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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Nature. 2021 Oct 27;599(7884):273–277. doi: 10.1038/s41586-021-04037-6

Figure 2. Auxin-triggered H+-ATPase phosphorylation and activation counteract auxin-mediated apoplast alkalinisation and growth inhibition.

Figure 2

a, Representation of the phospho-sites in the model of the Arabidopsis thaliana H+-ATPase 2 (AHA2) identified in the phospho-proteomic analysis of auxin-treated root samples (2 minutes of 100 nM IAA). The color reflects their impact on the H+-translocation activity (green for activation and orange for inhibition).

b, Quantification of ATP hydrolysis induction in root samples treated with 100 nM IAA for 1 hour in comparison to mock-treated roots. The IAA-treated sample was normalized by the corresponding mock-treated sample. Bars indicate means of 3 biological replicates + SD. Unpaired t-test. * p≤0.05.

c, Phospho-proteome detection of auxin-induced AHA2-Thr947 phosphorylation after 2 minutes of 100 nM IAA treatment. n= 4. Box plot depicts minimum to maximum, mean ± SD. Two-sample t-test (part of MaxQuant-Perseus analysis). **p≤0.01.

d, Time-course western blot analysis of auxin-induced Thr947-phosphorylated AHA2 levels in roots treated with 10 nM IAA using AHA2 and pThr947-specific antibodies. Total AHA2 protein levels were monitored in the same samples as a control. Band intensities of the different lanes are quantified by the Gels Analysis function in ImageJ.

e-f, Apoplastic pH (e) and root growth (f) analysis upon pharmacological activation of H+-pumps by 10 μM FC and 10 nM IAA in vRootchip. Apoplastic pH was monitored using HPTS. The shaded area represents the duration of the indicated treatment. Mean of 4 roots for each treatment + SD. p≤0.0001 between IAA and IAA+FC, from 0 – 32 min (e), from 0 – 31 min (f), Two-way ANOVA.

g-h, Apoplastic pH response in two independent AHA knock-down lines (AtTAS1c-AHA#2 and #4) (n > 9) (g), and the ost2-3D gain-of-function allele (n > 5) (h) in response to 30 minutes 5 nM IAA treatment. Apoplastic pH changes are determined by the HPTS dye and IAA treatment was normalized to the corresponding mock-treatment. **p≤0.01, ***p≤0.001, ****p≤0.0001, One-way ANOVA.

i, Dose-response of auxin-induced root growth inhibition of AtTAS1c-AHA lines and ost2-3D mutants reveals hypersensitivity and resistance respectively to IAA in comparison to WT (n > 15) (i). The relative GR is calculated by the ratio of GR for each IAA concentration relative to mock-treated GR of the same genotype. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, Welch ANOVA.