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

doi:10.21203/rs.3.rs-266395/v3

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

https://doi.org/10.21203/rs.3.rs-266395/v3

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published 26 Oct, 2021

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Growth regulation tailors plant development to its environment. A showcase is response to gravity, where shoots bend up and roots 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 phospho-proteomics in Arabidopsis thaliana, we advance our understanding how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on the rapid regulation of the apoplastic pH, a causative growth determinant. 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 alkalinisation. The simultaneous activation of these two counteracting mechanisms poises the root for a rapid, fine-tuned growth modulation while navigating complex soil environment.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

auxin signalling

root growth

PM Hᐩ-ATPases

TMK1

Acid Growth Theory

Auxin, a major growth regulator in plants, acts oppositely in shoots and roots. In shoots, canonical/intracellular auxin TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) receptors by downstream transcriptional regulation activate H⁺-pumps to acidify the apoplast a promote cell elongation^3,4, in accordance with the Acid Growth Theory, which postulates that low apoplastic pH promotes growth⁵. In roots of many species including Arabidopsis, auxin inhibits growth. These contrasting responses are the basis for positive versus negative bending of roots and shoots in response to gravity and light¹. The inhibitory auxin effect in roots also involves TIR1/AFB receptors but its rapid timing points towards an unknown non-transcriptional signalling branch⁶. Besides, a cell surface-based pathway involving TMK1 regulates development⁷, including differential growth in the apical hook⁸, while its role in auxin-regulated root growth remains unclear. Hence, the auxin signalling mechanism and the downstream processes for regulating root growth remain elusive.

In this study, we revealed antagonistic action of intracellular TIR1/AFB and cell surface TMK1 auxin signalling converging on regulation of apoplastic pH, which we confirm as the key cellular mechanism allowing immediate and sensitive root growth regulation.

Growth inhibition correlates with H⁺-influx

Auxin rapidly inhibits root growth through a non-transcriptional branch of TIR1/AFB signalling⁶. Although several cellular processes, including cortical microtubule (CMT) reorientation^9,10, vacuolar fragmentation¹¹ and apoplastic pH changes^12-14 have been implicated, the causal mechanism remains unidentified.

We critically re-evaluated the kinetics of these processes using the vRootchip⁶ (Extended Data Fig. 1a) in combination with vertical confocal microscopy¹⁵. Growth inhibition by 10nM natural auxin indole-3-acetic acid (IAA) was observed within 30s⁶. In contrast, less than 5% CMTs in elongating epidermal cells reoriented after 1min even at 100nM IAA and pharmacological inhibition of this reorientation had no effect on auxin-induced growth inhibition (Extended Data Fig. 1b-f). Similarly, we could not detect changes in vacuolar morphology in elongating cells even after 30min of 100nM IAA treatment (Extended Data Fig. 1g). These results argue against direct involvement of CMT reorientation and vacuole constriction in the rapid auxin-triggered growth inhibition.

To evaluate the kinetics of apoplastic pH, we applied a membrane-impermeable ratiometric pH indicator: 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and imaged apoplastic pH, while simultaneously tracking root tip growth. We detected an apoplastic pH gradient¹² in the root, decreasing from transition to elongation zone but regardless of their position, all cells showed a rapid (30s) apoplastic pH increase (Fig. 1a, b and Extended Data Fig. 2a). This provides higher temporal and spatial resolution to previous observations of auxin-induced apoplast alkalinisation¹² and reveals that auxin-triggered alkalinisation and root growth inhibition occur simultaneously (Fig. 1b). The pH increase was robust and extended to the external medium (Extended Data Fig. 2b). Using the PM-Cyto reporter for monitoring intracellular pH¹⁶, we also detected simultaneous (30s) decrease in the PM-adjacent cytosolic pH after 5nM IAA treatment (Fig. 1c). Concomitant apoplastic increase and intracellular pH decrease implies H⁺-influx into the cells. This was confirmed using non-invasive microelectrodes monitoring direct net H⁺-exchange across the PM of elongating root epidermis cells after IAA treatment (Extended Data Fig. 2c), consistent with similar observations in root hair cells¹⁷.

