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Cell surface and intracellular auxin signalling for H+ fluxes in root growth

Abstract

Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments.

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Fig. 1: Auxin rapidly inhibits root growth by apoplast alkalinization.
Fig. 2: Auxin-triggered H+-ATPase activation counteracts auxin-triggered apoplast alkalinization.
Fig. 3: TMK1 directly mediates auxin-induced H+-ATPase activation.
Fig. 4: Antagonistic TIR1/AFB and TMK1 signalling converge on apoplastic pH for growth regulation.

Data availability

The data and full blots are available within the paper and its Supplementary InformationSource data are provided with this paper.

Code availability

All codes used in the manuscript are provided in the Supplementary Information.

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Acknowledgements

We thank N. Gnyliukh and L. Hörmayer for technical assistance and N. Paris for sharing PM-Cyto seeds. We gratefully acknowledge the Life Science, Machine Shop and Bioimaging Facilities of IST Austria. This project has received funding from the European Research Council Advanced Grant (ETAP-742985) and the Austrian Science Fund (FWF) under I 3630-B25 to J.F., the National Institutes of Health (GM067203) to W.M.G., the Netherlands Organization for Scientific Research (NWO; VIDI-864.13.001), Research Foundation-Flanders (FWO; Odysseus II G0D0515N) and a European Research Council Starting Grant (TORPEDO-714055) to W.S. and B.D.R., the VICI grant (865.14.001) from the Netherlands Organization for Scientific Research to M.R. and D.W., the Australian Research Council and China National Distinguished Expert Project (WQ20174400441) to S.S., the MEXT/JSPS KAKENHI to K.T. (20K06685) and T.K. (20H05687 and 20H05910), the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 665385 and the DOC Fellowship of the Austrian Academy of Sciences to L.L., and the China Scholarship Council to J.C.

Author information

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Authors

Contributions

L.L., I.V. and J.F. conceived and designed the experiments. L.L. and I.V. carried out most of the experiments and analysis. M.R. and D.W. performed the phosphoproteomics analysis and TIR1/AFB1 IP–MS/MS. I.V., W.S. and B.D.R. performed TMK1 IP–MS/MS experiments and statistical analysis. MS/MS analysis was performed by the VIB Proteomics Core. L.S. and S.S. performed MIFE experiments. K.T. and T.K. performed the ATP hydrolysis assays. J.C. and S.V. created and shared the AtTAS1c-AHA lines. L.R. and L.L. created transgenic lines and crosses. H.R. and W.M.G. conducted [γ-32P]ATP kinase assays and leaf wilting phenotype analysis, shared plasmids and seed materials, and contributed to discussion of the results. J.M. and L.L. modified the microfluidic chip. L.L., I.V. and J.F. wrote the manuscript.

Corresponding author

Correspondence to Jiří Friml.

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The authors declare no competing interests.

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Peer review information Nature thanks Malcolm Bennett, Anthony Bishopp and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Investigation of CMT and vacuolar morphology in auxin-induced rapid root growth inhibition.

a, Scheme of the modified vRootchip, with added valve 6 and adjusted valve routes. b, c, Dynamic cortical microtubule (CMT) transversal to longitudinal reorientation in response to 100nM IAA treatment. CMT were imaged at 6.25s intervals in elongating root epidermal cells in the pEB1b::EB1b-GFP marker line in vRootchip. Max Z-projection of 10 subsequent time frames was analysed using the FibrilTool. Average orientation of CMT is represented by the slope of the red line and the length of the line represents its anisotropy (b). (c) Quantification of CMT reorientation as in b. CMT reorientation at every time point is calculated as the difference in angle of that time point minus the initial time point angle divided by the difference in the angles of the initial time point and end time point (42min). Mean of 5 elongating cells±s.d. (c). df, Analysis of CMT reorientation in elongating root epidermal cells (d, e) and root growth (f) of p35S::MAP4-GFP in response to 10nM IAA, 10µM taxol and IAA+taxol co-treatment. CMT orientation was analysed with the Bioline script. Green-colored CMTs mark transversal oriented CMT (angle between −45° and +45°), while red-colored CMTs indicate longitudinal orientation (angle between +45° and 135°). Scale bar=15µm (d). Percentage of longitudinal CMT. n>11 roots, One-way ANOVA (e). Growth on respective treatments after 2h. n>10 roots. Box plots depicts minimum to maximum, mean±s.d. One-way ANOVA without modifications for multiple comparison (f). *P≤0.05, ****P≤0.0001. g, Vacuolar morphology tracked using pSYP22::SYP22-YFP (green signal) in elongating cells before and after 30min of 100nM IAA. Scale bar=15µm. Magenta signal represents propidium-iodide stained cell walls.

