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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

In Situ Molecular Identification of the Plastid 3 Fatty Acid Desaturase FAD7 from Soybean: Evidence of Thylakoid Membrane Localization¹

Department of Plant Nutrition, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50059 Zaragoza, Spain (V.A., R.C., R.P., M.A.); and Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain (P.S.T., M.d.C.R.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
3 fatty acid desaturases are the enzymes responsible for the synthesis of trienoic fatty acids in plants. These enzymes have been mainly investigated using molecular, biochemical, and genetic approaches but very little is known about their subcellular distribution in plant cells. In this work, the precise subcellular localization of the 3 desaturase FAD7 was elucidated by immunofluorescence and immunogold labeling using a monospecific GmFAD7 polyclonal antibody in soybean (Glycine max) photoautotrophic cell suspension cultures. Confocal analysis revealed the localization of the GmFAD7 protein within the chloroplast; i.e. signals from FAD7 and chlorophyll autofluorescence showed specific colocalization. Immunogold labeling was pursued on cryofixed and freeze-substituted samples for convenient preservation of antigenicity and ultrastructure of membrane subcompartments. Our data revealed that the FAD7 protein was preferentially localized in the thylakoid membranes. Biochemical fractionation of purified chloroplasts and western analysis of the subfractions further confirmed these results. These findings suggest that not only the envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants.

Relatively little is known about the subcellular localization of fatty acid desaturases in plants, largely due to the lack of monospecific antibodies against them. In the only previous study concerning this question, immunocytological evidence of the subcellular localization of FAD2 and FAD3, the 6 and 3 desaturases of the reticulum, respectively, was obtained by developing an epitope-tagging scheme that allowed the immunological detection of transiently expressed FAD2 and FAD3 proteins in plant suspension-cultured cells (Dyer and Mullen, 2001). There are no evidences of the subcellular localization of the plastid desaturases, to our knowledge. In cyanobacteria, prokaryotic ancestors of plants, immunogold studies provided evidence that the 6, 9, 12, and 3 desaturases were located in the regions of both cytoplasmic and thylakoid membranes (Mustardy et al., 1996). However, to our knowledge, in plants, there are no clear evidences showing which proportion of the total desaturase activity in the chloroplast takes place in each specific membrane. In a series of elegant experiments, Schmidt and Heinz (1990b) obtained a membrane fraction with high desaturase activity. This fraction was a mixture of thylakoid and envelope membranes. Further purification of this fraction resulted in envelope membranes that retained high and stable oleate desaturase activity while it was more difficult to reproduce linoleic acid desaturation (Schmidt and Heinz, 1990b). More recently, proteomic analysis of the chloroplast envelope identified both FAD6 and FAD7 desaturases as part of the proteins that were detected in inner envelope-enriched fractions (Ferro et al., 2003; Froehlich et al., 2003). However, other components of the machinery for fatty acid biosynthesis like the ACP, which is a cofactor of acyl-ACP:glycerol-3-P acyltransferase, are bound to the thylakoid membranes (Slabas and Smith, 1988), suggesting that thylakoids could also be sites of fatty acid biosynthesis and desaturation in plants.

We have recently described the obtention of a monospecific polyclonal antibody against the plastid 3 desaturase FAD7 from soybean (Glycine max; Collados et al., 2006). We used this antibody as a tool for immunofluorescence, immunogold labeling, and biochemical fractionation essays to precisely determine the subcellular localization of the FAD7 3 desaturase in soybean cultured cells. This simple and convenient culture system of photosynthetic cells displays most of the structural and functional characteristics of mesophyll cells (Rogers et al., 1987) and has proven successful for different studies of the chloroplast function. Our results indicate that the FAD7 protein was detected preferentially in the thylakoid membranes, suggesting that these membranes could be sites of lipid desaturation in higher plants. These findings provide new information concerning the role of plant fatty acid desaturases, not only in the lipid synthesis metabolism but also in the environmental stress response and in plant defense-signaling pathways.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Subcellular Localization of GmFAD7 by Immunofluorescence