Overall, auxin triggers rapid apoplast alkalinisation by increasing the net cellular H⁺-influx. Spatial and temporal correlation with root growth inhibition suggests apoplast alkalinisation as the underlying cellular mechanism.

Apoplastic pH regulates root growth

To investigate the causal relationship between apoplast alkalinisation and root growth inhibition, we manipulated the apoplastic pH by changing the medium pH (Extended Data Fig. 2d, e) and monitoring the impact on root growth. This extended previous observations of prolonged (2.5h) external extreme pH manipulation¹². Replacement of the basal medium at pH 5.8 by more alkaline (pH 6.15) medium caused instant reduction of root growth; the growth rate restored rapidly after washout with the original pH 5.8 medium (Fig. 1d, e). Gradual alkalinisation of the medium resulted in gradual root growth inhibition (Extended Data Fig. 2f). Replacing basal medium by more acidic (pH 5.1) medium increased root growth instantly and washout restored original growth (Fig. 1f, g).

Thus, exogenous manipulation of apoplastic pH has immediate and reversible effects on root growth, with alkaline pH inhibiting and acidic pH promoting growth. This strongly supports that auxin-induced apoplast alkalinisation is the key downstream cellular mechanism for rapid root growth inhibition.

Auxin triggers PM H⁺-ATPases activation

The auxin effect on apoplast alkalinisation occurs too fast to involve transcriptional regulation as also confirmed by pharmacological interference with translation (cycloheximide) or transcription (cordycepin) (Extended Data Figure 3a, b). To gain insights into the underlying mechanism, we mined recent datasets from Mass Spectroscopy (MS)-aided phospho-proteomics in WT root tips treated for 2min with 100nM IAA¹⁸. Among the differentially phosphorylated targets were two Plasma Membrane (PM) H⁺-ATPases: AHA1 and AHA2. Multiple putative auxin-regulated phosphorylation sites were identified in the auto-inhibitory C-terminal region, leading to both activation and deactivation of H⁺-pump activity¹⁹ (Fig. 2a and Extended Data Table 1).

To test whether auxin changes the activity of PM H⁺-ATPases in roots, we performed an ATP hydrolysis assay measuring the hydrolytic release of inorganic phosphate from ATP, representing the activity of PM H⁺-ATPases. After 1h treatment with 100nM IAA, we detected increased ATP hydrolysis activity in root protein extracts (Fig. 2b). This suggests that auxin activates H⁺-pumps, which should, however, lead to apoplast acidification instead of the observed alkalinisation (see Fig. 1b).

We next reanalysed the phospho-proteomics data specifically for the phosphorylation of Thr⁹⁴⁷ in AHA2, a well-known activation site¹⁹. Thr⁹⁴⁷ was significantly more phosphorylated after IAA treatment (Fig. 2c). To confirm this, we used an antibody against the AHA2 catalytic domain and the anti-pT947-AHA2 antibody revealing that 10nM IAA induced phosphorylation of Thr⁹⁴⁷ in 10min (Extended Data Fig. 3c). Thus, auxin induces AHA2 phosphorylation leading to its activation.

Our results show that auxin rapidly induces AHA phosphorylation leading to H⁺-pump activation in roots. This is similar to shoots^20,21, however, is opposite to the observed auxin-induced H⁺-influx (see Fig. 1), suggesting that in roots H⁺-pump activation may act antagonistically to auxin-triggered apoplast alkalinisation.

H⁺-ATPases counteract apoplast alkalinisation

To better understand the role of H⁺-pump activation during auxin-triggered apoplast alkalinisation, we used the fungal toxin Fusicoccin (FC), which stabilizes the pump in the activated form¹⁹ without affecting transcriptional auxin signalling (Extended Data Fig. 3d). FC caused rapid apoplast acidification and promoted root growth¹² (Extended Data Fig. 3e, f), opposite to auxin. When FC and IAA were applied simultaneously or sequentially, we observed an intermediate response proportional to the auxin/FC ratio (Fig. 2d and Extended Data 3e-k). These suggest that FC-triggered H⁺-ATPase activation and IAA-triggered apoplast alkalinisation act antagonistically.