Source data

Extended Data Fig. 2 Apoplastic pH in auxin-induced rapid root growth inhibition.

a, Apoplastic pH dynamics measured across the whole EZ (p1-p8) in vRootchip. The TL and blue-yellow scale image are from the same sample shown in Fig. 1a. Scale bar=30µm. The upper charts depict apoplastic pH in the indicated cells in response to 5nM IAA, and the lower charts represent the pH in response to washout. The right two charts show the speed at which each cell reaches its maximum pH change calculated as the difference between pH at a given time point and pre-stimulus pH, divided by the final pH change. b, Dynamics of root surface pH and medium pH in vRootchip. The left graph shows the elongation zone of the root. ROIs p1-p5 were chosen vertically along the root, 30µm away from the root surface indicated by the vertical white dotted line, while ROIs p6-p9 were distanced horizontally away from the root. The pH at the surface of the root (p1-p5) increased after IAA and recovered within 30s after washout. In contrast, the pH away from the root surface did not change significantly (p6-p9). c, H+-net influx measured by a non-invasive microelectrode before and after 10nM IAA treatment in the elongating zone of WT roots. Mean of 9 roots+s.e.m. d, e, Changes in medium pH (d) and apoplastic pH (e) after different medium pH exchanges in vRootchip. Sequentially used media: basal medium at pH 5.8, auxin-containing medium at pH 5.8, more acidic medium of pH 5.6, followed by pH 5.4 and again basal medium at pH 5.8. f, Quantification of root growth in response to gradual addition of KOH in the medium in the vRootchip. The greener the shade, the more KOH was added and followed by washout with initial pH 5.8 medium

Source data.

Extended Data Fig. 3 H+-ATPase activation counteracts auxin-mediated apoplast alkalinization and growth inhibition.

a, b, Apoplastic pH of WT elongating root cells pre-treated (yellow) with 1µM cycloheximide (CHX) for 3min (a), or 50µM cordycepin (CORD) for 32min (b) followed by addition of 5nM IAA (pink). Mean of 3 (a) or 4 (b) roots+s.d. c, 10nM IAA induced Thr947 phosphorylation in roots using AHA2 and pThr947 specific antibodies. Band intensities of the different lanes were quantified by the Gel Analysis function in ImageJ. d, Measurement of DR5::LUC luminescence intensity in the root tip after 10µM FC, 10nM IAA and IAA+FC co-treatment. n>3 roots. IAA and IAA+FC are significantly different from the mock (P ≤0.0001). No significant difference between IAA and IAA+FC (ns, P>0.05). Two-way ANOVA. ek, FC and IAA counteract each other. In vRootchip, addition of IAA still increased apoplastic pH (e) and inhibited root growth (f) in presence of FC, while addition of FC decreased apoplastic pH (j) and promoted root growth (k) in presence of IAA. Upon simultaneous addition of 10µM FC and 10nM IAA, both apoplastic pH (Fig 2d) and root growth (g) were less affected than by IAA alone. Shaded area represents the duration of the treatments. Mean of 4 roots+s.d. ****P≤0.0001 between IAA and IAA+FC from 0–31min (g), Two-way ANOVA. (h-i) Steady-state 1h root growth after FC, IAA and co-treatment was obtained by scanner. 1µM FC and 10nM IAA were used in (h) while 10µM FC and 2nM IAA were used in (i). n>9 roots. Box plot depicts minimum to maximum, mean±s.d. ns P>0.05, *P≤0.05, **P≤0.01, ****P≤0.0001, One-way ANOVA (h, i). l, Dose-response of auxin-induced root growth inhibition of aha single mutants. n>22 roots. Relative GR is ratio between auxin-affected growth and mock for the same genotype. ns P>0.05, **P≤0.01, ***P≤0.001, Welch ANOVA. m, Quantitative Real-time PCR on the AHA1,2,7,11 expression in root tips of AtTAS1c-AHA#2 and #4. The expression level was normalized to EF1α as housekeeping gene. Mean of 6 biological replicates in 3 technical replicates+s.d. Box plot depicts minimum to maximum, mean±s.d. ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, One-way ANOVA. n, 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 roots). Relative GR is calculated as mentioned in (l). ns P>0.05, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001, Welch ANOVA

