Fatty Acid Oxidation by Lipoxygenase and ß-Oxidation in Germinatíng Barley
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Fatty acid oxidation by lipoxygenase and ß-oxidation in germinatÍng barley Wessel L. Holtman ISBN: 90-802521-7-4 Cover: in collaboration with Marit Boot and peter Hock rH? l---..l Printed by Offsetdrukkerij Ridderprint 8.V., Ridderkerk Fatty acid oxidation by lipoxygenase and ß-oxidation in germinatÍng barley Proefschrift ter verkrijging van de graad van Doctor aan de Rijksuniversiteit te Leiden, op gezag van de Rector Magnificus Dr. W.A. Wagenaar, hoogleraar in de faculteit der Sociale Wetenschappen, volgens het besluit van het College van Dekanen te verdedigen op donderdag26 juni 1997 te klokke 16.15 uur. door Wessel Luitje Holtman geboren te Ten Boer in 1962 Promotiecommissie Promotor Prof. dr, J.W. Kijne Co-promotor dr. A.C. Douma Referenten Prof. dr. G.A. Veldink (Universiteit Utrecht) Prof. dr. D. von Wettstein (V/ashington State University ) Overige leden Prof. dr. J.F. Bol Prof. dr. K.R. Libbenga Prof. dr. P.J. Lea (Lancaster University) Published by : Foundation for Single Cell Research, Leiden, The Netherlands Financial support for the publication of this thesis by the TNO Nutrition and Food Research lnstitute is greatfully acknowledged voor G.O. I. N. CONTENTS Chapter 1 Introduction Chapter 2 ß-Oxidation of fatty acids is linked to the glyoxylate cycle in the aleurone but not in ttre embryo of germinating barley Chapter 3 Monoclonal antibodies for differential recognition of catalase subunits in barley aleurone cells Chapter 4 The ratio of peroxidative to cat¿latic activity of catalase in leaves of barley seedlings is linked to the expression levels of two catalase subunits Chapter 5 Differential expression of lipoxygenase isoenzymes in embryos of germinating barley Chapter 6 Lipoxygenase-2 oxygenates storage lipids in embryos of germinating barley Chapter 7 General and summariztng discussion Samenvatting List of publications Curriculum vitae Chapter 1 Introduction INTRODUCTION Plant seeds store food reserves prior to a period of dormancy or of unfavourable envi¡onmental conditions. These reserves are used for germination and postgermi- native growth of the seedling. Principally, the three classes of storage compounds are fatloils, carbohydrates and proteins, of which fatloils represent the most efficient way of energy storage (Huang, 1992). Fatloils are present mainly in the form of triacylglycerols (TAG), which are partitioned in small discrete intracellular organelles, called oil bodies (Tzen and Huang, 1992). Oil bodies, also called lipid bodies or spherosomes consist of TAGs, surrounded by a monolayer of phospholipids and oleosines (Tzen and Huang, 1992; Hills, Watson and Murphy, 1993). Following germination TAGs are mobilized, whereby oxidation of the fatty acids may occur by at least 4 different oxidative pathways, namely cr-oxidation, ß-oxida- tion, <,r-oxidation and lipoxygenase. a-Oxidation results in the ¡emoval of one carbon unit from the carboxyl terminus of the fatty acid chain and appears to be rest¡icted to long-chain fatty acids. Chain shortening by e-oxidation will not proceed beyond C,, in chain lengttr (Gerhardt, 1992). a-oxidation results in oxidation at the methyl-end of the fatty acid molecule, and fulfils a biosynthetic rather than catabolic function. ß-oxidation results in the removal of two carbon units at the carboxyl terminus of the fatty acid, and is the sole degradation process which may lead to complete degradation of fatty acids. The lipoxygenase pathway starts with the incorporation of oxygen in cis-double unsaturated fatty acids, a reaction catalyzed by the enzyme lipoxygenase. The primary reaction products, fatty acid hydroperoxides, may be metabolized, yielding products with significant physiological importance. This thesis will deal with fatty acid oxidation in germinating barley by ß- oxidation and lipoxygenase. In this chapter, a condensed overview of both pathways will be presented. ß.OXIDATION Localization and phy siolo gical function It is generally believed that in higher plant cells, ß-oxidation occurs solely within specific organelles, called microbodies (Kindr, l9B7), although a few reports suggest a dual location in both microbodies and mitochondria (wood et al., 19g6; Masterson et al., 1990; Masterson et al., 1992; Dieuaide et at., 1993). Microbodies exhibit a diameter from 0.2-l .7 pm, contain a coarsely granular or fibrillar matrix and have a single membrane. since, like in animal cells, microbodies in plants contain the enzyme catalase and at least one Hror-producing oxidase, they may be designated peroxisomes (Huang et at, L9g3). on basis of different functions plant microbodies can be subcategorized into glyoxysomes, leaf peroxisomes, and other unspecialized peroxisomes (Huang et