Figure 7

FRET between VHA-subunits co-expressed in protoplasts of A. thaliana and onion epidermis cells. (A) Mesophyll protoplasts of A. thaliana were simultaneously transformed with p35S::VHA-a/YFP and p35S::VHA-c/CFP using the polyethylene glycol method. After 20 h, fluorescence emission from protoplasts was measured following excitation at 458 nm and 514 nm, and image analysis in the range of 470 – 500 nm for CFP and 560 – 585 nm for YFP fluorescence, respectively. (B) Acceptor bleaching in dependence on scan numbers. For this experiment, protoplasts expressing VHA-a-YFP were excited at 514 nm and emission was recorded between 550 and 600 nm. (C) Emission spectra of VHA-c-CFP and VHA-a-YFP before (solid line) and after (broken line) acceptor bleaching. (D) FRET between VHA-A-YFP and VHA-B-CFP after co-expression in onion epidermis cells. Excitation was achieved at 458 nm, and 2D emission images were recorded in the range of 470 to 510 nm (CFP), and 550 to 600 nm (YFP).