Physiological effects induced by IPA-N3 and its localization in pea epicotyls. (a,b) Effects on pea plants induced by application of lanolin paste containing IAA or IPA-N3 (30 mM). (a) When applied on the decapitated hypocotyl, both IAA and IPA-N3 inhibited the outgrowth of lateral buds (arrowheads). The numbers indicate the cases of the bud outgrowth from the total number of plants (in brackets). (b) Unlike lanolin mock treatment, lateral application of lanolin with IAA or IPA-N3 to the pea stem induced bending (arrowheads mark the site of application). The experiment was repeated twice with at least five stems treated for each compound. (c–f) Resin sections of the pea epicotyls 5 mm below the decapitation wound treated by (c,d) lanolin only and (e,f) lanolin containing 30 mM IPA-N3. (c,e) Staining with Calcofluor White marking cell walls (CW; blue channel) and propidium iodide marking nuclei (PI; red channel). Note the ectopic cell divisions (closed arrowhead) and cell swelling of parenchymatic cells (open arrowhead) in (d). (d,f) Click chemistry detection of IPA-N3 in the section reveals cell wall and nuclear localization. (g) Quantification of the diameter of the parenchymatic cells (three layers from the epidermis) in control, IAA and IPA-N3 treated epicotyls. 500 cells were measured for each treatment. (h) Quantification of the IPA-N3 nuclear and cell wall signal as the ratio between the green channel and respective cellular markers (propidium iodide and Calcofluor White) in the control and IPA-N3 treated sample. The mean of 20 measurements ± SE. *p < 0.01 (Student’s t-test). (i–l) Properties of the cell wall auxin binding sites in pea epicotyl parenchyma. (i) Labelling of mock-incubated control. (j) Incubation with 5 μM IPA-N3 for 15 min is sufficient for labelling of cell walls. Note that the cytoplasm is washed out due to the nature of vibratome sectioning. (k,l) The binding of IPA-N3 can be inhibited by pre-treatment of the sections with (k) IAA (10 μM, 15 min) and (l) by degradation of proteins using proteinase K. Scale bars = 5 mm (a,b), 300 μm (c–f,i–l).