Localization of IPA-N3 in Arabidopsis root hairs. (a,b) The IPA-N3 localization in the outgrowing root hair (arrowheads) at the stage of (a) initiation and (b) elongation up to app. 100 μm in length. Upper panels: signal obtained after click reaction to alkyne functionalised Alexa Fluor 488. Middle panels: Counterstain with Calcofluor White (CW) marking cell wall. Bottom panels: overlay of the channels confirms the cell wall localization of IPA-N3. (c) The IPA-N3 localization (green channel) in the elongated root hair, co-stained by two cellular markers: COS647 (red channel) staining cell wall pectin components and DAPI (blue channel) staining nucleus. Overlay confirms the IPA-N3 localization in the cell wall. Some signal could be observed in the nucleus and a compartment surrounding the nucleus. (d–i) Controls for the IPA-N3 labelling in Arabidopsis root hairs. No visible cell wall labelling was observed in the case of seedlings incubated with (d) mock only or (e) control compound 2-azido acetic acid. (f) IPA-N3 labelling of root hair cell wall (10 μM, 1 h). (g) Labelling in the absence of copper catalyst, (h) when pre-treated with proteinase K, (i) or pre-incubated first with IAA (10 μM, 30 min). (j) Quantification of the cell wall signal (n = 5) from the labellings from (d–i). Scale bars = 10 μm.