Fig. 5: TOB1 regulates PIN2 levels in Arabidopsis cotyledon marginal cells.

a PIN2 levels increase in tob1 mutants. Confocal images of PIN2-GFP cotyledon in Col-0, tob1-1-/- and tob1-3-/- mutant background, generated by introgression. All lines are non-segregant F3 PIN2-GFP + /+. Images are z-stack maximum projection capturing all detectable fluorescence signal. Scale Bar = 100 µm. b Quantification reflecting PIN2-GFP levels in tob1 mutants. For quantification, a region of interest was defined around the entire file of MCs. Box plot shows all points, the box indicates the 25th and 75th percentile and the median value, the whiskers indicate minimum and maximum values. n = 6 cotyledons, 60 HAP. Unpaired two-tailed t-test, p-value = 0.0185 for Col-0 vs tob1-1, p-value = 0.0158 for Col-0 vs tob1-3, * p-value < 0.05. c Auxin transcriptional response distribution pattern revealed by DR5-GUS histochemical analysis in 96 HAP cotyledons of wild type, tob1-1 -/- and tob1-3 -/-. All lines are non-segregant F3 DR5-GUS +/+. Scale bar = 400 µm. d GUS activity in cotyledons tob1-1 -/- and tob1-3 -/- measured by fluorometric quantification of 4-MU. GUS activity is shown as nanomolar units of 4-MU per sample. Welch’s ANOVA with Dunnett’s T3 for multiple comparisons test. **** p-value < 0.0001. n = 22, 16, 23 samples for Col-0, tob1-1, tob1-3. Sample is defined as the 2 cotyledons in a seedling. e Auxin induced TOB1 accumulation. TOB1p::YFP-TOB1 cotyledons grown for 24 HAP were treated during 4 h with mock or 100 nM IAA. Scale bar = 100 µm. f Quantification of fluorescence intensity of YFP-TOB1 along the cotyledon’s MCs upon auxin treatment. Images are z-stack maximum projection capturing all detectable fluorescence signal. n = 9 cotyledons. Unpaired two-tailed t-test. p-value = 0.0471, * p-value < 0.05.