Abstract
20 min readA cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with aK 0.5 of 30 μm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria. A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with aK 0.5 of 30 μm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria. reactive oxygen species alternative oxidase glycine decarboxylase complex 4-hydroxy-2-nonenal malondialdehyde 2-oxoglutarate dehydrogenase complex pyruvate dehydrogenase complex thiobarbituric acid-reactive substances N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid The polyunsaturated fatty acids of membrane lipids are susceptible to reactive oxygen species (ROS)1-induced peroxidation yielding various aldehydes, alkenals, and hydroxyalkenals, including the cytotoxic compounds malonaldehyde (MDA) and 4-hydroxy-2-nonenal (HNE). These two end products are commonly measured in studies of lipid peroxidation by the thiobarbituric acid-reactive substances (TBARS) assay (1Hodges D.M. DeLong J.M. Forney C.F. Prange R.K. Planta. 1999; 207: 604-611Crossref Scopus (3046) Google Scholar). Interest in the biological effect of HNE was stimulated by studies showing cytotoxicity due to rapid reaction with sulfydryl groups via Michael addition (2Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (6027) Google Scholar). A number of stress conditions, including cardiac reperfusion injury, increase ROS production and the rate of membrane peroxidation and ultimately lead to inhibition of the respiratory rate in mammals (3Lucas D.T. Szweda L.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 510-514Crossref PubMed Scopus (236) Google Scholar). HNE has been shown to directly inhibit respiration of isolated mammalian mitochondria through modification of the lipoic acid moieties of 2-oxo acid dehydrogenases, forming HNE-Michael adducts (Fig. 1) (4Humphries K.M. Yoo Y. Szweda L.I. Biochemistry. 1998; 37: 552-557Crossref PubMed Scopus (200) Google Scholar). These targets have also been shown to be damaged in vivo under conditions that induce lipid peroxidation (5Lucas D.T. Szweda L.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6689-6693Crossref PubMed Scopus (130) Google Scholar). A range of biotic and abiotic stresses also raise ROS levels in plants due to perturbations of chloroplastic and mitochondrial metabolism and the generation of ROS in defense responses (6Van Camp W. Van Montagu M. Inze D. Trends Plant Sci. 1998; 3: 330-334Abstract Full Text Full Text PDF Scopus (232) Google Scholar). Such accumulation of ROS in plants will clearly result in a wide variety of deleterious effects through oxidation reactions involving proteins, lipids, and nucleic acids. Lipid peroxidation in plants has been known for many years, but direct evidence of the pathways involved and the identification of HNE production in plants has only been reported recently (7Takamura H. Gardner H.W. Biochim. Biophys. Acta. 1996; 1303: 83-91Crossref PubMed Scopus (44) Google Scholar). However, the exact molecular targets, the relative sensitivity of target enzymes, the mechanisms of repair, and the impact on plant function have not been extensively investigated. Research on the impact of plant lipid peroxidation on metabolism has centered on investigations of sensitive reactions in the chloroplast and the antioxidant systems present in this organelle (8Mano J. Ohno C. Domae Y. Asada K. Biochim. Biophys. Acta. 2001; 1504: 275-287Crossref PubMed Scopus (125) Google Scholar, 9Kraus T.E. McKersie B.D. Fletcher R.A. J. Plant Physiol. 1995; 145: 570-576Crossref Scopus (170) Google Scholar). Our understanding of the impact of lipid peroxidation on plant mitochondrial functions is still very limited. Recently we have shown that HNE specifically inhibits both pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes through modification of lipoic acid moieties (Fig. 1) in mitochondria from potato tubers. However, in photosynthetic plant tissues, these tricarboxylic acid cycle enzymes are dwarfed by the presence of the glycine decarboxylase complex (GDC). This enzyme also contains a lipoic acid moiety, can account for up to 50% of matrix protein, and is responsible for the most prominent metabolic activity in the mitochondria of illuminated leaves, photorespiration (10Douce R. Bourguignon J. Neuburger M. Rebeille F. Trends Plant Sci. 2001; 6: 167-176Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). GDC is a multienzyme complex composed of four component enzymes, the P-, H-, T-, and L-proteins and is responsible for the conversion of glycine produced in the peroxisome to serine in the mitochondria during operation of the photorespiratory cycle (11Vauclare P. Diallo N. Bourguignon J. Macherel D. Douce R. Plant Physiol. 