Extended Data Figure 6. High auxin levels rapidly inhibit cell division and expansion, but not differentiation.

a, First signs of expansion zone differentiation 7 h after 5 μM IAA application marked by appearance of protoxylem elements (arrow). By 24 h ubiquitous differentiation of the expansion zone is evident. 0 h image used from Fig. 1a. Scale bar, 50 μm. b, Auxin application rapidly inhibits growth while xylem differentiation, monitored by green fluorescence of S18 marker³², proceeds towards meristem. Snapshots from a video of the same root before and after application. Right panel, S18 signal is tightly associated with protoxylem differentiation (arrow). c, Root growth (μm h⁻¹) and mitosis (below the x-axis) of two roots over time (min). IAA applied at t = 0. d, e, Application of 5 μM IAA and inhibition of auxin signalling by 30 μM PEO-IAA leads to decreased accumulation of 5-Ethynyl-2′-deoxyuridine (EdU) stain (red fluorescence), marking DNA replication. Asterisks in e, Mann-Whitney U test P < 0.05, after Bonferroni correction of multiple comparisons; reduction of number of EdU-stained nuclei compared with DMSO control. Error bars show s.d. f, Application of moderate levels of IAA (30 nM) still inhibited cell expansion (Supplementary Notes) but did not inhibit cell division. Root growth (μm h⁻¹) and mitotic events (below the x-axis) of two roots over time (min). IAA (30 nM) was applied at t = 0. g, h, To measure the duration of the differentiation process, individual cells were followed as they left the meristem, expanded and entered the differentiation zone. g, Tracking of a GFP clone²⁰ consisting of four cells. Arrows highlight a cell just entering the expansion zone in the first panel and in the last panel the same cell entering the differentiation zone. For this particular cell it took approximately 6 h 45 min to travel through the expansion zone. Six clones located in six roots were followed through the expansion zone, and it took 6–8 h for these clones to travel through the expansion zone. h, Snapshots from a video recording the growth of wild-type root in the presence of 30 nM IAA. The cells entering the expansion zone (arrows in left panel) were traced in the video to record the time it takes to enter the differentiation zone (arrows in the right panel). For the marked cell it took approximately 7 h 10 min to travel through the expansion zone. Tracking of cells through the expansion zone was carried out for nine cells located in three different roots, and it took 6–8 h for these cells to travel through the expansion zone.