With the purpose to develop a specific immunobased method for the determination of the precise subcellular localization of the plastid 3 fatty acid desaturase FAD7, we used our monospecific antibody raised against the GmFAD7 protein. The obtention and properties of this antibody have been already described in a previous article of our group (Collados et al., 2006). In brief, we raised an antiserum against a synthetic oligopeptide corresponding to residues 69 to 84 of the deducted mature FAD7 protein. This region was specific for FAD7 and presented very low homology with the microsomal FAD3 protein and the cold-inducible FAD8 plastid enzyme. The GmFAD7 antiserum reacted with a polypeptide of approximately 39 kD, which corresponds well with the predicted M_r of the GmFAD7 protein (Collados et al., 2006).

The structural organization of soybean photosynthetic cells, as visualized in toluidine blue-stained semithin sections (Fig. 1A ), was similar to that of mesophyll cells from young leaves. They showed a large and central cytoplasmic vacuole, numerous chloroplasts distributed along the peripheral layer of cytoplasm, and an ellipsoid nucleus (Fig. 1A). Most chloroplasts contained large granules that appeared as clear inclusions in I₂KI stained sections observed under phase contrast (Fig. 1C) or visualized under bright field (Fig. 1D). The content of these inclusions was revealed by iodide-based cytochemical methods as starch (Fig. 1, C and D).

View larger version (78K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Subcellular localization of GmFAD7 by immunofluorescence. A, Structural organization of soybean photosynthetic cell cultures. Historesin semithin sections after toluidine blue staining. Vacuole (V), cytoplasm (C), nucleus (N), and chloroplasts (arrows). B, Anti-GmFAD7 inmunofluorescence showing green positive signal in rounded cytoplasmic structures (arrows). C and D, Structural organization of a similar cytoplasmic area than in B; historesin semithin sections visualized under phase contrast after I2IK staining for starch (C) with chloroplasts containing clear starch deposits (arrows). D, Starch deposits revealed as dark inclusions in the chloroplasts by iodide-based cytochemistry, observed under bright field. E to M, Confocal laser microscopy observations of the same vibratome sections. The images represent projections of 15 to 20 optical sections. E, Inmunofluorescence with anti-GmFAD7 antibody (green) in soybean photosynthetic cells. Nuclear DNA was stained with DAPI (blue). F, Autofluorescence from chlorophyll (red). G, Overlap of autofluorescence from chlorophyll (red) with anti-GmFAD7 immunofluorescence (green). Yellowish-orange colors indicate signal colocalization. The image shows the chloroplast localization of GmFAD7. H, Control with anti-GmFAD7 antibody blocked with synthetic peptide. Control showed no labeling. Nuclei are visualized by DAPI (blue; I) autofluorescence from chlorophyll. J, Differential interference contrast image of the corresponding section. K, Control without anti-GmFAD7. Nuclei are visualized by DAPI (blue). L, Autofluorescence from chloroplyll. M, Differential interference contrast image of the corresponding section. Bars in figures B to D represent 5 µm; bars in A and E to M represent 10 µm.

The specificity of the anti-GmFAD7 antibody in the immunofluorescence assays was supported by the immunodepletion experiments. These controls were performed by preincubating the anti-GmFAD7 antibodies with the oligopeptide used as immunogen to raise them. Immunofluorescence assays with the preblocked anti-GmFAD7 antibodies abolished the green signal in intact chloroplasts on soybean cells, as identified by their chlorophyll autofluorescence (red signal; Fig. 1, H–J). These results provided additional support to the specificity of the antibodies that were specifically titrated away by this known protein fragment. No significant labeling was obtained in control experiments with the preimmune serum (data not shown) or in the absence of anti-GmFAD7 antibody (Fig. 1, K–M).