1996; 112: 1523-1530Crossref PubMed Scopus (52) Google Scholar). The H-protein plays a pivotal role as a mobile substrate that commutes between the other subunits, allowing its lipoic acid “arm” to visit the active sites of the other three components (10Douce R. Bourguignon J. Neuburger M. Rebeille F. Trends Plant Sci. 2001; 6: 167-176Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Here we show that HNE rapidly inhibits glycine-dependent respiration of isolated pea leaf mitochondria and that the site of this inhibition is the H-protein of GDC. This site is much more sensitive to oxidative damage than those of related 2-oxo acid dehydrogenases. Further, we show that in vivolipid peroxidation, induced by the herbicide paraquat and by the environmental stresses of chilling and water deficit, specifically inhibits the ability of mitochondria to oxidize glycine. GDC is, therefore, a major target for oxidative damage in leaf mitochondria. Tryquat 200 (Crop Care Australasia Pty. Ltd., Pinkenba, Australia) is a commercially available herbicide combination of paraquat (437.5 mg/L) and diquat (225 mg/L). Studies on the mode of action of these two components appear to indicate that both function identically (12Dodge A.D. Endeavour. 1971; 30: 130-135Crossref PubMed Scopus (181) Google Scholar), and this combination is referred to here simply as paraquat. HNE was obtained from Calbiochem, and all other chemicals were at least reagent-grade. Pea (Pisum sativum L. cv Green Feast) plants were germinated in vermiculite and grown in controlled environment chambers with a light intensity of 700 μmol/m2/sec at 24 °C and 65% humidity for 10 days on a 16/8-hour day/night cycle. Mitochondria were isolated according to the methods of Day et al. (13Day D.A. Neuburger M. Douce R. Aust. J. Plant Physiol. 1985; 12: 219-228Crossref Google Scholar) and Zhang and Wiskich (14Zhang Q. Wiskich J.T. Arch. Biochem. Biophys. 1995; 320: 250-256Crossref PubMed Scopus (13) Google Scholar) from ∼60 g of leaves. Leaves were disrupted with a Polytron (Kinematica, Kriens, Switzerland) in 250 ml of cold extraction medium (0.3 M sucrose, 25 mm tetra-sodium pyrophosphate, 10 mm KH2PO4, 2 mm EDTA, 1 mm glycine, 1% (w/v) PVP-40, 1% (w/v) bovine serum albumin, 20 mm ascorbate, pH 7.5). The homogenate was filtered through 4 layers of miracloth and centrifuged for 5 min at 1100 × g. The supernatant was centrifuged for 20 min at 18,000 × g and the pellet resuspended in 200 ml of wash medium (0.3 M sucrose, 10 mm TES, 1 mmglycine, 0.1% (w/v) bovine serum albumin, pH 7.5) and centrifuged for 5 min at 1100 × g. The supernatant was centrifuged for 20 min at 18,000 × g and the pellet resuspended in 10 ml of wash medium. 5-ml aliquots were then layered over 27.5 ml of solution (0.3 M sucrose, 10 mm TES, 1 mmglycine, 0.1% (w/v) bovine serum albumin, 28% (v/v) Percoll, and a linear gradient of 0–10% (w/v) PVP-40, pH 7.5) in a centrifuge tube and centrifuged for 40 min at 40,000 × g. The mitochondria were found as a tight light yellow-brown band near the bottom of the tube. The mitochondrial fraction was removed and diluted in 250 ml of wash medium and centrifuged at 31,000 × gfor 15 min. The supernatant was removed, and this wash was repeated. The final mitochondrial pellet was resuspended in ∼1 ml of wash medium. Isolated pea mitochondria were treated with HNE and a number of co-factors and substrates before assays were carried out. Mitochondria for glycine oxidation assays were incubated for 60 min on ice with 10 mm glycine and 0.5 mm ATP to maximize GDC activity as described by Zhang and Wiskich (14Zhang Q. Wiskich J.T. Arch. Biochem. Biophys. 