aI.,l9g3). Fatty acid ß-oxidation has been characterized to be a basic function of each microbody class. Highest activities of ß-oxidation enzymes have been detected in lipid-rich tissues (Gerhardt, 1986). glyoxysomes, In ß-oxidation is coupled to the glyoxylate cycle, resulting in the formation of sugars (cooper and Beevers, 1969; Gerhardt, 19g6; Kindl, l9g7). These organelles are commonly found in lipid-rich tissues of seeds, and play a major role in the mobilization of fat reserves. During the greening of germinating oil-seed cotyledons, a functional transition occurs, whereby glyoxysomes are transformed directly into leaf peroxisomes (Titus and Becker, l9g5; Nishimura el al., 1986). Leaf peroxisomes, present in green leaves and cotyledons, contain enzymes of ttre glycolate cycle and participate in the photorespiration process. They show ß-oxidation activity (Gerhardt 1986), but lack an active glyoxylate cycle (Kindl, 1987). However, in dark-induced senescing or naturally senescing leaves (Pistelli et aL,,1991) or detached darkened leaves and cotyledons (De Bellis et al., 1990), glyoxylate cycle enzymes could be detected together with ß-oxidation enzymes, which is consistent with a peroxisome-glyoxysome transition. Finally, ß- oxidation activity has been detected in unspecialized peroxisomes (Macey arid Snrmpf, 1982 Gerhardr, 1983; pistelli et al., l9g9) which are found in achlorophyllous and non-oil storage tissues. These organelles contain peroxisomal enzymes such as catalase, glycolate oxidase and urate oxidase. In conclusion, in fatty tissues the function of ß-oxidation is well understood, namely mobilization of fat reserves enabling sugar formation, which benefits the growth of the germinating embryo. However, the function of ß-oxidation in non- fatty tissues is not yet clear. [n these tissues, where ß-oxidation is located in non- specialized'peroxisomes and leaf peroxisomes, ß-oxidation may be used for generation of energy (Kindl, 1987) or may be linked to the turnover of membrane lipids or proteins (Gerhardt, 1986). Also, a ¡ole in growth of coleoptiles has been suggested (Pistelli et al., 1996), while an involvement in met¿bolism in senescent tissues (Wanner et al., 1991; Pistelli et al, 1995) suggests a general role of peroxisomes in the metabolism of membrane components (Pistellli et al., 1996). In studies in which both a mitochondrial and microbody location of ß-oxidation have been described, ß-oxidation in microbodies was suggested to function for gluconeogenesis and energy production, while at ttre mitochondrial site it may function to shorten fatty acids to mid-chain length (Masterson et al, 1992). This is in fact contrary to the situation in mammalian cells, where mitochondrial ß- oxidation proceeds to completion, whilst microbody ß-oxidation serves to shorten fatty acids, which are then transported (Bieber 1988) Alternatively, plant mitochondrial ß-oxidation may function during glucose starvation, a situation which results in a shift from carbohydrate metabolism to fatty acid and protein utilization (Dieuaide et al., 1992). ln this case, mitochondrial ß-oxidation may function as a spillover for the peroxisomes with the increased availability of substrates (Dieuaide et aL.,1993). Enzymes Peroxisomal ß-oxidation The biochemistry of peroxisomal ß-oxidation in plants has been studied extensively (reviewed by Kindl, 1987; Gerhardt 1992). Fatty acid degradation starts with the release of a fatty acid from a structural lipid or a triglyceride (Kindl, 1987), stored in lipid bodies. This reaction is catalyzed by ttre enzyme lipase. ß- Oxidation of fatty acids derived from storage lipids principally occurs in glyoxysomes. ß-Oxidation encompasses activation of a fatty acid, followed by l0 oxidative steps and removal of acetyl-CoA, together shortening the carbon chain by a C-2 moiety (Figure l). HSCoA ATP I l7-FAO1 ,H2O2 Acvl-CoA oxidase l' Y lt roo*rAo, ,-."=tr-L""o ( l.-uzo B¡funoionat I q"{ enoyt-CoAhydrat"." 3 and JJ nJH_CH2_€_SCoA r--s-nffiy..yr- ') l7urc* CoA dehydrogenase | | t þ*oo, ? R-€-{H2-æCoAIt lr-nscoe Thiolase I *&".o^r'\-c'ylscon Figure 1. Peroxisomal ß-oxidation pathway in plants (adapted from Gerhardr, 1986) l1 The frst step in the ß-oxidation pathway is energy (that is ATP) dependent and is catalyzed by the enzyme acyl-CoA synthetase. In this step, a CoA-moiety is linked to the fatty acid by the enzyme, resulting in an acyl-CoA ester. Substrate specificity studies showed that short-chain fatty acids (<n:8) are poor substrates for the erzyme (Gerbling and Gerhardt, 1987). While other ß-oxidation enzymes in higher plant peroxisomes are soluble matrix proteins, or are loosely associated with the organelle membrane (Frevert and Kindl, 1980; Gerbling and Gerhardt, 1987; Miernyk and Trelease, 1981), the acyl-CoA synthetase is tightly associated with ttre peroxisome