To test this genetically, we analysed auxin response of loss- and gain-of-function aha mutants. Single aha1 and aha2 mutants showed no growth defects (Extended Data Fig. 3l), while the double mutant is embryo-lethal¹⁹. To overcome the redundancy, we used a synthetic trans-acting siRNA targeting AHA1/2/7/11 (AtTAS1c-AHA), expressed from the PIN2 promoter²². AHAs were downregulated in two independent transgenic lines (Extended Data Fig. 3m) and both were hypersensitive to auxin for apoplast alkalinisation (Fig. 2e) and root growth inhibition (Extended Data Fig. 3n). In contrast, constitutive activation of AHA1 in the ost2-3D mutant resulted in decreased auxin sensitivity of apoplastic pH (Fig. 2e) and root growth (Extended Data Fig. 3n).

These observation show that H⁺-ATPase activation antagonizes auxin-induced apoplast alkalinisation in roots.

TMK1 interacts with H⁺-ATPases

To address how auxin signalling regulates apoplastic pH, we performed co-immunoprecipitation (co-IP) followed by MS-assisted identification of proteins associated with either the TIR1/AFB1 receptor or PM-localized TMK1 auxin signalling component⁷ (Extended Data Fig. 4a). For TIR1/AFB1 this approach did not reveal any relevant components, while for TMK1, AHAs were among the top enriched associated peptides (Extended Data Fig. 4b-d and Extended Data Table 2, 3).

We verified the interaction between AHAs and TMK1 by co-IP from pTMK1::TMK1-FLAG (Fig. 3a) and pAHA2::AHA2-GFP (Extended Data Fig. 4e) roots. From TMK1-FLAG pulldowns, we detected associated AHA2 and reciprocally from the AHA2-GFP pulldowns, we detected TMK1. Additional in vivo verification was provided by bimolecular fluorescent complementation (BiFC) in tobacco leaves co-transformed with TMK1 and AHA2 (Fig. 3b and Extended Data Fig. 4f).

These observations show that TMK1, the component of cell surface auxin signalling, interacts with PM H⁺-ATPase.

TMK1 mediates auxin effect on H⁺-ATPases

To test the role of TMK1 in H⁺-ATPase phosphorylation, we performed phospho-proteomic analysis in tmk1-1 roots compared to WT and detected strong hypo-phosphorylation of AHAs (Fig. 3c and Extended Data Table 1) suggesting TMK1 involvement in H⁺-ATPases phosphorylation.

To verify this, we cloned p35S::TMK1-HA and two kinase-dead versions with mutations in the ATP binding site: TMK1^K616E or TMK1^K616R. Transient overexpression of the wild type (TMK1^WT), but not the kinase-dead constructs resulted in rapid wilting of tobacco leaves (Extended Data Fig. 5a), an effect consistent with PM H⁺-ATPase activation²³. We further generated Arabidopsis dexamethasone (DEX)-inducible gain-of-function lines. Compared to TMK1^WT, root extracts of TMK1^K616R did not show IAA-induced phosphorylation of AHA^Thr947 (Extended Data Fig. 5b). Importantly, in vitro [g-³²P]-ATP kinase assays confirmed that TMK1^WT, but not kinase-dead TMK1^K616E directly phosphorylates the AHA2 C-terminal domain (Fig. 3d and Extended Data Fig. 5c).

Next, we analysed different tmk loss-of-function mutants. We detected less auxin-induced AHA2 phosphorylation in tmk1-1 single, tmk1,3 and the stunted tmk1,4 double mutant roots (Fig. 3e and Extended Data Fig. 5d, e). Besides, the ATP hydrolysis assay showed that auxin-stimulated H⁺-ATPase activity diminished in the tmk1-1, tmk4-1 and tmk1,4 roots (Fig. 3f).