Ultrastructural Localization of GmFAD7 by Immunogold Labeling

The only previous report concerning the subcellular localization of two integral membrane-bound plant fatty acid desaturases was elucidated by immunofluorescence microscopic analyses of tobacco (Nicotiana tabacum) suspension cells transiently transformed with different epitope-tagged versions of the reticular FAD2 and FAD3 enzymes (Dyer and Mullen, 2001). To our knowledge, no data are available for plastid desaturases like FAD7. Moreover, the resolution of a confocal microscope and immunofluorescence studies could not inform about the precise localization of the GmFAD7 antigen inside the different chloroplast subcompartments. To precisely define the subcellular distribution of the GmFAD7 protein, electron microscopy immunogold labeling was performed (Fig. 2 ). An accurate electron microscopy immunolocalization of membrane-associated antigens requires specific sample processing methods to preserve the chemical integrity, antigenic reactivity, and membrane ultrastructure, which are highly affected by some chemically fixing and most dehydrating procedures. Cryofixation and freeze substitution have been reported as very convenient processing methods for immunogold assays of membrane-associated antigens in plant cells (Risueño et al., 1998; Seguí-Simarro et al., 2003, 2005; for review, see Koster and Klumperman, 2003). Our results showed that the cryoprocessing and freeze-substitution method followed by low-temperature embedding in an acrylic resin, was fully adequate for soybean cells, which displayed an excellent ultrastructural preservation of the different subcellular compartments, including chloroplasts and thylakoid membranes (Fig. 2, A and B). Immunogold labeling with the GmFAD7 antiserum was mostly found decorating the chloroplasts, labeling appearing either as isolated or clustered particles (Fig. 2, C–E). Some labeling could be found on the chloroplast envelope (Fig. 2C), but most gold particles were observed preferentially over thylakoid membranes, whereas the stroma appeared mostly free of labeling (Fig. 2, C–E). Interestingly, most part of the gold particles that appeared in the thylakoid membranes were located in regions that had access to the stroma (black arrowheads in Fig. 2, C–E), while only a low proportion of them (18% of the gold particles over thylakoids) were found inside the grana stacks (white arrowheads in Fig. 2, C–E). A few gold particles could be found over the rest of the cytoplasm, constituting the nonspecific background provided by the antibody. Immunogold control experiments replacing the first antibody by the preimmune serum or phosphate-buffered saline (PBS) did not provide any significant labeling (data not shown).

View larger version (130K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Anti-GmFAD7 immunogold labeling on soybean photosynthetic cell cultures. Ultrathin Lowicryl sections. A, General view of soybean cells. B, General view of chloroplasts. C, D, and E correspond to sections showing immunogold labeling distribution. Gold particles were localized in envelope (C; black arrow) and thylakoid membranes (C, D, and E; arrowheads). Difference is shown between those particles located in the thylakoid in regions that had access to the stroma (black arrowheads) and those located inside the grana stacks (white arrowheads). V, Vacuole; N, nucleus; R, stroma; T, thylakoid membranes; S, starch granules. Bars in A and B represent 0.5 µm, and in C, D, and E represent 200 nm.

Biochemical Fractionation Studies for Subcellular FAD7 Protein Localization

To complete the subcellular localization study and to obtain additional information about the distribution of FAD7 in the different chloroplast subcompartments, we performed western-blot experiments on purified plastid subfractions (i.e. envelope membranes, stroma, and thylakoid). To purify plastid subfractions, Percoll-purified intact chloroplasts were lyzed in hypotonic medium and the three fractions were separated on a Suc gradient. The results obtained from the western-blot analysis of the purified fractions are shown in Figure 3 . Several antibodies were used as specific markers of the different chloroplast subfractions. The Tic 110 protein was used as a specific envelope marker (Jackson et al., 1998). Antibodies against the light-harvesting complex II (LHCII) antenna protein, one of the components of PSII, were used for thylakoid membrane validation. Rubisco, the major stroma protein, was used as a marker of this chloroplast subfraction. As shown in Figure 3, the Tic 110 protein was only detectable in the envelope fraction while it was hardly visible in any of the other fractions. Tic 110 was not detectable in chloroplasts probably because its low abundance with respect to other plastid proteins. Rubisco was present in chloroplasts and stroma fractions while it was almost undetectable in the other fractions, indicating that the fractionation procedure was correct. The LHCII, which is a major thylakoid protein, was highly enriched in the thylakoid fractions as expected (Fig. 3). A significant amount was also detected in the envelope fraction (Fig. 3). The presence of LHCII in envelope fractions has been reported as a contaminant even in very pure preparations used for proteomic studies (see Ferro et al., 2003). However, we cannot discard that a part of this LHCII pool found repeatedly in envelope preparations could reflect a certain pool of LHCII in transit through its import route to the thylakoid. Being the most likely source of membrane contamination of the purified envelope fraction, thylakoid cross contamination was precisely assessed. Chlorophyll being the most conspicuous thylakoid component, we analyzed the presence of this pigment in the envelope membrane preparations. On a protein basis, the ratio of chlorophyll content detected in the purified envelope and thylakoid membranes was 1:25. This value is within the range reported by other groups during the isolation of envelope membranes for proteomic studies (Ferro et al., 2003; Froehlich et al., 2003). According to this, we can estimate thylakoid cross contamination of envelope membranes between 5% to 10%, depending on the preparations. This result indicated that the envelope fraction was highly pure. Interestingly, when the antibody against the GmFAD7 protein was tested, the major amount of FAD7 protein was detected in the thylakoid membranes while very low amounts were detectable in the envelope fractions (Fig. 3). No FAD7 protein was detected in the stroma fraction. These results further confirmed the results obtained by immunogold labeling, indicating that the FAD7 3 plastid desaturase is preferentially located in the thylakoid membrane.

