Fig. 4: TOB1-mediated vacuole internalization of IBA terminates with PIN2 polar accumulation at cotyledon’s marginal cells.

a TOB1 consolidates its tonoplast localization in MCs overtime. TOB1p::YFP-TOB1 (YFP-TOB1) reticular signal at 48 hours after plating (HAP) changes to tonoplast (ring-like) signal at 60 HAP. To the right of each micrograph, a schematic shows the relationship between PIN2 and TOB1 protein levels. At 48 HAP, PIN2-GFP is abundantly expressed whereas TOB1 displays weak reticular expression pattern. At 60 HAP, PIN2-GFP levels are substantially lower and TOB1 shows clear tonoplast accumulation. Similar results were observed in 4 independent experiments. The complete time course analysis from 24 to 96 HAP can be found in Supplementary Fig. 5. Scale bar size = 20 µm. b Tonoplast marker CFP-vac-ck signal co-localize with YFP-TOB1 signal in 60 HAP cotyledon’s MCs. White empty arrowhead indicates the superimposition of CFP-vac-ck with YFP-TOB1 signal. Similar results were observed in 3 independent experiments. Scale bar size = 10 µm. c TOB1 and PIN2 accumulation pattern are inversely correlated. Spectral confocal microscopy image of PIN2-GFP + / + X YFP-TOB1 +/+ cotyledons grown for 48, 60, 72 hours after plating (HAP). White arrowheads indicate the progressive disappearance of PIN2-GFP signal. Empty red arrowheads denote the reticular YFP-TOB1 expression. Red arrowheads indicate the progressive appearance of tonoplast YFP-TOB1 signal. Similar results were observed in 5 independent experiments. Scale bar size = 100 µm.