1995; 320: 250-256Crossref PubMed Scopus (13) Google Scholar). These mitochondria were then incubated with 250 μl of assay buffer, glycine (10 mm), NAD (0.5 mm), ATP (0.5 mm), ADP (0.25 mm), and various concentrations of HNE (1.5–250 μm) for 10 min. 750 μl of assay buffer was then added and electrode traces started, with additions of ADP at appropriate times. For pyruvate (+ malate) oxidation assays, mitochondria were incubated for 10 min with pyruvate (10 mm), malate (1 mm), NAD (0.5 mm), TPP (0.05 mm), CoA (0.06 mm), ATP (0.5 mm), ADP (0.25 mm), and HNE (1.5–250 μm). For 2-oxoglutarate oxidation assays, mitochondria were incubated with 2-oxoglutarate (10 mm), NAD (0.5 mm), TPP (0.05 mm), CoA (0.06 mm), ATP (0.5 mm), ADP (0.25 mm), and HNE (1.5–250 μm). Again, 750 μl of assay buffer was then added and electrode traces started, with additions of ADP at appropriate times. Plants were sprayed evenly with paraquat (662.5 mg/L), allowed to dry, and then returned to controlled environment chambers and exposed to light until harvest. For low temperature treatment, plants were placed at 4 °C for 36 h prior to harvest while maintaining a normal day/night light cycle. For drought treatment, plants were not watered for 7 days prior to harvest while maintaining a normal humidity, temperature, and day/night light cycle. O2 consumption was measured in an O2 electrode (Rank Bros., Cambridge, UK) in 1 ml of reaction medium containing 0.3 m sucrose, 10 mm TES-KOH, pH 7.5, 5 mmKH2PO4, 10 mm NaCl, 2 mm MgSO4, and 0.1% (w/v) bovine serum albumin. Glycine (10 mm), pyruvate (10 mm), malate (0.5 mm), 2-oxoglutarate (10 mm), succinate (10 mm), NADH (1 mm), NAD (0.5 mm), TPP (0.05 mm), CoA (0.06 mm), ATP (0.5 mm), ADP (0.1–1 mm), and HNE (1.5–250 μm) were added as indicated. Protein concentrations were determined by the method of Peterson (15Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7333) Google Scholar) using bovine serum albumin as standard. We used the method of Hodges et al.(1Hodges D.M. DeLong J.M. Forney C.F. Prange R.K. Planta. 1999; 207: 604-611Crossref Scopus (3046) Google Scholar), which takes into account the presence of anthocyanins and sucrose. Approximately 1 g of tissue was in (v/v) and in a and by at × g for 10 min. aliquots of the supernatant were placed in and 1 ml of solution (w/v) (w/v) was added to three and (w/v) thiobarbituric (w/v) (w/v) added to the other The were and to °C for 25 by on ice for 5 min. The were then at × g for 10 min and of at and The of was from 1 A and Mitochondria from in were treated with (10 to HNE and at × g for 10 min. The supernatant was removed and the pellet resuspended in the mitochondria 5 μl of mitochondrial proteins were by under conditions on 0.1% (w/v) (w/v) according to PubMed Scopus Google Scholar). mitochondria from abiotic stress were under conditions on 0.1% (w/v) (w/v) according to PubMed Scopus Google Scholar). For proteins were from and of the acid serum K.M. Szweda L.I. Biochemistry. 1998; 37: PubMed Scopus Google Scholar), of the of GDC and of the oxidase serum T.E. L. Plant Physiol. PubMed Google Scholar) were used as antibodies. was used for of and using a The were using the with the band as the other were relative to that of pea mitochondrial was according to et al. P. Plant Physiol. 2001; PubMed Scopus Google Scholar). and were as was on an using an to be were from the at °C in a and at For the the proteins were with P. Plant Physiol. 2001; PubMed Scopus Google Scholar) and in 50% and were by and by and were with is as the of the by its and the acid on of inhibition of lipoic acid-containing tricarboxylic acid cycle enzymes by in both mammalian (4Humphries K.M. Yoo Y. Szweda L.I. Biochemistry. 1998; 37: 552-557Crossref PubMed Scopus (200) Google Scholar, K.M. Szweda L.I. Biochemistry. 1998; 37: PubMed Scopus Google Scholar) and plant PubMed Scopus Google Scholar) to the effect of this lipid peroxidation product on the photorespiratory in pea leaves. Isolated pea leaf mitochondria were incubated for 10 min in the presence of various substrates and HNE at concentrations from to 250 μm. O2 consumption was concentrations up to HNE effect on 2-oxoglutarate-dependent respiration but inhibited GDC by 250 pyruvate and 2-oxoglutarate-dependent respiration by while glycine-dependent respiration was inhibited by more than more the of this we incubated isolated mitochondria with in the presence of and various substrates for of before O2 consumption 30 min of 2-oxoglutarate-dependent respiration was by O2 consumption by and glycine-dependent respiration by more than The in 2 indicate that the inhibition of respiration by HNE with This that oxidative damage will be in We also showed that lipoic acid these lipoic acid-containing proteins from inhibition by of lipoic acid during of isolated mitochondria with HNE for 10 min inhibition to only not with inhibition in lipoic acid (Fig. 2 of pea mitochondrial proteins, with to lipoic showed four of molecular and in potato these proteins, as the two of the of and the H-protein of GDC. The GDC H-protein can be from the by and antibody reaction and from the of and by with the and from potato PubMed Scopus Google Scholar). on pea has shown that this enzyme also contains two of and J. Biochem. 