View larger version (62K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. GmFAD7 protein distribution in chloroplast subfractions. Percoll-purified intact chloroplasts were lyzed and subjected to Suc gradient fractionation into envelope, stroma, and thylakoid fractions. Proteins were separated by SDS-PAGE and blotted against the GmFAD7 antibody and antibodies specific of each chloroplast subcompartment. Anti-Tic110 was specific of the envelope membranes, Rubisco was specific of the stroma, and the anti-LHCII was used as marker of the thylakoid membranes. 19, 16, 12.5, and 19 µg of total proteins from purified chloroplasts, thylakoids, stroma, and envelope fractions, respectively, were loaded per lane.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The presence in the thylakoid of FAD7 suggests that thylakoid membranes could be sites of TA fatty acid production in plants. In the chloroplast, the major reservoir of polyunsaturated fatty acids are the galactolipids present in the thylakoid membranes. In fact, they represent about 70% to 80% of the total amount of lipids in the chloroplast. Interestingly, our results indicate that the 3 desaturase responsible for TA fatty acid production in the plastid is preferentially located in the same membrane where its product (18:3) is accumulated. In that sense, fatty acid desaturases need electron donors for their function. In the plastids, reduced ferredoxin provides the electrons necessary for FAD6 and FAD7 activity (Schmidt and Heinz, 1990a). Alternatively, several electron paramagnetic resonance signals, corresponding to iron-sulfur clusters, semiquinones, or flavins were detected in envelope preparations and proposed as components of an alternative electron transport chain that would facilitate electrons to the desaturases in the envelope (Jäger-Vottero et al., 1997). However, no specific components of these chains have ever been isolated or unambiguously identified. In principle, there would be no constraint for ferredoxin to donate its electrons to the desaturases present in the envelope. Similarly, our immunogold results suggest that most part of the FAD7 protein detected in the thylakoid is located in regions that have access to the stroma and thus are also accessible to ferredoxin. In fact, our results place the desaturase in close vicinity with its major electron donor (ferredoxin), making it unnecessary to invoke the presence of any other alternative electron carriers.