Collectively, this demonstrates that active TMK1 mediates auxin-triggered phosphorylation and activation of H⁺-ATPases in roots.

TIR1 and TMK1 converge on pH regulation

Our results show that TMK1 directly phosphorylates and activates PM H⁺-ATPases leading to apoplast acidification (see Fig. 2 and 3). This is opposite to the observed auxin-induced apoplast alkalinisation leading to growth inhibition (see Fig. 1) prompting us to address the underlying signalling mechanism.

aux1-100 mutants in the auxin influx transporter AUXIN RESISTANT1 (AUX1) that are impaired in uptake of IAA²⁴ were less sensitive to auxin, both for growth inhibition⁶ (Extended Data Fig. 6b, d) and apoplast alkalinisation (Extended Data Fig. 6a, c) suggesting requirement of intracellular auxin perception.

Given that intracellular TIR1/AFB receptors mediate auxin-triggered rapid growth inhibition⁶, we evaluated apoplastic pH in parallel to growth in the tir1 afb2 afb3 (tir triple) mutant. tir triple roots were resistant to IAA in both apoplast alkalinisation and growth inhibition (Fig. 4a and Extended Data Fig. 6e). AFB1 with its predominant cytosolic localization was proposed to be the major auxin receptor mediating auxin effect on growth and membrane depolarization^25,26. We found that both TIR1 and AFB1 contribute to this regulation with tir1-10 more auxin-resistant in the long term, while afb1-3 showed pronounced resistance for rapid auxin effects (Extended Data Fig. 6h-k). We also applied the PEO-IAA anti-auxin to block downstream TIR1/AFB signalling²⁷. Simultaneous addition of 10µM PEO-IAA and 5nM IAA prevented apoplast alkalinisation and growth inhibition (Extended Data Fig. 6f, g). We also took advantage of the cvxIAA/ccvTIR1 system, in which the engineered concave (ccv) TIR1 receptor cannot interact with natural IAA, but only with a synthetic convex (cvx) IAA, allowing specific activation of TIR1/AFB signalling²⁸. Application of 50nM cvxIAA resulted in apoplastic alkalinisation in ccvTIR1 plants (Fig. 4b), confirming that specific activation of TIR1 is sufficient to trigger apoplast alkalinisation. These approaches demonstrate involvement of intracellular TIR1/AFB receptors in auxin-induced apoplast alkalinisation.

This effect is counteracted by the cell surface TMK1-mediated H⁺-ATPase activation for apoplast acidification and growth promotion. Indeed, in the steady state, TMK1 is redundantly required for root growth as demonstrated by shorter roots in tmk mutants⁷ (Extended Data Fig. 6l). In response to low concentrations of auxin, tmk1-related mutants were hypersensitive (Fig. 4c), while overexpressing TMK1 (pUBQ10::TMK1-3HA) led to a slight auxin resistance (Extended Data Fig. 6m). This resembles the corresponding loss- and gain-of-function aha mutants (see Fig. 2) providing additional support for the antagonistic, growth-promoting role of TMK-mediated AHA activity.

We also created a tmk1 tir1 double mutant and analysed the auxin effect on apoplastic pH and root growth. As expected, tmk1 tir1 mutants showed intermediate auxin sensitivity compared to the single mutants both for growth and apoplastic pH (Fig. 4d, e and Extended Data Fig. 6n, o).

Collectively, we propose that auxin activates two antagonistic signalling pathways: (i) cell surface TMK1-mediated H⁺ export acidifying apoplast and (ii) more dominant, intracellular TIR1/AFB-dependent apoplast alkalinisation leading to rapid growth inhibition (Fig. 4f).

Our findings provide novel insights into a long-standing question how plant root growth is regulated. In particular, we address the old mystery of opposite growth regulation in shoots and roots by the phytohormone auxin and we also clarify the downstream cellular mechanism of auxin-triggered root growth inhibition.