Source data.

Extended Data Fig. 4 TMK1 interacts with PM H+-ATPase.

a, TMK1 expression pattern in the elongation zone (EZ), meristematic and transition zone (MZ-TZ) in the primary root shown by pTMK1::TMK1-GFP. Scale bar=60 μm. b, c, IP-MS/MS on pTIR1::TIR1-VENUS in tir1-1 (b) and pAFB1::AFB1-VENUS in afb1-3 (c) under mock condition compared to 1h 50µM MG132 pre-treatment and 2min 100nM IAA treatment. Proteins surpassing the threshold FDR of 0.05 are marked. Green depicts the respective bait protein and red depicts known members of the SCF E3 ubiquitin ligase complex. Pulldowns were performed in triplicate, LFQ analysis. d, IP-MS/MS on pTMK1::TMK1-GFP. Peptides corresponding to AHA1/2 are shown in red. p-values are calculated based on three biological replicates using two-sided t-tests. e, Co-IP of pAHA2::AHA2-GFP roots, followed by Western blot detection of TMK1 and Thr947-phosphorylated AHA2 after 100nM IAA for 30min. Auxin did not affect interaction, but induced AHA2-phosphorylation. Input of pAHA2::AHA2-GFP roots was the control. f, Bimolecular Fluorescent Complementation (BiFC) in Nicotiana benthamiana leaves transiently transformed with the reciprocal controls for Fig. 3b: YFPN-TMK1, AHA2-YFPC or both. Scale bar=10µm. g, Demonstration of specific interaction between YFPN-TMK1K616R and AHA2-YFPC as no complementation was observed in the leaves expressing YFPN-TMK1K616R and AUX1-YFPC or leaves expressing YFPN-AHA2 and AUX1-YFPC. Scale bar=100µm

Source data.

Extended Data Fig. 5 TMK1 directly phosphorylates PM H+-ATPases.

a, Wilting N. benthamiana leaves that transiently express TMK1WT and ATP-site mutated forms TMK1K616E or K616R. b, Western blot analysis of the AHA2 levels and the Thr947 phosphorylation in roots of DEX::TMK1WT or K616R-HA treated +/− DEX (30µM for 24h) and +/- IAA (100nM for 1h). c, Ponceau-stained SDS-PAGE gel as loading control for in vitro kinase assay with [γ-32P]-ATP, substrate C-terminal AHA2 (AHA2-C) and the intracellular kinase domain of TMK1WT or kinase dead TMK1K616E. d, e, Western blot detection of AHA2 levels and Thr947 phosphorylation in tmk1,3 roots (d) or tmk1,4 roots (e) treated with 100nM IAA for 1h. WT control for (d) is shown in Fig. 3e.

