased in these lipids (Carlsson et al. 1994, 1996). It was Introduction The phytotoxicity of ozone has been known for more than 4 decades (Richards et al. 1958), but the intracellular mech- anisms behind the observed injuries are not yet fully under- stood. Because ozone is a highly reactive compound, it most likely reacts with plant components already at the interface between the leaf and the environment, that is the cuticle or, since ozone enters plant leaves through the stomata, the cell walls of the leaf mesophyll. It has been shown that when ozone is passed over a buffer containing components of the apoplastic fluid, namely 0.1 mM of either caffeic acid, phenol, or cinnamic acid, hydroxyl radicals are formed (Grimes et al. 1983). Thus, if the antioxidant defense in the apoplast is insufficient, hydroxyl radicals and ozone will react with components of the symplast. Abbreiations – 16:0, hexadecanoic acid; 18:0, octadecanoic acid; 18:1, cis -9-octadecenoic acid; 18:2, all-cis -9,12-octadecadienoic acid; 18:3, all-cis -9,12,15-octadecatrienoic acid; excGP, excitation generalized polarization; Laurdan, 6-lauroyl-2-dimethylaminonaphtalene; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PC aldehyde, 1-hexadecanoyl-2-(9-oxononanoyl)-sn -glycerol-3-phosphocholine; PC- Criegee ozonide, 1-hexadecanoyl-2[8-5(octyl-1,2,4,-trioxolan-3-yl)-octanoyl]-sn -glycerol-3-phosphocholine; PE, phosphatidylethanolamine. Physiol. Plant. 111, 2001 165