Auxin regulates root growth very rapidly, utilizing a non-transcriptional branch of a signalling pathway downstream of intracellular TIR1/AFB receptors⁶. The same branch mediates apoplast alkalinisation, which we confirm as the causative cellular mechanism for root growth regulation, thus extending the classical Acid Growth Theory also for root growth inhibition.

Remarkably, auxin-induced apoplast alkalinisation in roots does not occur through regulation of PM H⁺-ATPases as observed in shoots, where the TIR1/AFB transcriptional auxin signalling leads to PM H⁺-ATPase activation and apoplast acidification^4,20. Instead, in roots, PM H⁺-ATPases are phosphorylated and activated by the cell surface TMK1-based auxin signalling, which leads to apoplast acidification. This mechanism, acts antagonistically to the more dominant TIR1/AFB-mediated alkalinisation.

A key open question concerns the downstream mechanism, by which TIR1/AFB signalling mediates apoplast alkalinisation. A plausible scenario would be a rapid increase in H⁺ permeability across the PM, which is intertwined with changes in PM potential²⁵ (Extended Data Fig. 7a-c). Such auxin-triggered H⁺-influx cannot be easily explained by IAA^-/2H⁺ symport via the AUX1 influx carrier as proposed¹⁷ (Extended Data Fig. 7d) and seems to require previously reported¹⁴ auxin-triggered cytosolic Ca²⁺ transients (Extended Data Fig. 8). Another persistent mystery is the auxin perception mechanism for the TMK1 pathway. Does this occur via direct activation of TMK1 by auxin or through another yet to be established auxin receptor?

With cell surface-based TMK1 activating H⁺-pumps and intracellular TIR1/AFB signalling causing net cellular H⁺-influx, two auxin-triggered mechanisms converge on regulation of extracellular pH, which directly determines root growth. This seemingly counterproductive simultaneous ‘gas and brake’ action presumably poises the root tip for rapid and flexible directional growth changes during the challenging task to navigate through complex soil environments.

Acknowledgements

We thank Nataliia Gnyliukh and Lukas Hörmayer for technical assistance and Nadine Paris for sharing PM-Cyto seeds. We gratefully acknowledge 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) 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.), the 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 the Marie Skłodowska-Curie Grant Agreement No. 665385 and the DOC Fellowship of the Austrian Academy of Sciences to L.L., the China Scholarship Council to J.C.

Author 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 phospho-proteomics 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. did 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 [g-³²P]-ATP kinase assay, leaf wilting phenotype analysis, shared plasmids, seeds 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.

Competing interests.

The authors declare no competing interests.

Correspondence

Additional information is available from the corresponding author upon request.

Data availability statement

The data and full blots are available within the paper and the Supplementary Information. All source data used for graphs are available.

Code availability statement

All codes used in the manuscript are provided in SI.

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Plant materials and growth conditions

