JXB Advance Access originally published online on July 2, 2004
Journal of Experimental Botany 2004 55(403):1655-1662; doi:10.1093/jxb/erh197
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RESEARCH PAPER |
Biochemical and immunochemical evidences for the presence of lipoxygenase in plant mitochondria
1Section of Plant Biology, Department of Biology and Agro-industrial Economics, University of Udine, via Cotonificio 108, I-33100 Udine, Italy
2Department of Chemical Science and Technology, University of Udine, via Cotonificio 108, I-33100 Udine, Italy
* To whom correspondence should be addressed. Fax: +39 0432 558784. E-mail: biolveg{at}dbea.uniud.it
Received 26 March 2004; Accepted 10 May 2004
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Abstract |
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In this paper, both biochemical and immunochemical evidence for the presence of lipoxygenase (LOX) in plant mitochondria is presented. Highly purified pea (Pisum sativum L., cv. Alaska) mitochondria show LOX activity, evaluated as conjugated diene formation, oxygen consumption, and hydroperoxide formation. Both 9- and 13-hydroperoxy-octadecadienoic acids are produced by the oxidation of linoleic acid. LOX activity is particularly evident in swollen mitochondria; it is inhibited by nordihydroguaiaretic acid, a pea anti-LOX B antibody, and has two pH optima (6.0 and 7.5). A mitochondrial protein of
97 kDa cross-reacts with a pea seed anti-LOX B antibody. This reaction is detectable in both soluble (matrix fraction) and membrane-bound (submitochondrial particles) proteins. Considering that pea mitochondria were extracted from actively growing stems that were differentiating tube elements, it is suggested that the presence of LOX in these organelles may be related to their degradation linked to xylem differentiation. Key words: Lipoxygenase, mitochondria, Pisum sativum L.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Lipoxygenases, LOXs (linoleate:oxygen oxidoreductase, EC. 1.13.11.12 [EC] ) constitute a large family of non-haem, iron-containing dioxygenases widely diffused in both plant and animal kingdoms (Brash, 1999
). They catalyse the addition of molecular oxygen to unsaturated fatty acids containing a (Z,Z)-1,4-pentadiene system to produce the corresponding fatty acid hydroperoxides. The major substrates of plant LOXs are linoleic and linolenic acids, while arachidonic acid is that preferred by animal LOXs. Oxygen can be added to either end of the pentadiene system of linoleic and linolenic acids, generating two possible products, the 9- and 13-hydroperoxy fatty acids. The latter are substrates for at least seven different enzyme families, whose products are a group of cyclic and acyclic compounds, collectively named oxylipins, which play different roles in plant cells (Feussner and Wasternack, 2002).
The major bulk of LOXs is localized in the cytoplasm and vacuole (storage proteins termed VSPs) of the plant cell (Porta and Rocha-Sosa, 2002). However, LOXs have also been found in chloroplasts (Boudnitskaya and Borisova, 1972; Schaffrath et al., 2000) and lipid bodies (Feussner and Kindl, 1992). In some cases, LOXs have been associated with plasma membrane (Droillard et al., 1993; Macrì et al., 1994; Nellen et al., 1995), chloroplast envelope (Blée and Joyard, 1996), thylakoid (Bowsher et al., 1992), and lipid body membrane (May et al., 2000).
The presence of LOXs in plant mitochondria is still controversial. In some cases, LOX has been associated with these organelles (Boudnitskaya and Borisova, 1972; Djebar and Moreau, 1990; Braidot et al., 1993), where it can also be induced by ethylene (Janes and Wiest, 1982). On the other hand, this activity has frequently been described as due to a contamination of mitochondria, which is particularly relevant during their extraction (Siedow and Girvin, 1980). With the aim of clarifying the latter aspect, in this work LOX activity was checked in highly purified pea stem mitochondria.
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Materials and methods |
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Plant material
Pea (Pisum sativum L., var. Alaska) seedlings were grown on sand for periods of 5–10 d, in darkness at 25 °C. Stems were cut into small pieces and then used for the isolation of mitochondria.
