Figure 4

Windowing the time allowed for patch-clamp recordings. (A) Confocal microscopy sections of tobacco mesophyll protoplasts transformed with empty "localisation" plasmids (control protoplasts). Left panel: protoplast analysed for GFP detection. Middle and right panels: images of protoplasts bathing in calcofluor dye solution respectively without and with wall. Bar = 20 μm. (B) Time-course of GFP expression in transformed tobacco mesophyll protoplasts. The number of cells displaying GFP expression at a given time after transformation is expressed as a percentage of the number of cells which finally (55 hours after transformation) expressed GFP. Closed symbols: 3 independent transformations with empty pLoc ("GFP"). Open symbols: 2 independent transformations with pFunct+Tag-KAT1 ("channel+GFP"). About 500 transformed cells were considered for each experiment. Line represents exponential fit of the data. (C) Time-course of cell wall regeneration. The cell wall was marked with calcofluor dye. A cell was considered to have a wall if part of its surface showed blue staining. Each point represents about 200 protoplasts. Triangles and circles represent protoplasts transformed respectively with empty pLoc and pFunct+Tag-KAT1. Dark symbols represent all the protoplasts and open symbols those displaying green fluorescence. Line represents sigmoidal fit of the data. (D) Operational time window for patch-clamp recordings. Superimposition of the GFP apparition and cell wall synthesis fitted curves allows determination of a time frame for patch-clamp experiments (see text). (E) Time-course of the amplitude of steady-state currents recorded at -200 mV in tobacco mesophyll protoplasts transformed with pFunct+Tag-KAT1 (dark symbols) or pFunct+Tag-AKT1 (open symbols).