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Correspondence to Maryse A. Block: maryse.block{at}cea.fr
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Abstract |
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Introduction |
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In leaves, the most abundant membrane glycerolipids are not phospholipids, but glycolipids such as galactolipids, i.e., monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). They represent up to 80% of leaf lipids (Joyard et al., 1996). Galactolipids were reported to be localized specifically in plastids and trace amounts of these lipids, which have been detected in the past in other isolated fractions of the cell, such as tonoplast (Haschke et al., 1990), were cautiously considered as possible contamination by plastid membranes. MGDG is synthesized from DAG and UDP-galactose by MGDG synthases, and this enzyme activity is located in the plastid envelope (Douce, 1974). In Arabidopsis thaliana, there are two types of MGDG synthases differing in their NH2-terminal portion: type A with MGD1 and type B with MGD2 and MGD3 (Awai et al., 2001). In MGDG produced by these enzymes, galactose is linked to DAG via a ß-glycosidic bond (Carter et al., 1956). On the other hand, two different mechanisms have been reported for the formation of DGDG: either by addition of galactose from UDP-galactose on MGDG with DGD1 or DGD2 enzymes (Kelly and Dormann, 2002; Kelly et al., 2003) or by reaction of two MGDG to form one DGDG and one DAG by the galactolipidgalactolipid galactosyltransferase enzyme (van Besouw and Wintermans, 1978). With DGD1 and DGD2, the inserted galactose is linked by an -glycosidic bond (Kelly and Dormann, 2002; Kelly et al., 2003), leading to the
-ß DGDG structure reported by Carter et al. (1956). The galactolipidgalactolipid galactosyltransferase enzyme generates a ß-ß DGDG structure because this enzyme activity correlates with the presence of oligogalactolipids carrying several galactose residues with ß-glycosidic bonds (Kojima et al., 1990; Xu et al., 2003).
During Pi deprivation, the cellular DGDG content increases (Essigmann et al., 1998; Härtel et al., 1998) and the expression of genes encoding type B MGDG synthases (MGD2 and MGD3; Awai et al., 2001) and DGDG synthases (DGD1 and DGD2; Kelly and Dormann, 2002; Kelly et al., 2003) is stimulated. The induced synthesis of DGDG involves several compartments of the cell. DAG backbone of DGDG was traced back from extraplastidial phosphatidylcholine (PC) (Roughan, 1970; Williams et al., 2000; Kelly et al., 2003), and lipid analyses during the first steps of Pi deprivation indicated that indeed PC was transformed into DGDG via DAG (Jouhet et al., 2003). Galactose insertion for synthesis of newly formed DGDG is expected to occur in plastids because enzymes encoded by MGD2, MGD3, DGD1, and DGD2 can all be addressed to the plastid envelope, very likely to the outer envelope (Awai et al., 2001; Froehlich et al., 2001; Kelly et al., 2003). Consistently, newly synthesized DGDG was proposed to replace missing PC (Härtel and Benning, 2000; Härtel et al., 2000) because (1) PC content is highly reduced; and (2) PC and DGDG adopt similar bilayer conformation in the membranes. These lipid changes cannot be limited to plastid membranes because the bulk of PC is located outside plastids (Dorne et al., 1985). Indeed, a recent report has shown that DGDG accumulates in oat plasma membrane during Pi deprivation (Andersson et al., 2003). However, the plasma membrane represents a low proportion of the cellular membrane surface, and therefore the plasma membrane lipid change cannot solely explain the high amounts of cellular PC and DGDG being affected by Pi deprivation.
Mitochondria are organelles limited by a double membrane like plastids. In plant cells, they represent 10% of cell membranes and contribute to 1020% of total cellular PC (Douce, 1985). During Pi deprivation, mitochondria seem relatively protected from the induced stress. The cellular proportion of diphosphatidylglycerol (DPG), a specific mitochondrial phospholipid, remains relatively constant (Jouhet et al., 2003). Moreover, respiration is not affected except for the cyanide-resistant pathway that is enhanced (Rébeillé et al., 1984). A possible response of the plant cell to Pi deprivation is that DGDG could be transferred to mitochondria as well, and therefore contributes to adaptation of this organelle to the stress conditions. To probe this hypothesis, we cultivated A. thaliana cells with defined levels of Pi and set up a procedure to isolate the mitochondria. In this article, we report that under Pi deprivation, mitochondria contain a high concentration of DGDG in contrast with mitochondria from cells grown with sufficient supply of Pi. The
-ß anomeric structure of DGDG present in mitochondria is characteristic of DGDG synthesized through a DGD type enzyme. We further point out that a transfer of DGDG occurs between plastid envelope and mitochondria. This transfer is apparently dependent on contact between plastids and mitochondria. The contacts seem to be favored at early stages of Pi deprivation when DGDG cell content is just starting to respond to Pi deprivation.