1999; PubMed Scopus Google Scholar, T.E. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and that a was to be the of pea J. Biochem. 1999; PubMed Scopus Google Scholar). that the of the two proteins in pea is the of and that the 17-kDa is the H-protein of we and identification of these lipoic acid-containing from the 17-kDa were to the H-protein of GDC from P. number from the with a these aliquots of mitochondria were during oxidation of substrates in the presence of added treated with (10 to by and with lipoic acid and the pea H-protein (Fig. acid a in antigenicity of and treatment with with the in respiratory rate in A. The in lipoic acid antigenicity of the H-protein was much more A of acid antibody reactivity of H-protein glycine-dependent respiration showed a with an of not This that the of active lipoic acid with H-protein has a very over the activity of GDC in mitochondria. with to the pea H-protein that was direct loss of H-protein following HNE very HNE all lipoic acid been H-protein to into on (Fig. the of of H-protein These which appear to have a molecular from the of HNE to acid of H-protein of an produced at HNE The exact of these of the H-protein is to be The herbicide paraquat as an from chloroplast (12Dodge A.D. Endeavour. 1971; 30: 130-135Crossref PubMed Scopus (181) Google Scholar) and also inhibits and the mitochondrial Biochim. Biophys. Acta. 1995; PubMed Scopus Google Scholar), the production of in both a of reactions stimulated by Free in and Scholar) and by reaction with this which lipid peroxidation Free in and Scholar). We used paraquat to induce oxidative stress in pea leaves. Pea plants sprayed with paraquat (662.5 mg/L) were in the and were at h Leaves from all were used for assays to the of lipid peroxidation end products Leaves from and h were also used for of mitochondria for of respiratory during the h before and at 10 h and 2-oxoglutarate-dependent respiration were by this treatment of leaves, but glycine-dependent respiration to of with the used in in H-protein lipoic acid reactivity to of the The of H-protein not during the h but between and of paraquat on acid and antibody reactivity pea mitochondrial Mitochondria were isolated from pea plants at the and their proteins were by on the molecular in those the of herbicide treatment, and those the relative band The and are the of the is the of and the 17-kDa is the H-protein of all using the acid antibody K.M. Szweda L.I. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). The is the H-protein of GDC with an The and in the are proteins with the antibody T.E. L. Plant Physiol. PubMed Google with the of following paraquat treatment, protein, which was at very low (Fig. is as a defense in induced to from the mitochondrial Y. L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, Physiol. Google Scholar, K. Physiol. 1995; 95: Scopus Google Scholar). in pea by paraquat with this but was to GDC from A number of environmental stresses have been shown to induce oxidative stress in These M. F. Physiol. Scopus Google Scholar, J.M. A. F. Free 1999; PubMed Scopus Google Scholar), chilling Plant 6: PubMed Scopus Google Scholar), and water deficit R. C. M. Plant Physiol. 1998; Scopus Google Scholar). The of the production of ROS under these conditions but of both ROS and lipid peroxidation as as responses of antioxidant enzymes, have the oxidative of the exposed We the effect of two environmental stresses on pea leaf low temperature and For the low temperature treatment, plants were at 4 °C for 36 h prior to harvest. For drought treatment, plants were for 7 days prior to harvest. Leaves were from three of plants from treatment, as as from three of and used for of mitochondria for of respiratory respiration was inhibited by all by low temperature, by water and by the herbicide treatment. These mitochondria were also for with the treatment groups showing to the not with the used in only minor in H-protein lipoic acid reactivity to of the in in and in the (Fig. The of not in of the The effect of these on was also in pea was with the oxidative conditions by the environmental stresses as in the its not GDC from Plant mitochondrial lipid peroxidation has been reported A. F. L. A. J. Plant Physiol. Scopus Google Scholar, A. F. A. Plant Physiol. Scholar, A. P. Plant Physiol. 