In situ molecular identification of the plastid omega3 fatty acid desaturase FAD7 from soybean: evidence of thylakoid membrane localization

Abstract

omega3 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 omega3 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.

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.

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.

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.