All Arabidopsis thaliana mutants and transgenic lines used are in Columbia-0 (WT) background. The pEB1b::EB1b-GFP¹, p35S::MAP4-GFP², pSYP22::SYP22-YFP³, DR5::LUC⁴, PM-Cyto⁵, GCAMP⁶ marker lines were described previously. The tir1-10⁷, afb1-3⁸, tir1-1 afb2-1 afb3-1 mutant⁹, pTIR1::ccvTIR1 in tir1-1 afb2-3¹⁰, pTIR1::TIR1 in tir1-1 afb2-3 (we called it control for ccvTIR1)¹⁰ and aux1-100¹¹, cngc14-2¹² were donated by the authors. The pTIR1::TIR1-VENUS in tir1-1¹³, pAFB1::AFB1-VENUS in afb1-3¹⁴ are shared by Stefan Kepinski. The aha mutants are the following: aha2-5 (SALK_022010)¹⁵, aha1-7 (SALK_065288)¹⁵, ost2-3D¹⁶ shared by Atsushi Takemiya. Two independent lines AtTAS1c-AHA#2 and #4 were generated by Jian Chen and Steffen Vanneste as follows: the syn-tasiRNA target sequence was inserted into pENTR-AtTAS1c-B/c¹⁷ using hybridized primers TAS-AHA pair (Extended Data Table 4) and was recombined into pH7m24GW¹⁸ together with pDONR P4-P1R¹⁹ carrying the pPIN2 promoter²⁰, to generate pPIN2:AtTAS1c-AHA. The pAHA2::AHA2-GFP²¹ seeds were donated by Anja T. Fuglsang. The tmk mutants are the following: tmk3-2 (SALK_107741) ordered from NASC; Tongda Xu^22,23 kindly contributed tmk1-1 (SALK_016360), tmk2-1 (SAIL_1242_H07), tmk4-1 (GABI_348E01), the complemented pTMK1::gTMK1-FLAG in tmk1-1 and tmk1-1 tmk4-1 (tmk1,4) double mutant seeds. The tmk1-1 tmk2-1 (tmk1,2) and tmk1-1 tmk3-2 (tmk1,3) were generated by crosses using alleles above. The transgenic plant lines carrying DEX::TMK1-HA and DEX-TMK1^K616R-HA were generated by Hong Ren and William M. Gray. The DEX::TMK1^WT (or TMK1^K616R/E)-HA constructs were done by cloning the cDNA of TMK1^WT (or TMK1^K616R/E) without stop codon (Extended Data Table 4) into pENTR/D-TOPO, and subsequently recombining into the pBAV154²⁴ binary vector used Gateway system. The pUBQ10::gTMK1-3HA and pTMK1::gTMK1-eGFP lines were generated by amplifying TMK1 full length gDNA without stop codon from WT genomic DNA using the primers indicated in Extended Data Table 4. TMK1 gDNA was inserted into pDONR221, subsequently recombined into pB7m34GW together with pDONR P4-P1R carrying the UBQ10 or TMK1 promoter and pDONR P2R-P3 3xHA or pDONR P2R-P3 eGFP, respectively. The constructs were transformed into the Agrobacterium tumefaciens strain pGV3101 by electroporation and further into WT plants by floral dip.

Seeds were surface-sterilized by chlorine gas, sown on half-strength Murashige and Skoog (½MS) medium supplemented with 1% (w/v) sucrose and 0.8% (w/v) phyto agar (pH 5.9), stratified in the dark at 4°C for 2d and then grown vertically at 21°C with a long-day photoperiod (16h light/8h dark). Light sources used were Philips GreenPower LED production modules [in deep red (660nm)/far red (720nm)/blue (455nm) combination, Philips], with a photon density of 140.4µmolm^-2s^-1±3%.

Treatment with inhibitors of gene translation, cycloheximide²⁵, or transcription, cordycepin²⁶ were done in the concentration and duration verified previously.

Microfluidics

The microfluidic vRootchip was used mostly to analyze root tip growth and apoplastic pH in real-time. The manufacturing of the chip, sample preparation procedure and data analysis of root tip growth were performed as described previously²⁵. Our new design contains an additional valve in the control layer that closes the ends of the root channels (Extended Data Fig. 1a). In case of air bubbles appeared in the root channels, the additional valve allows pressurizing the channel and air will be absorbed into the Polydimethylsiloxane (PDMS) chip material within 2-10min. Afterwards, experiments started after adaptation of at least 2h. Besides, we introduced a graphical user interface (Extended Data Script 1) using the Processing software (https://processing.org/) with the ControlIP5 package (http://www.sojamo.de/libraries/controlP5/) that sends serial commands to the Arduino. A sketch (Extended Data Script 2) runs on the Arduino to operate the electronics and receive commands. For one vRootchip, maximum 8 samples were used. When comparing two genotypes, 3-4 seedlings were used for each genotype and mounted in alternating channels to minimize the time difference between imaging two genotypes. For each root, we imaged one ROI containing early elongating epidermal cells and the other ROI covering the root tip. As these two ROIs were captured sequentially, we imaged the apoplastic pH and the growth of the same root close to simultaneously.