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Results |
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Mitochondria isolated from Pi-deprived cells contain DGDG
Lipids were extracted from both types of mitochondria and their composition was compared with that of cells. Fig. 3 A shows results of glycerolipid analyses normalized to the total amount of glycerolipid in each fraction. The composition of cells grown with or without Pi was consistent with data published earlier (Essigmann et al., 1998; Härtel et al., 1998; Jouhet et al., 2003). DPG was found at a relatively high level in both types of cells, indicating that mitochondria lipids represent a fair proportion of total cell lipids. In Pi-deprived cells, we mainly observed a decrease in phospholipids and a high increase in DGDG and sulfoquinovosyldiacylglycerol (SQDG). Lipid composition of mitochondria isolated from control cells was similar to that reported earlier, containing mostly phosphatidylethanolamine (PE) and PC (Douce, 1985; Harwood, 1987). Only traces of MGDG and DGDG were detected. The mol percentage of DPG was three times higher in isolated mitochondria than in whole cells. In mitochondria isolated from Pi-deprived cells, the levels of phospholipids (i.e., PC, PE, and PG) were all lower except for DPG, which was present in higher proportion. Contents in MGDG and SQDG were slightly higher than in mitochondria from control cells, but both remained at a low level. By contrast, the level of DGDG was remarkably higher, representing >18% of the Pi mitochondria lipids. By analyzing the lipid contamination attributable to chloroplast envelope with mitochondrial lipid data, we calculated that the amount of DGDG issued from envelope contamination is much lower than the amount of DGDG measured in the mitochondria fraction (Table S1), indicating that most of the DGDG detected in mitochondria upon 3 d of Pi deprivation was indeed located in mitochondria.
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Discussion |
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All types of phospholipids (with the notable exception of DPG) seem to be replaced by DGDG in mitochondria during Pi deprivation. Our data show that this is not only the case of PC, but also of PE and PG. Although DGDG and PC are both bilayer-forming neutral lipids, PE and PG are negative lipids and, in addition, PE preferentially organizes in hexagonal-II phase. Therefore, the lipid changes induced by Pi deprivation in mitochondria mean a strong modification of the structure and of the charge of the lipid bilayer subsequently altering the environment of membrane proteins. Our results primarily question the possible role of DGDG in mitochondria membranes. In thylakoids, it has been shown that DGDG is required for structure and stability of LHCII in vitro (Reinsberg et al., 2000). A preservation of the mitochondrial electron transport machinery by changing phospholipids for DGDG when Pi is on shortage is a possibility. Indeed, isolated mitochondria were preserved in their respiratory activity (this paper and Rébeillé et al., 1984). Whether DGDG can take the place of phospholipids in the fine structures of the plant respiratory complexes is an open question. One should have expected that the level of DPG, being a Pi-rich phospholipid, can be decreased during Pi deprivation. Obviously this is not the case, and DPG appears to be quite protected. One possible explanation is that DPG fulfils a key function that is maintained or even enhanced in the Pi-deprived cells, and consequently preserved. In support to this hypothesis, several reports indicate that DPG is required for structure and stability of respiratory chain complexes in animal cells (Zhang et al., 2002; Pfeiffer et al., 2003).
