Figure 3. TMK1 directly mediates auxin-induced H⁺-ATPase activation.

a, Co-immunoprecipitation (co-IP) of the pTMK1::TMK1-FLAG from roots mock or 100 nM IAA-treated for 30 minutes, followed by Western blot detection of AHA2 and Thr⁹⁴⁷-phosphorylated AHA2. The control is the co-IP of WT (Col-0) roots.

b, Co-IP of pAHA2::AHA2-GFP from roots, followed by Western blot detection of TMK1 and Thr⁹⁴⁷-phosphorylated AHA2 on roots mock or 100 nM IAA-treated for 30 minutes. Interaction does not depend on auxin presence, but auxin-induced phosphorylation of the interacting AHA2 was observed. As a control sample we include input of of pAHA2::AHA2-GFP roots.

c, Bimolecular Fluorescent Complementation (BiFC) in Nicotiana benthamiana leaves transiently transformed either with TMK1-YFP^N (TMK1 in pSPYNE), YFP^C-AHA2 (AHA2 in pSPYCE) or both. Scale bar = 10 μm.

d, Differentially detected phospho-sites of AHAs in tmk1-1 normalized to WT (Col-0) MS detection levels. We observe less phosphorylation on the indicated phospho-sites in AHA1, AHA2 and AHA3 in the tmk1-1 mutant. Green indicates the sites with known activation function, while orange is used for known inhibitory function. n= 4. Box plot depicts minimum to maximum, mean ± SD. Two-sample t-test (part of MaxQuant-Perseus analysis).

e, Western blot analysis of auxin-induced AHA2 Thr⁹⁴⁷ phosphorylation in WT and tmk1-1 roots treated with 100 nM IAA for 1 hour. AHA2 levels were determined as a control.

f, Quantification of the auxin effect on AHA2 phosphorylation in (d) by normalising the intensity ratio of pThr⁹⁴⁷ to AHA2 in auxin-treated root samples to the same ratio in mock-treated samples of the respective genotypes.

g, Western blot detection of the AHA2 levels and its Thr⁹⁴⁷ phosphorylation in full seedlings shows reduced AHA2 phosphorylation in tmk1,4 compared to WT.

h, Auxin-induced ATP hydrolysis activity is impaired in tmk mutants relative to WT roots (1 hour mock or 100 nM IAA treatment). The IAA-treated sample was normalized to the mock-treated WT. Means of 3 biological replicates + SD. *p≤0.05, ns p>0.05, One-way ANOVA.