Extended Data Fig. 6 Cytosolic TIR1/AFB mediates rapid apoplast alkalinization and root growth inhibition.

a, b, Apoplastic alkalinization (a) and root growth inhibition (b) in response to IAA measured in aux1-100 mutant compared to WT roots in vRootchip. Mean of 3 roots+s.d. ****P ≤ 0.0001, Two-way ANOVA. c, d, Apoplastic alkalinization (c) and root growth inhibition (d) in response to 2,4-D in aux1-100 mutant compared to WT roots. Steady state pH measured 30min after 100nM 2,4-D treatment. Mean of >6 roots+s.d., One-way ANOVA (c). (d) Growth obtained in 2h was captured by scanner. Mean of >4 roots+s.d., One-way ANOVA. ns P>0.05, **P≤0.01, ***P≤0.001. e, Root growth of tir triple mutants compared to WT in response to 5nM IAA in the vRootchip. Mean of 3, 2 roots+s.d. ****P≤0.0001, two-way ANOVA. f, g, Apoplastic pH (f) and root growth (g) after 10µM PEO-IAA and 5nM IAA. The steady state pH was measured 30min after treatments, while the root growth obtained in 1h was recorded by scanning. Mean of >7 roots+s.d.. ns P>0.05, **P≤0.01, ****P≤0.0001, One-way ANOVA. h, i, Dose-response of auxin-induced root growth inhibition of tir1-1, tir1-10 and afb1-3 mutants reveals slight resistance to 5nM IAA in comparison to WT (n>6 roots). Relative GR is calculated as the ratio of GR at 1h (h) or 6h (i) after IAA treatments relative to mock-treated GR of the same genotype. Mean+s.d. *P≤0.05, ***P≤0.001, One-way ANOVA. j, k, Apoplastic pH (j) and root growth (k) analysis comparing tir1-10 null mutant and afb1-3 mutants in response to IAA in vRootchip. Shaded area represents the duration of the treatment. Mean of 4 roots for each treatment+s.d. P≤0.0001 (j) and P≤0.05 (k), Two-way ANOVA. l, Steady-state root growth over 6h in tmk1-related mutants. n=6 roots for tmk1,4; n>26 for others. Mean+s.d. Box plot depicts minimum to maximum, mean±s.d. ****P≤0.0001, One-way ANOVA. m, Dose-response of auxin-induced root growth inhibition of pUBQ10::TMK1-3HA compared to WT and tmk1-1. Relative GR is the ratio between auxin-affected growth to the mock growth in the same genotype. Mean of >7 roots+s.d.. ns P>0.05, *P≤0.05, Welch ANOVA. n, o, Raw data for Fig. 4d, e, respectively. n>16 roots. Box plot depicts minimum to maximum, mean±s.d. ns P>0.05, **P≤0.01, ****P≤0.0001, One-way ANOVA

Source data.

Extended Data Fig. 7 PM potential and AUX1 involvement in auxin-induced apoplast alkalinization.