The second intriguing question is the origin of mitochondrial DGDG. To date, all the DGDG synthase enzymes, galactolipidgalactolipid galactosyltransferase, DGD1, and DGD2, were reported to be localized in chloroplast envelope membranes (Dorne et al., 1982; Froehlich et al., 2001; Kelly et al., 2003). Our NMR analyses indicated that the structure of mitochondrial DGDG, 1,2-diacyl-3-O-(-D-galactopyranosyl-(1
6)-O-ß-D-galactopyranosyl)-sn-glycerol, is consistent with a synthesis via a DGD type enzyme and not via the galactolipidgalactolipid galactosyltransferase. Indeed, working on dgd1 dgd2 double mutants, Kelly et al. (2003) have shown that both DGD1 and DGD2 contribute to DGDG synthesis during Pi deprivation and only them. The fatty acid composition and positional distribution of mitochondrial DGDG can give some indications about which of the DGD1 and DGD2 enzymes is involved in its synthesis. Considering analyses of dgd1 and dgd2 mutants (Härtel et al., 2000; Klaus et al., 2002; Kelly et al., 2003), the slight increase in 16:0 in DGDG compared with control cell DGDG suggests the involvement of DGD2, whereas the strong amount of 18:3 is indicative of the DGD1 functioning. Therefore, mitochondrial DGDG likely results from both DGD1 and DGD2 enzyme activities present in the chloroplast envelope. Eventually, the presence in DGDG of high proportion of 18:3, which was shown not to depend on ER FAD3 desaturation (Klaus et al., 2002), is an additional indication of the synthesis of mitochondrial DGDG in chloroplast envelope membranes. As additional evidence, we verified that the level of galactolipid synthesis in fractions enriched in mitochondria was strictly related to the level of mitochondria cross-contamination by envelope membranes.
Several models were proposed to explain net transfers of lipid between membranes and organelles (for review see Voelker, 2003). Particularly, lipid movements can occur via specialized zones of apposition between subcellular membranes. For example, in yeast and in mammalian cells, a movement of phosphatidylserine (PS) from ER to mitochondria inner membrane was clearly demonstrated to occur through specialized region of ER referred to as mitochondria-associated membranes (MAM; Vance, 1990; Ardail et al., 1991; Achleitner et al., 1999). To investigate how DGDG transfers from chloroplast envelope to mitochondria membranes, we surveyed cell structures during the course of adaptation to Pi deprivation. We failed to observe any formation of vesicles. Rather, we noticed numerous tight appositions of membranes from envelope and mitochondria during early phases of Pi deprivation that could sustain contact-favored transfer. In the case of PS transfer through MAM, physical interactions between mitochondria and ER were not easily disrupted (Voelker, 1990). Similarly, we observed that mitochondria were more difficult to separate from envelope after 12 h of Pi deprivation than after 3 d. Together, our data indicate that DGDG uptake by mitochondria seems to require a physical contact between mitochondria and chloroplast envelope membranes. Although we could not detect a transfer of DGDG from isolated chloroplasts toward isolated mitochondria, we did observe an in vitro transfer of DGDG from mitochondria-associated envelope membranes to mitochondria. In addition, the transfer was selective for DGDG compared with MGDG or TriGDG. The association of DGDG synthesis with the transfer process is possible, but could not be investigated. The same kind of association was considered for PS transfer to mitochondria to encounter the fact that a selected pool of PS was transferred (Vance, 1990).
In conclusion, this work provides evidence that in plant cells starved for Pi, DGDG is present in several specific membranes outside plastids and becomes rapidly highly abundant in mitochondria membranes. A future challenge includes elucidation of the molecular mechanisms involved in the synthesis and transfer of DGDG from plastid envelope to mitochondria membranes. The high stability observed in lipid composition of mitochondrial membranes under standard situation, even when comparing different plants and the triggering of the DGDG transfer upon Pi deprivation, will have to be considered. A last challenging question is the role of DGDG in mitochondria, specifically whether this role extends further than building up primary membrane blocks.
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Materials and methods |
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Preparation of cell fractions
About 70 g of Arabidopsis cells were filtered and resuspended in 50 ml ice-cold grinding buffer (0.3 M mannitol, 15 mM MOPS, 2 mM EGTA, 0.6% [wt/vol] polyvinylpyrrolidone 25, 0.5% BSA, 10 mM DTT, 1 mM PMSF, 5 mM -aminocaproic acid, and 1 mM benzamidine, pH 8.0). The cells were disrupted by adding 10 ml of sand and by crushing with a mortar. Cell extract was collected as a supernatant after centrifugation at 150 g for 10 min.
