Localization of IPA-N3 in the Arabidopsis root tip using click chemistry. Detection of IPA-N3 in Arabidopsis roots after 1 h incubation (10 μM concentration of the compounds) followed by in vivo coupling to the alkyne functionalized fluorophore Alexa Fluor 488 in comparison with a set of controls scanned using LSCM with the same settings. (a) Fixed and non-treated seedlings, (b) seedlings incubated with in vitro conjugate of IPA-N3 to alkyne Alexa Fluor 488, (c–i) seedlings subjected to a click reaction after incubation in (c) mock, (d,e) control azido group-containing compounds (d) 2-azido acetic acid and (e) 2-azido-3-benzyloxypropionic acid. (f–i) Seedlings incubated with IPA-N3. (f) Seedlings incubated with IPA-N3 only. Note the signal in epidermis (arrowhead) and outer cell layer of root cap. (g) IPA-N3 signal can be reduced when seedlings are co-incubated with IAA (10 μM). (h) Absence of IPA-N3 signal can be observed when the copper catalyst of the reaction is omitted or (i) when the seedlings are pre-treated with the proteinase K. (j–o) Analyses of the IPA-N3 signal in the transition zone of the Arabidopsis roots. Cells before elongation are marked with open arrowheads and those elongating are marked with a closed arrowhead. (j) Scan of the transition zone with the lower photo multiplier (PMT) intensity as in (f) reveals that the majority of the IPA-N3 signal appears in the root epidermal cells starting to elongate. (k) Plasmolysis and co-localisation with cell wall marker Calcofluor White (CW) confirms the cell wall signal of IPA-N3. An open arrow marks the cell wall and a closed arrow marks the cytoplasm. (l,m) Analysis of IPA-N3 signal in semi-thin resin sections. Click labelling of the resin sections of the root tip in the area of the elongation zone of seedlings (upper panels) treated with (l) mock and (m) IPA-N3. Bottom panels: counterstain with propidium iodide marking nuclei (PI; red channel) and Calcofluor White marking cell walls (CW; blue channel). The position of the nuclei is marked with asterisks. Note the cell wall signal in epidermal cells and the enhanced nuclear signal in the IPA-N3 treated sample. (n,o) The effect of IAA on IPA-N3 binding in the transition zone of Arabidopsis roots. (n) Seedlings incubated with IPA-N3 only and (o) seedlings co-incubated with 10 μM IAA. Note the reduction of the signal in (o). (p) Quantification of the cell wall signal from both treatments. The reduction of the IPA-N3 signal by co-incubating with IAA has been observed in three independent experiments. The mean values of 7 measurements ± SE. *p < 0.01 (Student’s t-test). Scale bars = 20 μm (a–i), 10 μm (j–o).