Since auxin causes simultaneously membrane depolarization25 and apoplast alkalinization, both of which are interdependent and required for growth, we addressed which of them mediates the auxin effect on root inhibition. By manipulating the external pH, we found that pH and growth were correlated (Fig. 1d–g) while membrane potential (MP) was uncoupled (a, b). Additionally, we observed that K+ efflux (c) compensates auxin-induced H+ and Ca2+ influx (Extended Data Fig. 2c, Extended Data Fig. 8a), suggesting that auxin-induced MP change is the result of complex ion fluxes, while H+ influx and resulting apoplastic pH change for growth regulation is just a subset of those. a, b, Membrane potential recorded by invasive micro-electrode in root elongating cells with IAA treatment (magenta). 4 roots+s.e.m. (a). Membrane potential measured in root elongating cells after 40min incubation in different pH medium. n>5 roots±s.e.m. *P≤0.05, One-way ANOVA (b). Alkaline medium, which alkalinized the apoplast and inhibited root growth (Fig. 1d, e) mimicking the auxin effect, did not result in membrane depolarization. Acidic medium, which acidified the apoplast and promoted root growth (Fig. 1f, g) depolarized membrane. MP is thus uncoupled from growth and apoplastic pH. c, PM net K+ efflux measured by a non-invasive microelectrode before and after 10nM IAA treatment in the elongating zone of WT roots. 16 roots+s.e.m. d, Scheme showing AUX1/LAX-mediated IAA-/2H+ symport and mechanistically elusive H+ influx. IAA-/2H+ symport by AUX1 auxin influx carrier was proposed17 a possible mechanism of auxin-induced H+ influx and apoplast alkalinization. Comparison of H+ influx rates in root hair cells17, or elongating root epidermal cells (Extended Data Fig. 2c) and conservative estimates of AUX1-mediated 3H-IAA transport in Xenopus oocytes24,64 argue against this. Below we show that calculations based on data of Xenopus oocytes, primary root and root hairs suggest that AUX1-mediated H+ symport is not sufficient to account for the auxin-induced H+ fluxes: (1) 3H-IAA transport in the AUX1 overexpressing Xenopus oocytes after 100nM 3H-IAA24 is ca. 2.6x10−14molmin−1. The min. diameter of the Xenopus oocyte at stage V/VI64 is ca. 1.0mm, so the surface area is minimally 3.142x10−6m2. The max. speed of IAA uptake across the membrane is calculated as: 2.6 x 10−14mol/(60s x 3.142x10−6m²) = 1.38x10−10molm−2s−1. Based on 2 H+ per IAA-, the max. speed of AUX1-symported H+ is 2.76x10−10 molm−2s−1. (2) H+ uptake after 10nM IAA (ten times less than in Xenopus) in Arabidopsis root elongating cells: 1.7x10−8molm−2s−1 (Extended Data Fig. 2c). This is still 62 times more than the conservatively estimated max. speed in (1). (3) H+ uptake NAA in Arabidopsis root hairs17 is ca. 1.0x10−7molm−2s−1. This is 362 times more than the conservatively estimated max. speed in (1)

Source data.

Extended Data Fig. 8 FER does not mediate auxin-induced rapid growth inhibition.

Root growth of fer-4 compared to Col-0 in response to application and washout of 100nM IAA in vRootchip. Shaded area indicates IAA treatment. Mean of 5 roots for Col-0 and 3 for fer-4+s.d. ns, P>0.05, Two-way ANOVA.

Source data

Extended Data Fig. 9 TIR1-mediated Ca2+ signalling contributes to auxin-induced apoplast alkalinization.

Another rapid output of TIR1/AFB perception mechanism are cytosolic Ca2+ transients in root hairs17. Therefore, we evaluated Ca2+ transients in apoplast alkalinization and root growth inhibition. Using vRootchip, GCaMP3 Ca2+ marker33, non-invasive microelectrodes and cvxIAA-ccvTIR1 system28, we confirmed that auxin via TIR1/AFB triggered rapid Ca2+ influx correlates with root growth inhibition (a-d). We noted a distinct Ca2+ response measured by the microelectrode (a-b) and GCaMP3 (c). Namely, Ca2+ channels are activated at the plasma membrane resulting in net influx, while the GCaMP3 reported more complex responses possibly involving intracellular Ca2+ storage and release. Moreover, the use of cvxIAA-ccvTIR1 (d) provided additional proof that TIR1-mediated auxin perception activates Ca2+ signalling. Further, we verified that mutants in the Ca2+ permeable cation channel Cyclic NUCLEOTIDE-GATED CHANNEL 14 (CNGC14) have delayed auxin-induced apoplast alkalinization and root growth inhibition (e) similarly as reported14 Furthermore, depletion of external Ca2+ resulted in attenuated auxin-induced Ca2+ spike, delayed apoplast alkalinization and growth inhibition (b, f, g). Ca2+ addition resulted in rapid growth inhibition (h, i). These observations collectively suggest that TIR1/AFB-mediated Ca2+ signalling is part of the mechanism for auxin-induced rapid apoplast alkalinization and growth inhibition. a, PM net Ca2+ influx measured by a non-invasive microelectrode before and after 10nM IAA treatment in the elongating zone of WT roots. 9 roots+s.e.m. b, Normalized fluorescence intensity of GCaMP3, cytosolic Ca2+ marker, in elongating cells responding to 5nM IAA treatment in vRootchip. The intensity was normalized to the initial intensity of the same root. Mean of 7 roots+s.d. Note the three peaks in cytosol compared to the single major peak outside of cells (a). c, d, Root growth (c) and fluorescence intensity in elongating cells (d) in GCaMP3 crossed into ccvTIR1 compared to control. Growth rate and intensity are normalized to the pre-stimulus value. Mean of 7 for ccvTIR1 and 2 for control+s.d. ****P≤0.0001, Two-way ANOVA. e, Root growth (upper graph) and apoplastic pH (lower graph) analysis in cngc14-2 and WT in response to IAA in vRootchip. Mean of 5 roots for WT and 3 for cngc14-2+s.d. ****P≤0.0001, Two-way ANOVA. f, g, Root growth (upper graph in f) and apoplastic pH (lower graph in f) in WT, as well as cytosolic Ca2+ analysis in GCaMP3 reporter marker line in vRootchip (g) with 140min pre-treatment of Ca2+ free medium (grey) followed by 5nM IAA addition (magenta for growth, blue for pH in f and yellow for Ca2+ in g). Auxin induced significant less Ca2+ response in Ca2+ free medium, compared to normal medium in (b). The red dotted square marked the non-responsive delay after auxin. Mean of 5 (f) and 6 (g) roots+s.d. h, i, Root growth (h) and apoplastic pH (i) analysis in WT upon Ca2+ addition after 140min Ca2+ free medium in the presence of 5nM IAA in vRootchip. Mean of 5 roots+s.d.

