Figure 5. Cell wall stiffening and inhibition of root cell elongation under −Pi are peroxidase-dependent.

(a–c) Dose–response relationship of the Δ primary root length plotted as a function of concentration of peroxidase inhibitors (mean±s.d.). For each data point, 3-day-old WT (Col^er105) seedlings were transferred for 7 days to −Pi plates containing different concentrations of inhibitor. The structure of the inhibitors is shown above the corresponding curves. (a) Salicylhydroxamic acid (SHAM). Blue data points: −Pi; yellow data point, +Pi without inhibitor (n=12–14 seedlings per condition). (b) 3,3′-diaminobenzidine (DAB) (n=8–12 seedlings per condition). (c) Methimazole (n=11–13 seedlings per condition). (d) Picture of the effect of peroxidases inhibitors on WT seedlings. Three-day-old WT seedlings were transferred for 7 days to +Pi or −Pi plates containing the indicated inhibitors. Top: picture of one representative seedling for each treatment (Methi., methimazole). Arrowheads indicate the primary root tips. Scale bar, 1 cm. Bottom: pictures of the root tips corresponding to the seedlings shown above. Note that inhibitors of peroxidases suppress the −Pi-induced early differentiation of root hairs. (e) Three-day-old WT seedlings were transferred for 30 min to +Pi or −Pi medium with or without 15 μM SHAM prior to measuring the stiffness of the cell surface by AFM in the transition zone of the primary root (median±interquartile, Tukey whiskers, Mann–Whitney test: ****P<0.0001). (f) Effect of SHAM and DAB on root epidermal cell length. Three-day-old WT seedlings were transferred for 7 days to +Pi or −Pi medium with or without 15 μM SHAM or 100 μM DAB prior to measuring the root epidermal cell length (median±interquartile; Tukey whiskers, Mann–Whitney test: ****P<0.0001, n=number of cells). (g) Peroxidase activity revealed by 4-chloro-1-naphtol staining of the WT primary root tip (the colourless dye turns black with peroxidases activity). Four-day-old WT seedlings were transferred for 48 h to +Pi or in −Pi plates with or without 15 μM SHAM (see also Supplementary Fig. 9a). (h) Peroxidase activity revealed by 4-chloro-1-naphtol staining of the primary root tip of the indicated genotypes. Four-day-old seedlings were transferred for 48 h to +Pi or in −Pi plates prior to staining. Single and double-headed arrows indicate the position of the elongation zone. The experiments were performed three times with consistent results and one representative experiment is shown (a–c,g,h). Scale bars, 100 μm (g,h).