Figure 2.

Subcellular localization of Chlase in ethylene-treated citrus fruit peel by in situ immunofluorescence. Mature green lemon fruit were treated with ethylene (20 μL L⁻¹) at 25°C in the dark for 24 h in a 4-L sealed container. The container was ventilated once after 12 h, followed by injection of fresh ethylene, to maintain CO₂ levels below 1%. The flavedo of the treated fruit was dissected and used for obtaining cross sections (70 μm thick) using a vibratome (see “Materials and Methods”). Tissue sections were fixed and dressed with Alexa-488-conjugated goat anti-rabbit secondary antibody as a control (A) or affinity-purified anti-citrus Chlase antibody (rabbit) followed by Alexa-488-conjugated goat anti-rabbit secondary antibody (B and C). Fluorescence was visualized using a laser scanning confocal microscope as described in “Materials and Methods.” Images of representative cells (A and B) are presented as follows: 1, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by anti-Chlase antibody; 2, red fluorescence corresponds to chlorophyll autofluorescence; 3, confocal image recorded simultaneously in transmitted and red fluorescence mode (i.e. chlorophyll fluorescence superimposed on the bright-field image); and 4, confocal image recorded simultaneously for red and green fluorescence (i.e. immunofluorescence resulting from detection by anti-Chlase superimposed on the chlorophyll autofluorescence). Chloroplasts demonstrating green fluorescence (i.e. detection by anti-Chlase antibody) were further analyzed by the confocal microscope colocalization program analysis tool. Images of representative chloroplasts (C) are presented as follows: 1, red fluorescence corresponds to chlorophyll autofluorescence; 2, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by anti-Chlase antibody; and 3, sectors of yellow color indicate colocalization of chlorophyll autofluorescence and immunofluorescence resulting from detection by anti-Chlase antibody. Colocalized sectors in the fluorescing chloroplasts account for 76% of the total fluorescence observed. Scale bar = 10 μm (A and B) and 2 μm (C).