Mitochondria were enriched by a three-step centrifugation from cell extract: two centrifugations at 3,000 g for 5 min and a centrifugation at 18,000 g for 15 min. The mitochondrial fraction was resuspended in washing buffer (0.3 M mannitol, 10 mM MOPS, 1 mM PMSF, 5 mM -aminocaproic acid, and 1 mM benzamidine, pH 7.4) and layered on top of a three-layer Percoll (Amersham Biosciences) gradient (18% [vol/vol], 23% [vol/vol], and 40% [vol/vol] Percoll in 0.3 M mannitol, 10 mM MOPS, 1 mM EGTA, and 0.1% BSA, pH 7.2). After centrifugation for 45 min at 70,000 g the mitochondria were isolated from the 23%/40% interphase. To remove Percoll, the mitochondria were centrifuged twice in washing buffer for 15 min at 17,000 g. The crude mitochondria fraction was resuspended in washing buffer and layered on top of continuous 28% Percoll gradients in 0.3 M mannitol, 10 mM MOPS, 1 mM EGTA, and 0.1% BSA, pH 7.2. After centrifugation for 1 h at 40,000 g the mitochondria were isolated in the medium of the gradient. To remove Percoll, the purified mitochondria were centrifuged three times in washing buffer for 15 min at 17,000 g. Purified mitochondria pellet was resuspended in washing buffer and frozen at 80°C.
Chloroplasts were purified from cell extract by centrifugation at 3,000 g for 5 min. The chloroplast fraction was resuspended in washing buffer, and 6 ml was layered on top of a two-step Percoll gradient (33% [vol/vol] and 50% [vol/vol] Percoll in 0.3 M mannitol, 10 mM MOPS, 1 mM EGTA, and 0.1% BSA, pH 7.2). After centrifugation for 15 min at 3,000 g chloroplasts were collected at the 33%/50% interphase. To remove Percoll, the purified chloroplasts were centrifuged twice in washing buffer for 5 min at 2,000 g. Purified chloroplast pellet was resuspended in a minimum volume of washing buffer and conserved at 80°C.
Mitochondria respiration measurement
O2 electrode (Oxygraph Hansatech) was calibrated in water and equilibrated for 5 min in 1 ml of 0.3 M mannitol, 20 mM MOPS, 10 mM Pi, 10 mM KCl, 5 mM MgCl2, and 0.1% BSA, pH 7.5. O2 consumption was measured after addition of 10 µl of mitochondria fraction, 10 µl succinic acid 1 M, and 10 µl ADP 100 mM. 10 µl KCN 100 mM was added to measure level of sensitivity to cyanide.
Western blotting and immunoblotting
Protein quantification was done by the Lowry method (Lowry et al., 1951). Proteins were solubilized in 0.1 M Tris, pH 7.8, 10% glycerol (vol/vol), 2% SDS (wt/vol), 25 mM DTT, and 0.1% bromophenol blue (wt/vol). Samples were analyzed by SDSPAGE (Broglie et al., 1980). After electrophoresis, proteins were stained in isopropanol/acetic acid (3:1, vol/vol) or Coomassie brillant blue (R-250; Sigma-Aldrich) 0.25% (wt/vol), or electrophoretically transferred to nitrocellulose membrane for immunoblotting. The proteins were visualized with a rabbit primary antibody against a specific protein and an antirabbit IgG-HRP conjugate and the ECL Plus Western blotting detection system (Amersham Biosciences). The anti-OEP21 antibodies were prepared by rabbit immunization using the recombinant protein corresponding to the full-length sequence At1g76405.2.
Antibodies against Nad9 were provided by Dr. Grienenberger (IBMP-CNRS, Strasbourg, France), antibodies against TOM20 and TOM40 by Dr. Braun (Universität Hannover, Hannover, Germany), antibodies against LHCII by Dr. Vallon (IBPC, Saclay, France), antibodies against BCCP1 by Dr. Alban (INRA, Grenoble, France), and antibodies against HPPK by Dr. Ravanel (Université Grenoble, Grenoble, France).
Lipid analysis
Lipids were extracted from 2 g of harvested cells, according to Folch et al. (1957) or from mitochondria fraction, according to Bligh and Dyer (1959). They were analyzed as described in Jouhet et al. (2003).
Galactolipid synthesis in organelle fraction
Organelle fractions were incubated at 25°C in washing buffer with 1 mM DTT, 1 mM MgCl2, and 10 mM UDP-[14C]galactose, 10,000 dpm/nmol. Reaction was stopped by a Bligh and Dyer lipid extraction (Bligh and Dyer, 1959). A sample was taken after 30 min and after 1 h to verify reaction linearity. We verified that addition of DAG in the incubation buffer and sonication did not increase galactose incorporation into lipids. Lipids were separated by 2D-TLC as described before, scraped of the plate, and radioactivity in each lipid was counted by scintillation. Activity was determined in dpm incorporated into lipids/h and mg of protein and converted in nmol.h1.mg1.