Mitochondria isolation and purification
Pea stem mitochondria were isolated as previously described (Vianello et al., 1997), except for the addition of 1 mM dithioerythritol and 10 mM histidine in the homogenizing buffer. The pellet (crude mitochondrial fraction) was suspended in 2 ml of resuspending medium containing 20 mM MOPS–KOH (pH 7.2), 0.3 M mannitol, 1 mM EDTA, 10 mM histidine, and 0.1% (w/v) fatty acid-free BSA. Crude mitochondria were then purified on Percoll gradients and diluted in 1 ml of resuspending buffer (20 mM HEPES–TRIS, pH 7.5, 0.4 M sucrose, and 10 mM histidine). The suspension, containing 0.5–1.5 mg protein ml–1, depending on the age of seedlings, was stored on ice for LOX assays or frozen for western blotting.
Submitochondrial particle (SMP) preparation
Purified mitochondria were resuspended in 2 ml of sonication buffer, containing 20 mM HEPES–TRIS (pH 7.5), 0.25 M sucrose, and 10 mM histidine, and subjected to one sonication for 30 s at 100 W on ice in a Labsonic 1510 water bath sonifier (Laboratory Supplies Co., New York, USA). The samples were centrifuged at 28 000 g for 10 min to remove unbroken mitochondria, and the resulting supernatant was centrifuged for 1 h at 100 000 g by a Beckman ultracentrifuge (70ti rotor). The supernatant (soluble proteins, SP) and the final pellet (SMP), resuspended in 0.5 ml of the above-mentioned resuspending buffer, were further utilized for LOX assays or alkaline and salt treatments.
Alkaline carbonate and salt treatments
SMPs were subjected to both alkaline and salt treatments in order to separate contaminating soluble or peripheral proteins (entrapped inside the vesicles or unspecifically absorbed) (Millar et al., 2001; Bérczi and Møller, 2000). Three aliquots of submitochondrial suspension were diluted (1:350) in 20 mM HEPES–TRIS (pH 7.5) and treated with 0.35 M KCl or 0.5 M Na2CO3 (pH 10). They were then subjected to three cycles of freeze and thaw and, after ultracentrifugation for 1 h at 100 000 g, each pellet was analysed by western blotting.
Marker enzyme activities and chlorophyll content determination
Vanadate-sensitive (plasma membrane marker enzyme), bafilomycin A1-sensitive (tonoplast marker enzyme), and oligomycin-sensitive (mitochondrial marker enzyme) ATPase activities were assayed as previously described (Macrì et al., 1994). Glucose-6-P dehydrogenase (cytoplasmic and plastidial marker enzyme) was detected as described by Green (1983). Cytochrome c reductase (endoplasmic reticulum marker enzyme) and latent IDPase (Golgi marker enzyme) were evaluated following the methods of Lord (1983) and Bergmeyer et al. (1974), respectively. Chlorophyll content was determined according to the method of Leegood and Walker (1983).
Transmission electron microscopy analysis (TEM)
TEM analysis was performed as previously described in Petrussa et al. (2001).
LOX assays
LOX activity was measured as conjugated diene formation or oxygen consumption, as previously described (Fornaroli et al., 1999). The incubation mixture was 20 mM TRIS–HCl (pH 7.5) and 50–100 µg of mitochondrial protein ml–1, as indicated. The reaction was started with the addition of 250 µM of linoleic or linolenic acid. The following incubation buffers were used to measure LOX activity as a function of pH: 0.1 M sodium acetate for pH 5.0–5.5; 0.1 M MES–KOH for pH 6.0–7.5; 0.1 M EPPS–KOH for pH 7.5–8.5; 0.1 M borate–KOH for pH 9.0–9.5.
An extinction coefficient (
m) of 25 000 M–1 cm–1 was used to estimate the amount of conjugated dienes.
Straight phase–high pressure liquid chromatography (SP–HPLC) analysis of LOX products
Hydroperoxy-octadecadienoic acids (HPOD), deriving from LOX activity, were extracted as previously described in Fornaroli et al. (1999). After dehydration, reaction products were redissolved in 2 ml HPLC mobile phase and identified by SP–HPLC on a Luna Silica column (5 µm, 4.6x250 mm, Phenomenex, CA, USA), performing an isocratic elution with hexane/2-propanol/acetic acid, (97/3/0.1, by vol.) at a flow rate of 1 ml min–1. Conjugated dienes were measured at 234 nm by the means of an UV–VIS (UV–visible) detector. The amount of HPOD was determined by integration of the chromatogram peaks, using the Star chromatography workstation version 5.5. The standards used were 9-E,Z HPOD and 13-Z,E HPOD, purchased by Cayman Chemicals (MI, USA).