Another situation in which the thylakoidal localization of FAD7 should be relevant is related with the implication of this enzyme in the biosynthesis of JAs. JA was shown to be synthetized in the plastids from linolenic acid (18:3). Linolenic acid is first oxygenated by lipoxygenase (LOX) to yield 13(S)-hydroxyperoxy linolenic acid. Further cyclization is achieved by the consecutive action of two enzymes, namely, allene oxide synthase and allene oxide cyclase (Vick and Zimmerman, 1979; Schaller et al., 2005). There are evidences that either free fatty acids or fatty acids esterified to membrane lipids could be substrates for LOX; a soybean 13-LOX has been shown to act on polyunsaturated fatty acids esterified to neutral membrane lipids (Fuller et al., 2001; Feussner and Wasternack, 2002). In fact, it was shown in Arabidopsis that most 12-oxophytodienoic acid (an intermediate of jasmonic acid synthesis) was bound to the sn-1 position of monogalactosyldiacylglycerol (Stelmach et al., 2001). It has been proposed that these differences could contribute, with temporal differentiation of activity, to an orchestration of the formation of hydroxyl PUFAs (Liavonchanka and Feussner, 2006). In addition, proteomic studies in Arabidopsis have identified allene oxide synthase (At5g42650.1) and two isoforms of the allene oxide cyclase family (At3g25760.1 and At3g25770.1) as components of the thylakoid proteome as thylakoid peripheral proteins at the stromal side (Peltier et al., 2004; accessible via Plant Proteome DataBase). The preferential localization within the thylakoid of the GmFAD7 protein, as evidenced with our results, is consistent with a similar location of these enzymes that are at the early beginning of JA biosynthetic pathway. Altogether, these data suggest that in addition to the envelope, thylakoid membranes should play a role in JA synthesis and thus in plant defense-signaling responses where oxylipins like JA are involved.

In conclusion, the results presented in this article provide experimental evidence for the thylakoidal localization of FAD7 in the plastids. These data strongly suggest that thylakoid membranes could be sites of lipid desaturation in plants as it seems to be the case in cyanobacteria. These findings provide new information concerning the role of plant fatty acid desaturases, not only in the lipid synthesis metabolism but also in the environmental stress response and in plant defense-signaling pathways.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Growth Conditions

Photosynthetic cell suspensions from soybean (Glycine max var. Corsoy) SB-P line were grown as described by Rogers et al. (1987) with some modifications (Alfonso et al., 1996). Liquid cultures were grown in KN¹ medium with a supplement of 1% Suc under continuous light (30 ± 5 µE m^–2 s^–1) and an atmosphere with 5% CO₂ at 24°C on a rotatory shaker at 110 rpm. For immunolocalization experiments, soybean cells were grown in 1.5% (w/v) agar plates with KN¹ medium at 24°C and an atmosphere with 5% CO₂. Cells cultured under these conditions were easier to handle during the fixation and sectioning procedures than liquid suspensions. For biochemical fractionation purposes and because of the difficulties for obtaining enough material, soybean plants instead of cultured cells were used. Soybean plants were grown hydroponically in one-half Hoagland solution in a growth chamber with 350 µE m^–2 s^–1 and 70% relative humidity at 24°C.

Purification of Chloroplast and Chloroplast Subfractions from Soybean

All procedures were carried out in a cold chamber (4°C–8°C) under a dim green light. Isolation of intact chloroplasts from soybean on Percoll gradients was carried out essentially as described in van Wijck et al. (1995). For subchloroplast fractionation, intact chloroplasts isolated from 50 g of soybean leaves were resuspended in 15 mM NaCl, 5 mM Mg Cl₂, 50 mM MES, pH 6.0. Then, an equal volume of Tris-EDTA (TE) buffer (2 mM EDTA, 10 mM Tris-HCl, pH 7.5) was added and the chloroplasts were kept on ice for 10 min. The chloroplasts were subjected to Suc step gradient fractionation as previously described (Li et al., 1991). The discontinuous Suc gradient was prepared with a 1.2, 1, and 0.46 M Suc solutions in TE buffer. After loading the chloroplasts, the samples were centrifuged at 39.000 rpm for 70 min in a Beckman centrifuge using a swinging bucket rotor (SW41Ti). After gradient separation, the envelope (a mixture of inner and outer membranes) was obtained as a yellow-green band in the interface of the 0.46 to 1 M Suc cushion; the stroma was retrieved from the supernatant and the thylakoid fractions were collected as a green pellet at the bottom of the tube. The envelope fraction was carefully removed, diluted with three volumes of TE buffer, and centrifuged at 20.000 rpm for 45 min in a 70.1 Ti rotor. After centrifugation, the envelope pellet was resuspended in 50 µL of TE buffer. Proteins in the stromal fraction were concentrated by acetone precipitation. Thylakoid membranes were resuspended in TE buffer and centrifuged at 7.500 rpm for 10 min. The pellet was again resuspended in TE buffer. All solutions contained a cocktail of protease inhibitors: antipain (2 µg mL^–1, leupeptin 2 µg mL^–1, and Pefabloc 100 µg mL^–1). To verify recovery and purity of the Suc density fractions several antibodies were used as markers: Tic110 was used as envelope marker (Jackson et al., 1998), anti-RuBisCO was used as stromal marker, and anti-LHC II was used as thylakoid membrane marker.