Agglutination assays
Antibodies raised against plastid envelope, mitochondria outer membrane polypeptides, and DGDG were used to probe the outer surface of isolated intact mitochondria or chloroplasts. For agglutination assays, mitochondria or chloroplast suspension corresponding to 15 nmol O2/min respiration or 18 µg chlorophyll, respectively, were incubated 10 min on a glass slide with 4 µl of the washing buffer (0.3 M mannitol, 10 mM MOPS, 1 mM PMSF, 5 mM -aminocaproic acid, and 1 mM benzamidine, pH 7.4) and 6 µl antibodies. The suspensions were then examined at RT under light microscopy (Axioplan 2, Carl Zeiss MicroImaging, Inc.) using an immersion 100x objective and a CCD camera (Hamamatsu Corporation) to follow agglutination.
Epifluorescence
Arabidopsis cells were fixed 20 min by 5% methanol-free formaldehyde (Polyscience) in TBS (1.3 M NaCl, 13 mM KCl, and 15 mM Tris-HCl, pH 7.4), washed in TBS, permeabilized 15 min with 0.002% saponin in TBS, and saturated 1 h with TBS, 5% FBS, and 5% goat serum. After incubation for 1 h with 1/10 rabbit serum against DGDG (Maréchal et al., 2002) and 1/100 guinea pig serum against BCCP1 or HPPK in TBS, 1% FBS, three washes of 10 min in TBS, 1% FBS, incubation for 1 h with 1/100 antirabbit IgG-goat BODIPY conjugate and 1/20 antiguinea pig IgG-goat Alexa 594 (Molecular Probes, Inc.) in TBS, 1% FBS in the dark, three washes of 10 min in the dark in TBS, 1% FBS, and one in TBS, cells were deposed on glass slide with ProLong AntiFade kit (Molecular Probes, Inc.) and stored in the dark. Before fixation, some cells were incubated for 1 h in the presence of MitoTracker orange CMTMRos (Molecular Probes, Inc.) and washed twice in TBS. Slides were observed with an immersion 40x objective at RT the following day by confocal laser scanning microscopy using a TCS-SP2 operating system (Leica). BODIPY, Alexa 594, and MitoTracker were excited and collected sequentially (400 Hz, line by line) using the 488-nm line of an argon laser for BODIPY and the 543-nm line of a He-Ne laser for Alexa 594 and MitoTracker. Fluorescences were collected between 498 and 533 nm, 580 and 650 nm, and 553 and 607 nm for BODIPY, Alexa 594, and MitoTracker, respectively.
Electron microscopy
Samples were fixed overnight in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 6.9). Next, they were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 h. After this double fixation, samples were exposed for 30 min to a solution of 1% tannic acid in the same buffer at RT, dehydrated in a graded series of ethanol and propylene oxide, and finally embedded in an Epon-Araldite mixture. Thin sections were cut with an ultramicrotome (Ultracut, Reichert-Jung), post-stained with lead citrate, and examined with a transmission electron microscope (model 300; Hitachi) operating at 75 kV.
1H-NMR spectroscopy
DGDG was isolated from TLC as described before and was extracted from silica by addition of 1.5 ml chloroform/methanol, 2:1, and 400 µl of NaCl 10 g/l. Extracts were dried under argon and dissolved in 440 µl deuterated chloroform and 220 µl deuterated methanol (Merck). 1H-NMR spectra were obtained on a Varian Unity+ 500 MHz spectrometer (Bruker) at 10°C with a 5-mm indirect detection probe. The methanol resonance was used as a lock signal. Acquisition parameters were as follows: 90° pulse, repetition time 3 s, spectral width 4993 Hz, number of scans = 64. Free induction decays were collected as 30-K data points, zero filled to 65 K, and processed with a 0.18-Hz exponential line broadening. A 1-s recycling time was used to obtain fully relaxed spectra.
Online supplemental material
Fig. S1 shows immunofluorescence localization of DGDG in A. thaliana cells grown with Pi. Analysis of galactolipid composition of mitochondria fraction is presented in Table S1, where the lipid contamination attributable to chloroplast envelope is compared with mitochondrial lipid data. Analysis of DGDG content in mitochondria fractions prepared from control A. thaliana cells subsequent to an extra purification step of mitochondria is presented in Table S2. Experiment was done as in Table III, except that mitochondria were purified from control cells. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200407022/DC1.
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Acknowledgments |
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Submitted: 6 July 2004
Accepted: 21 October 2004
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References |
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