Outer mitochondrial membrane intactness
Outer mitochondrial membrane integrity was determined as described in Chiandussi et al. (2002).
Western blot analysis
Proteins from purified mitochondria, SMP, or supernatants (soluble fraction) were separated by SDS–PAGE, electroblotted onto nitrocellulose membrane (Bio-Rad, CA, USA) and probed against pea anti-LOX B (dilution 1:2000) as primary antibody. Pea anti-LOX B was a generous gift of Dr C Domoney, John Innes Institute, UK.
Computer-assisted densitometric analysis of the western blot was performed by means of Quantity One Software (Bio-Rad, CA, USA, 4.2.3 release). Stained gel and western blotting membrane images were taken using a scanner.
Protein determination
The protein content was measured by the Bradford method (Bradford, 1976) with the Bio-Rad protein assay, using crystalline bovine serum albumin (BSA) as a standard.
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Results |
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The activity of some marker enzymes was detected in purified pea stem mitochondria to evaluate their purity, in comparison with a crude preparation (Table 1). Already crude mitochondria showed low levels of both vanadate- (plasma membrane marker enzyme) and bafilomycin A1-sensitive (tonoplast marker enzyme) ATPase activity. Nevertheless, their levels decreased in purified mitochondria to negligible values. The ATPase activity was only 50% inhibited by oligomycin (mitochondrial marker enzyme) in crude mitochondria, but this inhibitory effect strongly increased in the purified ones. The activity of the antimycin A-insensitive cytochrome c reductase (endoplasmic reticulum marker enzyme) was high in crude mitochondria and was only slightly lowered by purification. However, this activity was lower than that recovered in microsomes and could in part, be, attributed to a cytochrome c reductase localized on the outer mitochondrial membranes (Møller and Lin, 1986
). Latent IDPase (Golgi apparatus marker enzyme) was high in crude mitochondria, but strongly decreased after purification. As the starting material was constituted by etiolated pea stems, the chlorophyll content was not detectable. In addition, glucose-6-P dehydrogenase (cytoplasmic and plastidial stroma marker enzyme) was negligible, particularly in the purified mitochondria, when its activity was compared with that of etioplasts. In agreement, as previously shown (Petrussa et al., 2001), transmission electron micrographs of the purified mitochondria revealed no contamination by etioplasts (Fig. 1). Therefore, the mitochondria utilized in this work were substantially devoid of contamination and, in particular, from plastids.
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The purified mitochondria, isolated from 5-d-old pea stems and resuspended in 0.1 M MES–KOH (pH 7.5), showed an unusual kinetics of linoleic acid-dependent conjugated diene formation (Fig. 2). The addition of this fatty acid (trace a) induced an initial decrease of absorbance at 234 nm, which was followed, after 1 min, by a limited but progressive increase of absorbance. When the mitochondria were resuspended in 20 mM TRIS–HCl (pH 7.5), linoleic acid induced, after a lag phase, a typical increase of absorbance that could be related to conjugated diene formation (trace b). This different behaviour was associated with the state of mitochondria (Table 2), because the organelles resuspended in 0.1 M MES–KOH were in the orthodox state, as demonstrated by the high degree of integrity of the outer membrane (85.9%). On the contrary, mitochondria resuspended in 20 mM TRIS–HCl were swollen and had the outer membrane completely broken. These results indicate, therefore, that LOX activity became completely evident in purified mitochondria only when the organelles had the outer membranes disrupted, and thus the added fatty acids could more easily reach the inner mitochondrial membrane.