Production of the GmFAD7 Antiserum and Western-Blotting Analysis

Antiserum was obtained from rabbits that had been immunized with the synthetic oligopeptide VASIEEEQKSVDLTNG, which corresponds to residues 69 to 84 of the deduced amino acid sequence of the GmFAD7 protein at Sigma Genosys Custom Antibody Service (Sigma-Genosys). This antiserum (200-fold dilution) was used in western analysis for the detection of GmFAD7 protein. Total protein (approximately 20 µg, depending of the fractions) was loaded per lane. Protein content was estimated using the BIO-RAD protein assay reagent. Western-blot procedures were carried out essentially as described (Collados et al., 2006) except that detection was carried out using a highly sensitive chemiluminiscent reagent for peroxidase detection (SuperSignal West Pico, Pierce). Densitometry analysis was performed with the Quantity One Software (Bio-Rad).

Sample Processing for Structural and Cytochemical Analysis and for Immunofluorescence

Soybean cells were fixed overnight at 4°C in formaldehyde 4% (w/v) in PBS, pH 7.3, and washed with PBS. Some samples were stored at 4°C for direct sectioning in a vibratome and further use for immunofluorescence. Other samples were dehydrated through an acetone series (30%, 50%, 70%, and 100% [v/v]), infiltrated, and embedded in Historesin 8100 at 4°C. Semithin sections (1 µm thickness) were obtained and used for light microscopy observations. Toluidine blue-stained semithin sections were observed under bright and phase contrast field for structural analysis in a Leitz microscope fitted with a digital camera Olympus DP10.

Cytochemical Stainings for Starch and DNA

Starch was detected by I₂KI staining (O'Brien and McCully, 1981) on Historesin semithin sections and observed under bright field (Barany et al., 2005) in a Leitz microscope coupled to an Olympus DP10 digital camera. DAPI staining for DNA was applied to semithin sections (Testillano et al., 1995) and observed under UV light in a Zeiss Axiophot epifluorescence microscope fitted with a CCD camera.

Immunofluorescence and Confocal Laser Microscopy

Immunofluorescence was performed on vibratome sections as previously described (Fortes et al., 2004). Vibratome sections of 30 µm (Vibratome1000, Formely Lancer) obtained from fixed soybean cells (see above) were placed onto 3-aminopropyltrietoxysilane coated slices and treated for permeabilization purposes. First, they were dehydrated (30%, 50%, 70%, 100% [v/v]) and rehydrated (100%, 70%, 50%, 30% [v/v]) in PBS:methanol series. Second, sections were treated with 2% (w/v) cellulose (Onozuka R-10) in PBS for 45 min at room temperature. After three washes in PBS for 5 min, sections were incubated with 5% (w/v) bovine serum albumin (BSA) in PBS for 5 min and then in anti-GmFAD7 polyclonal antibodies, diluted 1:25, respectively, in 1% (w/v) BSA for 1 h at room temperature. After washing twice with PBS for 10 min each, the signal was revealed with either ALEXA 488 (green fluorescence)-conjugated anti-rabbit antibodies (Molecular Probes) diluted 1:25 in 1% (w/v) BSA for 1 h in the dark at room temperature. Finally, the sections were washed twice with PBS for 10 min, stained with DAPI (Serva) for 10 min, washed with milli-Q water, and mounted with Mowiol 4 to 88 (Polysciences). Confocal optical section stacks were collected using a confocal laser scanning microscope Leica TCS-SP and laser excitation lines of 348 (UV), 488 (blue), and 633 (far-red) nm wavelength to reveal DAPI (nuclei), anti-GmFAD7 immunofluorescence signal, and chlorophyll autofluorescence (chloroplast), respectively. Controls were performed by replacing the first antibody by preimmune serum or PBS.

