Figure 12. Auxin depletion alters boundary position.

(A and B) Confocal projections of the IMs showing expression pattern of REV-2 × YPet (red), KAN1−2 × GFP (green) and PIN1-CFP (blue) before (A) and 18 hr (B) after the application of 100 μM auxinole. Inset shows close-up of the primordium outlined with the dotted rectangle. Similar colored dots mark the same cells at 0 hr and 18 hr time-points. Note the presence of KAN1 expression in the proximity of the cells marked with colored dots in the inset in (A) and its absence in the inset in (B). Similar color arrowheads in (A and B) mark the same cells that showed REV expression at 0 hr but KAN1 expression at 18 hr after treatment with auxinole. (C and D) Confocal projections of the IMs showing expression pattern of REV-2 × YPet (red), KAN1−2 × GFP (green) and PIN1-CFP (blue) before (C) and 18 hr (D) after the combined application of 100 μM auxinole, 100 μM KYN and 100 μM Yucasin (auxin depleting drugs). Note KAN-2 ×GFP expression has expanded centrally at the expense of REV-2 × YPet expression (compare the cells marked by arrowheads in (C) with (D), similar colored arrowheads mark the same cells tracked over 18 hr) (n = 6/6). (E and F) Confocal projections of the IMs indicating the predicted auxin distribution (magenta) based on R2D2 expression along with PIN1-GFP expression (green) before (E) and 17 hr after the combined application of 100 μM auxinole, 100 μM kyn and 100 μM yucasin (auxin depleting drugs) (F). Note lack of detectable auxin based on R2D2 expression in (F) compared to (E) after the combined drug application (n = 3/4). Scale bars 20 μm (A–D), 30 μm (E and F).

Figure 12—figure supplement 1. Auxin depletion results in KAN expression at the expense of REV.

(A and B) Close-up views of cells marked with arrowheads in Figure 12A (A) and Figure 12B (B) showing PIN1 (blue), KAN (green) and REV expression (red). Similar colored arrowheads mark the same cells tracked over 18 hr (same as in Figure 12 (A and B). (C and D) Same as (A and B) but showing PIN1 expression alone to clearly highlight cell outlines for their identification after 18 hr of treatment with auxinole. (E and F) Close-up views of the cells marked with arrowheads in Figure 12C (E) and Figure 12D (F) showing PIN1 (blue), KAN (green) and REV expression (red). Similar colored arrowheads mark the same cells tracked over 18 hr (same as in Figure 12 (C and D). (G and H) Same as (E and F) but showing PIN1 expression alone to clearly highlight cell outlines for their identification, 18 hr after combined treatment with auxinole, yucasin and kyn. Cells marked with same colored dots are tracked over 18 hr in (A–H). Scale bars, 5 μm (A–D) and 10 μm (E–H).

Figure 12—figure supplement 2. Auxin depletion alters dorsoventral gene expression in the vegetative meristems.

(A and B) Confocal projections of the VMs showing expression pattern of REV-2 × YPet (red), KAN1−2 × GFP (green) and PIN1-CFP (blue) 48 hr after the combined application of 100 μM auxinole, 100 μM KYN and 100 μM Yucasin (auxin depleting drugs). Arrowhead in (B) marks an arrested leaf primordium expressing KAN throughout. Inset in (B) shows a longitudinal optical section of the leaf primordium ectopically expressing KAN1−2 × GFP. (C and D) Longitudinal optical sections of the VMs in (A) and (B) respectively. Note that the meristems grow as pins with no new organs initiating. Scale bars 30 μm (A-D, inset in B).

Figure 12—figure supplement 3. Auxin depletion does not results in the shrinking of the central zone of the meristem.

(A and B) Confocal projections of the IMs showing expression of pCLAVATA3::GFP-ER (green) before (A) and 18 hr after combined application of auxin depleting drugs. Cells expressing GFP-ER have been tracked using similar colored dots over 18 hr (A and B). (C and D) Longitudinal optical sections along the dashed white lines in (A) and (B) respectively. Note no significant reduction in the zone of CLAVATA3 expression (B and D) 18 hr after auxin depletion. Scale bars 20 μm (A–D) (n = 4).