Figure 6.

Subcellular localization of recombinant-oleosin-attached LDs, with emphasis on the relative locations of LDs, PSVs, and vacuoles, in tobacco cells after stable transformation. A, In linear portions (following those described in Fig. 2 legend), OLE and s-p-OLE-10. s-p-OLE-10 has at its N terminus an attached ER-targeting peptide (bean phaesolin) followed by a 12-residue PSV-targeting propeptide (p; SLLIRPVVPNFN, castor ricin; Frigerio et al., 2001). B, Images of portions of 2-week-old cells after stable transformation of DNA constructs encoding the two oleosins with the C terminus attached to GFP. Some cells were also cotransformed with a DNA construct encoding s-p-RFP (marker of PSVs). Fluorescence of GFP, RFP, and Nile Red (NR) was monitored with CLSM. Bars = 10 or 2 μm. C, TEM images of portions of transformed cells containing OLE or s-p-OLE-10 after chemical fixation with glutaldehyde and then osmium. Chemical fixation allowed for a clear distinction between PSV (clear) and LD (grayish) structures, whereas high-pressure freezing fixation (no osmium) resulted in fairly similar, clear background of PSVs and LDs but better preservation of membranes (used for Fig. 6). LDs and PSVs (V) in OLE cells were not associated but were often associated (including LDs inside vacuoles) in s-p-OLE-10 cells. Arrows indicate potential vacuole membrane. Bars = 0.2 μm.