Control Experiment by Antibody Preblocking (Immunodepletion Experiment)

Preblocking of the anti-GmFAD7 antibody with its corresponding immunogen, the synthetic oligopeptide used to raise the serum (Collados et al., 2006), was performed by incubating the antibody with a 1 mM solution of the peptide in a proportion of 1:2 (v:v) at 4°C overnight. The preblocked antibody solution was used as primary antibody for immunofluorescence on vibratome sections, following the same protocol and conditions as previously described.

Freeze Substitution and Low-Temperature Embedding for Immunoelectron Microscopy

Soybean cells were fixed overnight at 4°C in formaldehyde 4% (w/v) in PBS, pH 7.3, and washed with PBS. Fixed cells were stored in a 0.1% paraformaldehyde solution in PBS at 4°C to prevent reversion of the fixation. Fixed soybean cells were cryoprotected by immersion in Suc:PBS solution at the following concentrations and times: 0.1 M for 1 h, 1 M for 1 h, and 2.3 M overnight, at 4°C. Then, the specimens were put in cryoultramicrotomy pins and cryofixed by rapid plunging into nitrogen liquid at –190°C. After that, the samples were dehydrated by freeze substitution in an Automatic Freeze-substitution system (AFS, Leica) essentially as described in Seguí-Simarro et al. (2005). Cryofixed samples were immersed in pure methanol containing 0.5% (w/v) uranyl acetate at –80°C for 3 d, and then the temperature was slowly (during 18 h) warmed to –30°C. After three washings in pure methanol, 30 min each at –30°C, samples were infiltrated and embedded in Lowicryl K4M at –30°C under UV. Ultrathin sections were obtained in an ultramicrotome (Ultracut Reichert) and collected on 200-mesh nickel grids having a carbon-coated Formvar-supporting film.

Immunogold Labeling

Nickel grids carrying ultrathin Lowicryl sections were sequentially floated in PBS, 5% (w/v) BSA in PBS, and undiluted anti-GmFAD7 antibody, for 1 h. After several washes in 0.1% (w/v) BSA in PBS, the grids were incubated with a secondary antibody, anti-rabbit IgG conjugated to 10 nm gold particles (Biocell) diluted 1:25 in 1% (w/v) BSA, for 1 h at room temperature, washed in PBS and water, air dried, counterstained with uranyl acetate and lead citrate, and observed in a JEOL 1010 EM at 80 kV. Controls were performed excluding the primary antibody.

Quantitative Analysis of Immunogold Labeling Density

Sampling was carried out over selected samples on each grid. The number of micrographs to be taken was determined using the progressive mean test, with a maximum confidence limit of = 0.05. The labeling density was defined as the number of gold particles per area unit (µm²). Particles were hand counted over the cellular compartment under study (chloroplast) and over other cytoplasm regions. The area in µm² was measured using a square lattice composed by squares of 15 x 15 mm each. The labeling density was expressed as the mean density ± SD.

	ACKNOWLEDGMENTS

 
The SB-P line was kindly provided by Professor Jack M. Widholm (Department of Agronomy, University of Illinois, Urbana). The Tic 110 antibody was kindly provided by Dr. J.E. Froehlich (Department of Energy, Plant Research Laboratory, Michigan State University).

Received September 24, 2007; accepted October 4, 2007; published October 19, 2007.

	FOOTNOTES

 
¹ This work was supported by the Aragón Government (grant no. PIP 140/2005) and the Ministry of Education and Culture of Spain (grant nos. BFU2005–07422–C02–01, AGL2005–05104, BFU2005–01094, and PIP2006–120). R.C. and V.A. were recipients of a predoctoral fellowship from the Aragón Government.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for authors (www.plantphysiol.org) is: Miguel Alfonso (alfonso{at}eead.csic.es).

www.plantphysiol.org/cgi/doi/10.1104/pp.107.109637

^* Corresponding author; e-mail alfonso{at}eead.csic.es.

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