1999; PubMed Scopus Google Scholar), but the of this damage for mitochondrial operation was not from these has also been shown that HNE has a effect on tricarboxylic acid cycle function through modification of lipoic acid moieties of enzymes (4Humphries K.M. Yoo Y. Szweda L.I. Biochemistry. 1998; 37: 552-557Crossref PubMed Scopus (200) Google Scholar, K.M. Szweda L.I. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Here we have shown for the that HNE has an effect on the lipoic acid-containing glycine a enzyme involved in photorespiration in photosynthetic plant and for the of cycle substrates in the photorespiratory The effect of HNE was much more dramatic on glycine-dependent respiration than on tricarboxylic acid that GDC is sensitive to HNE modification and be a target during oxidative stress in photosynthetic plant with this we a in GDC activity following in vivo oxidative stress induced by paraquat treatment, by and by water the with isolated mitochondria and plants that environmental stress directly to lipid peroxidation, the products of which can inhibit mitochondrial function in photorespiratory In pea plants treated with were only Lipid peroxidation is to following paraquat the of accumulation indicate that end products to to levels of the presence and function of pathways in the However, as these are and lipid peroxidation products The of damage to the H-protein of GDC before were by the methods here of the H-protein to low concentrations of lipid peroxidation end before takes this were effects on plant by the In this GDC inhibition be an of oxidative damage in photosynthetic plant Glycine metabolism by GDC is an in the photorespiratory and its inhibition in is under conditions A. R. Biol. Sci. PubMed Scopus Google Scholar). The sensitivity of GDC to the here therefore, that photorespiration is the of oxidative damage in leaves. The mechanisms for cytotoxic lipid peroxidation products in plants are only to be an A. M. K. L. A. D. Plant J. PubMed Google Scholar) has been in both the mitochondria and the that with A which HNE as a has been in mammalian mitochondria P. 1985; PubMed Scopus Google Scholar, M. Biochem. J. 1998; PubMed Scopus Google Scholar) and in the Plant Physiol. 1998; PubMed Scopus Google Scholar). has also been that a recently which an in mitochondria to the end products of lipid peroxidation as and HNE F. H. Plant 2001; PubMed Scopus Google Scholar, Trends Plant Sci. 2001; 6: Full Text Full Text PDF PubMed Google Scholar). In the of the pea plants used in the present the protein, was induced h herbicide treatment, with the in (Fig. This three that mitochondrial defense proteins can be and during the oxidative stress following paraquat treatment, that this treatment not simply lead to rapid and of the plant this of correlated with lipid damage than the damage of the to which is due to in mitochondria. that defense of at least as by is to susceptible sites as the H-protein of GDC. However, of have to damage to the susceptible tricarboxylic acid We have PubMed Scopus Google Scholar) that the inhibition of by in the light Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) in the lipoic acid moieties of this enzyme during of lipid peroxidation to be during be for glycine is to be the respiratory substrate in the light under photorespiratory conditions. of GDC will of the of lipoic which is for modification by HNE (4Humphries K.M. Yoo Y. Szweda L.I. Biochemistry. 1998; 37: 552-557Crossref PubMed Scopus (200) Google Scholar). The identification of pathways for lipoic acid to in mitochondria J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and the of a for lipoic acid in plant mitochondria from photosynthetic Macherel D. M. Douce R. Bourguignon J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) have been in of the of matrix However, an enzyme to damaged lipoic the and allowing of an to of P. sativum H-protein, a but as this to an role in damage Such an the for and of in complexes and the of lipoic acid We K. M. and P. of and of for the of acid antibodies. S. Research is for the of and of of is for the of antibodies. of is for with of is for during
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