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This contention was confirmed by the results reported in Fig. 3, using purified mitochondria isolated from 10-day-old pea stems. Fig. 3A shows a typical linoleic acid-dependent conjugated diene formation in mitochondria resuspended in 20 mM TRIS–HCl (trace a). After a lag phase, which can be abolished by hydrogen peroxide addition (not shown), the diene formation increased linearly. This activity was unaffected by 1 mM KCN (trace b) and completely inhibited by nordihydroguaiaretic acid (NDGA) (trace c), a typical LOX inhibitor, or partially inhibited by pre-incubation (
20 min) of mitochondria with a pea anti-LOX B antibody (dilution 1:2000) (trace d). Inset shows the double-reciprocal plot of diene formation versus linoleic acid concentration, which permits calculation of an apparent affinity constant (Km) of 250 µM for this fatty acid. This diene formation was paralleled by an oxygen consumption (Fig. 3B, trace a), which was again uninhibited by KCN (trace b) and inhibited by NDGA (trace c). The ratio between diene formed and O2 consumed was 1.3, a value close to the theoretical one.
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The products of the linoleic acid-dependent LOX activity (specific activity, 71 nmol HPOD · (mg protein · min)–1) were separated by a SP–HPLC (Fig. 3C). The chromatogram reveals nine peaks, which coincide with those previously identified as hydroperoxidation products of linoleic acid by pea seed LOX (Wu et al., 1995
). The first (peak 1) is an 
keto-octadecadienoic acid (KOD). Peaks 2 and 6 correspond to the two isomers of 13-hydroxyoctadecadienoic acid (13-Z,E HOD and 13-E,E HOD), whereas peaks 8 and 9 coincide with 9-E,Z HOD and 9-E,E HOD, respectively. The complete set of 9- and 13-HPOD was also detected. Peaks 3 and 4 are 13-Z,E HPOD and 13-E,E HPOD; peaks 5 and 7 correspond to 9-E,Z HPOD and 9-E,E HPOD, respectively. This identification was confirmed by two standards, namely 9-E,Z HPOD and 13-Z,E HPOD, whose elution time is perfectly coincident with the two above-described peaks (result not shown). The calculated HPOD relative concentrations, out of three different chromatograms, show that the 13-Z,E HPOD is the highest component (60%), while 13-E,E HPOD represents only 14%. Instead, 20% and 6% of the total HPOD produced can be ascribed to 9-E,Z HPOD and 9-E,E HPOD, respectively. This LOX activity was dependent on pH of the incubation medium (Fig. 4). The activity could be resolved into two peaks, indicating the presence of two isoforms with optimums at pH 6.0 and pH 7.5, respectively. At higher pH values (9.0–9.5), the activity strongly decreased. LOX activity at pH 7.5 was stimulated by calcium in the 0.1–0.5 mM range, while at 1 mM it was completely inhibited (result not shown).
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The presence of LOX protein in purified pea stem mitochondria was also confirmed by a pea anti-LOX B antibody. Figure 5 shows the SDS–PAGE of mitochondrial proteins (lane 1) and the relative western blot (lane 2). The pea anti-LOX B antibody cross-reacted with a protein of 97 kDa, whose presence appears to be related to the developmental stage of pea seedlings (Fig. 6). The cross-reactivity, after SDS–PAGE of mitochondrial proteins (Fig. 6a), increased in organelles from 10-d-old stems when compared with those extracted from 5-d-old seedlings (Fig. 6b). This increase was paralleled by a stimulation of linoleic or linolenic acid-dependent diene formation (Fig. 6c) and was also confirmed by the densitometric analysis (
30% increase) of the immunoblotted proteins (Fig. 6d).
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To determine the mitochondrial localization of LOX, the organelles were subjected to sonication. The broken membranes (SMP) were separated from the supernatant (SP) by ultracentrifugation. The proteins of the two samples were analysed by SDS–PAGE (Fig. 7a) and then by western blotting (Fig. 7b). A cross-reaction with the anti-LOX B antibody was again found with the protein of 97 kDa in both preparations (Fig. 7b). This suggests that LOX may be present in either the matrix or the mitochondrial membranes. The linkage of LOX with the latter membranes appears to be strict (Fig. 8). After SDS–PAGE (Fig. 8a), cross-reactivity with a protein with 97 kDa was again found in submitochondrial particles, obtained from 5- or 10-d-old seedlings and washed with KCl or Na2CO3 (Fig. 8b). However, the latter treatments tended to reduce the level of this cross-reactivity.
