Enzymatic and non-enzymatic lipid peroxidation in leaf development Susanne Berger a , Heiko Weichert b , Andrea Porzel a , Claus Wasternack a , Hartmut Ku «hn c , Ivo Feussner b; * a Leibniz-Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany b Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany c Institut fu «r Biochemie, Universita «tsklinikum Charite ¨, Humboldt Universita «t Berlin, Hessische Strasse 3^4, D-10115 Berlin, Germany Received 5 April 2001; received in revised form 23 July 2001; accepted 25 July 2001 Abstract Enzymatic and non-enzymatic lipid peroxidation has been implicated in programmed cell death, which is a major process of leaf senescence. To test this hypothesis we developed a high-performance liquid chromatography (HPLC) method for a simultaneous analysis of the major hydro(pero)xy polyenoic fatty acids. Quantities of lipid peroxidation products in leaves of different stages of development including natural senescence indicated a strong increase in the level of oxygenated polyenoic fatty acids (PUFAs) during the late stages of leaf senescence. Comprehensive structural elucidation of the oxygenation products by means of HPLC, gas chromatography/mass spectrometry and 1 H nuclear magnetic resonance suggested a non- enzymatic origin. However, in some cases a small share of specifically oxidized PUFAs was identified suggesting involvement of lipid peroxidizing enzymes. To inspect the possible role of enzymatic lipid peroxidation in leaf senescence, we analyzed the abundance of lipoxygenases (LOXs) in rosette leaves of Arabidopsis. LOXs and their product (9Z,11E,13S,15Z)-13- hydroperoxy-9,11,15-octadecatrienoic acid were exclusively detected in young green leaves. In contrast, in senescing leaves the specific LOX products were overlaid by large amounts of stereo-random lipid peroxidation products originating from non-enzymatic oxidation. These data indicate a limited contribution of LOXs to total lipid peroxidation, and a dominant role of non-enzymatic lipid peroxidation in late stages of leaf development. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Hydroperoxy polyenoic fatty acid ; Lipoxygenase ; 1 H Nuclear magnetic resonance ; Lipid peroxidation ; Senescence 1. Introduction Senescence, which is speci¢cally induced in di¡er- ent plant organs during the annual life cycle, is one of the most complex processes in plants [1]. In leaves it is characterized by a programmed degradation of constituents such as chlorophyll, proteins, nucleic acids and lipids. In the last years an increasing num- ber of senescence-related genes have been identi¢ed [2,3]. However, many of them are also activated in response to stress or pathogen attack and only few of them (e.g. cysteine protease, SAG-12) appear to be senescence-speci¢c [4]. A variety of metabolic reac- tions are altered during leaf senescence [5], but the 1388-1981 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S1388-1981(01)00161-5 Abbreviations : HOD, hydroxy octadecadienoic acid ; HOT, hydroxy octadecatrienoic acid; HPOD, hydroperoxy octadecadi- enoic acid ; HPOT, hydroperoxy octadecatrienoic acid ; HO(D/ T)E/PUFA ratio, hydroxy polyenoic fatty acid/polyenoic fatty acid ratio; JA, jasmonic acid; LA, linoleic acid; LeA, linolenic acid; LOX, lipoxygenase; PUFA, polyenoic fatty acid * Corresponding author. Fax : +49-39-482-5548. E-mail address : feussner@ipk-gatersleben.de (I. Feussner). Biochimica et Biophysica Acta 1533 (2001) 266^276 www.bba-direct.com