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Supplementary Information

This file contains Supplementary Fig. 1 and Codes 1 and 2. Supplementary Fig. 1: This figure contains the raw and uncropped data for every figure in which we show western blot results. The blots are labelled according to the corresponding figure, and the cropped area is indicated. Supplementary Codes 1 and 2: For easier manipulation of the Arduino device controlling medium flow in the vRootchip, two in-house-designed scripts were generated. These scripts were first used to generate the data shown in this manuscript, and we make them publicly available here.

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Supplementary Table 1

Phosphoproteomic data of rapid auxin effects in roots and phosphoproteomic analysis of H+-ATPases in tmk1-1 mutants. Differentially regulated phosphopeptides (FDR ≤ 0.05) in H+-ATPases in IAA-treated versus mock-treated roots. IAA treatment was at 100 nM for 2 min. QHF and LTQXL analyses are presented in tabs 1 and 2, respectively. Tab 3 shows the differentially phosphorylated phosphosites of AHAs in the tmk1-1 background compared with WT.

Supplementary Table 2

IP–MS analysis of pTIR1::TIR1-VENUS and pAFB1::AFB1-VENUS. Overview of the putative TIR1 and AFB1 interactors after MaxQuant and Perseus statistical analysis. Samples were TIR1- and AFB1-VENUS lines with mock treatment or 50 µM MG132 preincubation for 1 h and 100 nM IAA treatment for 2 min. Proteins passing the threshold of FDR 0.05 and specific fold change are included in the table. P values were calculated based on the three replicates using a two-sided t-test. Pulldowns were performed in triplicate. The respective bait proteins are highlighted in yellow.

Supplementary Table 3

IP–MS analysis of pTMK1::TMK1-GFP. Overview of the putative TMK1 interactors after MaxQuant and Perseus statistical analysis. The list is sorted based on the ratio of pTMK1::TMK1-GFP versus WT control. Proteins passing the threshold of FDR 0.05 and specific fold change are included in the table. P values were calculated based on the three replicates using a two-sided t-test. Yellow, bait; green, GFP; orange, selected proteins.

Supplementary Table 4

Primers used for cloning and quantitative PCR analysis

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Li, L., Verstraeten, I., Roosjen, M. et al. Cell surface and intracellular auxin signalling for H+ fluxes in root growth. Nature 599, 273–277 (2021). https://doi.org/10.1038/s41586-021-04037-6

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