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. 2018 Apr 23;9:430. doi: 10.3389/fpls.2018.00430

FIGURE 5.

FIGURE 5

Control experiments for the establishment of a protoplast esculin assay. (A–H,J) Protoplasts expressing different GFP-fusion constructs were incubated with 1 mM esculin in W5 buffer (pH5.6) for 40 min. GFP is given in green, esculin fluorescence in cyan and chlorophyll autofluorescence in red. (A) Overview image of Col-0 protoplasts transformed with p35S:AtSUC2c-GFP. (B) Bright field to (A). (C) Col-0 protoplast expressing p35S:GFP-STP10c. (D) Individual protoplast expressing p35S:AtSUC2c-GFP with esculin in the vacuole at higher magnification. (E) Protoplast of a Attmt1/tmt2 knockout-plant transformed with p35S:AtSUC2c-GFP. (F) Col-0 protoplasts with p35S:AtSUC2c-GFP. The arrowhead indicates a small non-transformed protoplast showing esculin uptake. (G) Companion cell protoplast of a pEPS1 line (stably transformed with pAtSUC2:GFP), labeled by cytosolic GFP. (H) GFP-labeled companion cell protoplast of pMH5a (stably transformed with a pAtSUC2:erGFP construct). (I) Leaf epidermal peel of Col-0 incubated with 1 mM esculin in W5 for 1 h. Overlay of bright field, esculin and chlorophyll fluorescence. The arrowhead points to a guard cell, the asterisk marks a neighboring subsidiary cell with esculin fluorescence. (J) WT protoplasts expressing p35S:AtSUC2c-GFP at different levels. (K) Correlation of GFP fluorescence in the plasma membrane and esculin fluorescence intensity in the vacuole of protoplasts transformed with p35S:AtSUC2c-GFP. Fluorescence intensities were normalized to the radius of the respective protoplast under the assumption of a spherical shape (n = 115). Scale bars: 50 μm in (A,B), 10 μm in (C–J).