(A–C) Scanning electron micrograph of a flower bud at 9 DAI, showing the dorsal (D, red shading), lateral (L, orange shading) and ventral (V, yellow shading) petals (A). Confocal images of PIN1 section-immuno (red signal) in calcofluor stained petal tissue (cyan signal) with a longitudinal section (B, position of section indicated by blue line in A) and a frontal section (C, position of section indicated by magenta line in A). The PIN1 signal is strong in the epidermis, where polarity points proximodistally towards the tip of the petal (white arrows in petal tip, yellow box inset in B) and in the internal tissues where the vascular tissues starts to develop. This PIN1 pattern is confirmed in a frontal section, where the epidermal PIN1 converges towards the most distal cells at the tip of the petal while internally, the PIN1 polarity in the sub-epidermal cells points towards the developing vascular strand (white arrows in green box inset of C). White line in B-C marks the petal edge. Px, proximal, Di, distal. Scale bar (A, B and C), 100 μm. Scale bar (D and E), 10 μm. (D-I) Confocal images of PIN1 whole-mount immuno in div (D, F and H) and wild-type (E, G and I) ventral-lateral sinus at 11 DAI (D and E), 13 DAI (F and G) and 15 DAI (H and I). Polarity of the PIN1 subcellular signal was estimated using the PinPoint software and is labelled with white arrow heads. Initially, PIN1 expression (red) is observed at the petal sinus and its cellular polarity is largely proximodistal at the sinus and proximomarginal along the marginal cells pointing towards the tips of the petals in both div and wild-type (D and E). Although the expression expands in div, its polarity pattern is maintained proximodistal (F). In wild type the extended PIN1 region is larger than in div (compare G to F) and is oriented proximodistally along the petal junction (3–4 central files) (files within green dashed line in H and zoomed-in image in blue box) while either side of this junction, polarity points diagonally, towards the central files and nearby sinus (G). At day 15, PIN1 expression at the sinus becomes restricted to the petal margins, both in div and wild-type (H and I). Scale bar, 10 μm. (J and K) PIN1 whole-mount immuno in wild-type lateral-dorsal sinus at 11 DAI (J) and 13 DAI (K). PIN1 signal is restricted to the sinus region where it shows a proximodistal polarity in the margin cells (next to white line) and a proximomarginal polarity in the cells adjacent to the marginal cells (J). The PIN1 expression at the lateral-dorsal junction is transient, as at 13 DAI no signal is observed around the sinus region (blue arrow) (K). Vascular strands have strong PIN1 signal (strands marked with asterisk). Scale bar, 10 μm. (L) Images of cells showing PIN1 in red (leftmost column), calcofluor cell wall staining in blue (second column), and composite image of PIN1 and calcofluor for use with the PinPoint software (third column). Sample cell segmentations are shown in yellow and polarity directions calculated from PinPoint software are indicated with yellow arrows (rightmost column).