Extended Data Fig. 3: Global, ultrafast auxin phospho-response and rapid cellular effects.
From: ABP1–TMK auxin perception for global phosphorylation and auxin canalization
a, Ultrafast phosphoproteomics in mock-treated (2 min) Arabidopsis roots. Distributions of log2 fold changes for P-peptides with significantly differential phosphorylation (FDR < 0.05) in tmk1-1 and abp1-TD1 as compared to WT. Both mutants exhibit global hypo-phosphorylation. b, Mock treatment (2 min) of Arabidopsis roots. The volcano plot depicts log2 fold changes (x-axis; tmk1-1 versus WT) and statistical significance (y axis). Highlighted in red is a subset of significantly regulated P-peptides (FDR < 0.05) which are also significantly regulated in abp1-TD1/WT. This shows global hypo-phosphorylation in tmk1-1 and extensive functional overlap between the two mutants. c, Mock treatment (2 min) of Arabidopsis roots. The volcano plot depicts log2 fold changes (x-axis; abp1-TD1 versus WT) and statistical significance (y axis). Highlighted in red is a subset of significantly regulated P-peptides (FDR < 0.05) which are also significantly regulated in tmk1-1/WT. This shows global hypo-phosphorylation in abp1-TD1 and extensive functional overlap between the two mutants. d, Relative MS intensity for P-peptides pertaining to various Arabidopsis MadB paralogs (MadB1, MadB/At5g20360, MadB2/PHOX2). Auxin (IAA, 100 nM, 2 min) treatment, 4 independent biological replicates, mean ± SD. Asterisks indicate FDR-controlled p-values collated from global phosphoproteomic comparisons as described in Materials and Methods. MadB1S391 (WT versus tmk1-1: no statistics due to low detection, WT versus abp1-TD1: P = 0.0354), MadB2S261 (WT versus tmk1-1: P = 0.1562, WT versus abp1-TD1: P = 1.17E-06), MadBS208 (WT versus tmk1-1: P = 0.0393, WT versus abp1-TD1: P = 0.0035), MadB2T215 (no statistics due to low detection), MadB2S216 (no statistics due to low detection), MadB2S263 (WT versus tmk1-1: P = 0.0926, WT versus abp1-TD1: P = 0.0056). * P ≤ 0.05, ** P ≤ 0.01, ****P ≤ 0.0001. e, Steady-state membrane potentials (MP) and IAA-induced depolarization (in mV) measured in the mature root zone of WT, abp1-c1 and tmk1-1 mutants after 100 nM IAA treatment. Values are means ± SEM (Depolarization amplitudes: WT, n = 9; abp1-c1, n = 10; tmk1-1, n = 3; Membrane potentials: WT, n = 26; abp1-c1, n = 35; tmk1-1, n = 11). f, Auxin sensitivity of abp1-c1 root growth. Data on the graph represent the normalized growth rate of abp1-c1 mutant in comparison to comp-c1 line and WT, mean ± SD (Col-0 DMSO, n = 54; Col-0 3 nM IAA, n = 45; Col-0 5 nM IAA, n = 54; abp1-c1 DMSO, n = 81; abp1-c1 3 nM IAA, n = 90; abp1-c1 5 nM IAA, n = 108; comp-c1 DMSO, n = 90; comp-c1 3 nM IAA, n = 81; comp-c1 5 nM IAA, n = 81). No statistically significant difference in sensitivity of growth rate to auxin was detected with two-way ANOVA. g, Auxin sensitivity of abp1-TD1 root growth. Data on the graph represent the normalized growth rate of abp1-TD1 mutant in comparison to comp-TD1 line and Col-4, mean ± SD (Col-4 DMSO, n = 81; Col-4 3nM IAA, n = 81; Col-4 5 nM IAA, n = 90; abp1-TD1 DMSO, n = 90; abp1-TD1 3 nM IAA, n = 72; abp1-TD1 5 nM IAA, n = 99; comp-TD1 DMSO, n = 81; comp-TD1 3 nM IAA, n = 72; comp-TD1 5 nM IAA, n = 90). No statistically significant difference in sensitivity of growth rate to auxin was detected with two-way ANOVA. h, Cytoplasmic streaming velocity increases with IAA concentration. WT seedlings were treated with 0, 10, 20, 50 and 100 nM IAA for 30 min. Fast-moving particles in epidermal cells of the root elongation zone were recorded. Error bars represent mean ± SD (n = 72 particles from 24 cells in 8 seedlings for each condition). One-way ANOVA (F4,354 = 16.32, P = 2.80E-12) with Dunnett’s multiple comparison tests indicated in the figure. * P ≤ 0.05, **** P ≤ 0.0001. i, Auxin-triggered acceleration of cytoplasmic streaming is not observed in abp1 mutant alleles but is restored in the complemented abp1 lines. Error bars represent mean ± SD (n = 63 particles from 21 cells in 7 seedlings for each condition). Two-way ANOVA (interaction effect: F4,608 = 2.29, P = 0.0585; genotype effect: F4,612 = 5.74, P = 0.0002; treatment effect: F1,612 = 37.26, P = 1.84E-09) with Šidák’s multiple comparison tests indicated in the figure. ** P ≤ 0.01, *** P ≤ 0.001. j. Auxin-triggered acceleration of cytoplasmic streaming is not observed in tmk1 and tmk4 mutants. Unlike tmk4, tmk1 roots have already accelerated cytoplasmic streaming but both mutants are largely auxin-insensitive. Error bars represent mean ± SD (n = 54 particles from 18 cells in 6 seedlings for each condition). Two-way ANOVA (interaction effect: F2,318 = 9.16, P = 0.0001) with Šidák’s multiple comparison tests indicated in the figure. **** P ≤ 0.0001.