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Discussion |
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The results indicate that at least two isoforms of LOXs can be described in mitochondria isolated from etiolated pea stems. The enzyme appears to be present in either the matrix or the inner membrane, because disruption by swelling of the outer membrane favours the detection of LOX activity. Therefore, it is possible to affirm that plant cells contain both soluble and membrane-bound LOXs, not only in chloroplasts (Boudnitskaya and Borisova, 1972
; Schaffrath et al., 2000) and lipid bodies (Feussner and Kindl, 1992), but also in mitochondria.
The mitochondrial LOX may be distinguished from the chloroplastic counterpart, because the reaction products of the first are both 9- and 13-HPOD isomers, whereas the chloroplastic LOX predominantly produces 13-HPOD (Peng et al., 1994; Royo et al., 1996). In agreement, the latter enzyme, expressed in Escherichia coli, catalysed the formation of 13-HPOD (Schaffrath et al., 2000). In the experiments in this study, the resulting ratio between 13- and 9-HPOD is about 3; this value is clearly significantly different from 20, which is that found with chloroplastic LOX (Peng et al., 1994; Royo et al., 1996).
To be targeted to mitochondria, LOXs, like other proteins, would have an N-terminal presequence able to recognize the organelle, which is removed after transfer (Emanuelsson and von Heijne, 2001). This targeting peptide has to be enriched in positively, but not in negatively, charged residues (arginine in particular) and have the ability to form amphiphilic -helices which are important for binding to receptors in the outer mitochondrial membrane. The net positive charge appears to be requested for the transmembrane electrical potential-driven import across the inner membrane. In this framework, LOXs could have a similar presequence. On the other hand, a similar targeting has been described in lipid bodies (Hause et al., 2000) and probably occurs in chloroplasts (Schaffrath et al., 2000).
Once LOXs have entered the mitochondrion, they are distributed in the matrix where they are stored. LOXs may then link to the inner membrane. In this way they may better utilize unsaturated free fatty acids (released from phospholipids or still esterified), because the latter cannot easily abandon the hydrophobic environment of the membrane (Brash, 1999). In this context, soybean LOX 1, a model for LOXs of different sources, is a protein composed of two domains, a 693-residue helical bundle and a 146-residue ß-barrel at the N-terminal, which could mediate the binding to membranes by the N-terminal ß-barrel (May et al., 2000; Walther et al., 2002). On the other hand, as shown in this work, LOXs are also strictly associated with the inner mitochondrial membrane.
The physiological significance of the presence of LOXs in plant mitochondria may be, at this stage, only a matter of speculation. In mammalian cells, at least, the presence of LOX in mitochondria has been associated with their degradation during reticulocyte differentiation (Koury et al., 2002). In this framework it must be considered that pea mitochondria have been isolated from actively growing and differentiating stems. In particular, xylem tracheary element formation is accomplished by a cell death involving mitochondria (Yu et al., 2002). Therefore, the presence of LOXs in mitochondria may be tentatively related to the activation of a cell death programme (van Leyen et al., 1998), which seems to accompany this differentiation and leads to organelle degradation. In agreement with this hypothesis, there is evidence, obtained by immunofluorescence, showing that LOXs migrate from the cortex and pith of soybean hypocotyls to the vascular cylinder as germination proceeds (Vernooy-Gerritsen et al., 1983). In addition, LOX 1 class transcripts accumulate in the apical and subapical regions of the newly formed potato tuber, specifically in the vascular tissue of the perimedullary region (Kolomiets et al., 2001).
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Acknowledgements |
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This research was supported by the ‘Ministero dell'Istruzione, dell'Università e della Ricerca’ (Cofin 1999–2000) in the section of the programme entitled: ‘Manipulation of redox signalling systems in plants to engineer resistance to pathogens’ and by the University of Udine. Purified antibody raised against pea LOX B was a generous gift by Dr Claire Domoney, Dept of Metabolic Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK. We also thank Dr Tiziana Populin, University of Udine, for her helpful collaboration during SP–HPLC analysis.
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Footnotes |
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Abbreviations: HOD, hydroxy-octadecadienoic acid; HPOD, hydroperoxy-octadecadienoic acid; KOD,
keto-octadecadienoic acid; LOX(s), lipoxygenase(s); NDGA, nordihydroguaiaretic acid; SP, soluble proteins; SP–HPLC, straight phase–high pressure liquid chromatography; SMP, submitochondrial particles; TEM, transmission electron microscopy. |
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