Fig. 1: PIN2 accumulation in MCs precedes and plays a role in coordinating PC morphogenesis.

a Polar PIN2-GFP in the apical side of marginal cells (MCs) of 48 Hours After Plating (HAP) Arabidopsis cotyledons. Image corresponds to a maximum intensity projection of a z-stack capturing all perceptible signal. Scale bar = 100 µm. Zoom-in indicates the predicted direction of auxin flow with the white arrows from the base to the tip of the cotyledon. Note: Green signal at the cotyledon tip is not GFP, see Supplementary Fig. 2a. b Schematic of the areas analyzed on each cotyledon. c, d PIN2 accumulates transiently prior to PC morphogenesis. c Cell shapes visualized by PI (upper panel) staining at the adaxial side of cotyledons expressing PIN2-GFP (lower panel) and grown for 24, 48, 72 hours after plating (HAP). Shown are representative images captured in the center (PI) and in the border of the cotyledon (PIN2-GFP). Scale bar = 10 µm. d Quantification of the interdigitation levels (magenta) and PIN2-GFP accumulation level (green) in 12 to 96 HAP cotyledons quantified as indicated in b. Interdigitation levels are expressed as 1-circularity, i.e., the greater the value, the greater the degree of interdigitation. Circularity is defined as 4π area/perimeter². PIN2 accumulation levels is quantified as the percentage in length of the border from base to tip that is expressing PIN2. Data is shown in arbitrary units (arb. units) as mean ± SD. n = 12 cotyledons from different seedlings. e, f Reduced auxin transcriptional response in 48 HAP eir1-1 (pin2) mutant. e Reduced DR5::GUS signal at the cotyledon’s tip in eir1-1 (pin2) compared to wild type/wt. Representative images are shown. The GUS signal was similarly displayed in 95% (19/20) and 84% (21/25) of the cotyledons assayed for wt and eir1-1, respectively. Scale bar =100 µm. f Reduced GUS activity in cotyledons eir1-1 (pin2) measured by fluorometric quantification of 4-MU. GUS activity is shown as nanomolar units of 4-MU per sample. Unpaired two-tailed t-test. **** p-value < 0.0001. n = 16 samples. Sample is defined as the 2 cotyledons in a seedling. g, h Loss-of-function mutant eir1-1 (pin2) showed reduced interdigitation in pavement cells at the cotyledon tip. g Representative images of wild type and eir1-1 (pin2) cotyledons. Boxes indicate areas to be quantified. Zoom-in shows the clear reduced interdigitation of pavement cells at the cotyledon’s tip. Scale bar =100 µm. h Interdigitation level (1-circularity) in 3 areas of identical dimensions: tip, middle, and base for wild type Col-0 (green dots) and PIN2 mutant allele eir1-1 (pin2) (gray dots) in 60 HAP cotyledons. n = 90 cells. Data was obtained from 5 different seedlings. two-way ANOVA using Šídák’s multiple comparisons test. ****p-value < 0.0001.