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Figure 2 | BMC Plant Biology

Figure 2

From: The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

Figure 2

AtAPY1 is present in intracellular dot-like structures, but absent from the extracellular space. CLSM (A, B, E-J) and fluorescence microscopy (C, D) images of various cell types are depicted. All WT control images were captured using the identical CLSM settings as for the corresponding images of the transgenic plants. The FITC, Alexa Fluor 488 and GFP fluorescence is shown in green, the chlorophyll autofluorescence in magenta. The fluorescence signals are overlaid with a bright field image in A, B, E and F. Scale bars = 20 μm. (A, B) Root hairs of DKO-SNAP and WT seedlings were fixed and successively incubated with α-SNAP antibodies and secondary α-rabbit IgG coupled with horseradish peroxidase. FITC tyramides were added to amplify the fluorescence signal. The parameters of the indirect immunofluorescence detection were modified in multiple independent experiments until FITC-specific fluorescence signals as shown here were obtained. (C, D)Two hundred-nm Tokuyasu cryo-sections through root tips of AtAPY1-GFP expressing seedlings were fixed and incubated with (C) or without (D) α-GFP antibodies. All samples were incubated with secondary α-rabbit Fab fragments coupled with Alexa Fluor 488. The pictures (C) and (D) were taken with the same exposure times. (E, F) Two guard cells of a 35S::AtAPY1-GFP transgenic and WT seedling are depicted. The dot-like green fluorescent signals were obtained in at least 20 independent live imagings. (G) A protoplast expressing AtAPY1-GFP and a WT protoplast (H), both prepared from cotyledons, were imaged. Overlays of the green and magenta fluorescent signals are shown. These images represent the results from three independent protoplastations. (I, J) The GFP fluorescence and the bright field image of the same epidermal section of a cotyledon from an AtAPY1-GFP overexpressing seedling grown under alkaline conditions are shown representative of the imaging results from three seedlings. Doughnut- or horseshoe-shaped fluorescent structures typical of Golgi stacks imaged from the top are indicated by white arrows.

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