Figure 2

Subcellular localization of green fluorescent protein and green fluorescent protein-elastin-like polypeptide targeted to the cytoplasm, chloroplasts and apoplast. The localization constructs were agro-infiltrated into Nicotiana benthamiana leaves and visualized by confocal microscopy. The pG (A) and pGE (B) proteins were both visible as diffuse expression surrounding variously shaped and sized organelles throughout the cell. Green fluorescent protein (GFP) fluorescence was most concentrated in the cytoplasmic strands and the nucleus. In mesophyll cells expressing chloroplast-targeted GFP (pRG), the GFP fluorescence was localized to the chloroplasts (C), which was confirmed by also detecting the chlorophyll autofluorescence (D). (E) Merged image of (C) and (D) showing complete co-localization of pRG and the chloroplasts. In the presence of an elastin-like polypeptide (ELP) fusion tag, the chloroplast-targeted GFP (pRGE) appeared to accumulate in the cytoplasm (F) and was excluded from the chloroplasts (G). (H) Merged image of (F) and (G), demonstrating that an ELP fusion tag prevents the accumulation of GFP in the chloroplasts. For both pPG (I) and pPGE (J), the images were taken from a cross-section of the cells showing a secreted pattern of fluorescence consistent with apoplast localization. Bar, 10 μm (A, B, I, J); 5 μm (C to H).