In vRootchip, we used basal liquid medium ¼MS+0.1% sucrose, pH 5.8 (adjusted with KOH). The media of different pH was prepared with basal medium adjusted pH by HCl or KOH. Besides, Ca²⁺-free liquid medium was prepared without CaCl₂.

Scanner growth assay

To complement the real-time imaging in vRootchip, growth analysis was performed on a vertical scanner with bigger sample sizes allowing more conditions to be evaluated. This growth measurement we called steady-state. 4d-old seedlings were transferred to 60×15mm petri dishes filled with 5ml ½MS medium with treatments as indicated. The petri dishes were placed on a vertically mounted flatbed scanner (Epson perfection V370) and seedlings were imaged through the layer of medium. Either wet black filter paper or ½MS medium containing activated charcoal was added in the lid to improve background contrast. The samples were automatically imaged every 10 or 30min using the AutoIt script described previously²⁷ and scans were taken at 1200dpi. The resulting image series were analyzed using StackReg stabilization and the Manual Tracking plugin in ImageJ, or using an in-house generated MATLAB-based application RootGrowth tracker²⁸.

Imaging and measuring apoplastic pH with HPTS dye

All apoplastic pH data were obtained using 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), a ratiometric fluorescent pH dye²⁹. pH measurements were done both in steady-state condition and real-time vRootchip imaging. For steady-state pH analysis, 4d-old seedlings were transferred to ½MS medium containing 1mM HPTS (Thermo Scientific 6358-69-6, dissolved in ddH₂O) and treatments were performed for 30 or 50min. Subsequently, seedlings on a slice of the treatment medium were mounted into a Lab-Tek Chambered Coverglass.

Real-time imaging of the apoplastic pH was done in vRootchip containing medium (¼MS+0.1% sucrose) supplemented with 1mM HPTS with or without treatment. All imaging was performed on the in-house established vertical Zeiss LSM 800 confocal microscope³⁰. Fluorescent signals for protonated HPTS (excitation 405nm, emission 514nm, visualized in red) and deprotonated HPTS (excitation, 488nm, emission 514nm, visualized in green) were detected with a 20x/0.8 air objective. Image analysis was performed on a cropped region of elongating epidermis cells using batch processing of a previously described the ImageJ macro²⁹. Relative pH value is calculated as the background-subtracted intensity of the deprotonated intensity divided by that of the protonated intensity. Resulting relative pH data were plotted over time and statistically evaluated in GraphPad Prism 6. Note that we did not transform the relative pH value to absolute pH values, which would require the generation of a calibration curve for each experiment.

Imaging and measuring cytosolic pH with PM-cyto reporter

Real-time imaging of the cytosolic pH near the PM was done by using PM-Cyto reporter line⁵ in vRootchip and a vertical Zeiss LSM 800 confocal microscope³⁰. Sequential illumination at 488 and 405nm with emission 514nm for both, corresponding to the two absorption peaks of pHluorin, were taken with a 20x/0.8 air objective. For each root in vRootchip, two ROIs were tracked over time with one containing elongating epidermal cells for measuring the cytosolic pH and the other covering the root tip for measuring the root growth rate. Image analysis was performed similar to the HPTS analysis described above.

Imaging microtubule orientation, vacuolar morphology and cytosolic Ca²⁺ spike

The pEB1b::EB1b-GFP maker line¹ was used to track the dynamics of CMTs orientation in vRootchip. Images were obtained every 6.25s and the analysis of the CMTs orientation was done in ImageJ by max Z-projection on every 10 frames and quantification by a for batch processing modified version of the Fibril Tool macro³¹. The p35S::MAP4-GFP marker line² was used for capturing the CMTs orientation after treatment for the indicated time period (steady state). The CMTs orientation angle was calculated using the Bioline script³². For both marker lines, the GFP (excitation 488nm, emission514 nm) signal was detected by Plan-Apochromat 20x/0.8 air objective in the vertical Zeiss LSM 800 confocal microscope³⁰.

The pSYP22::SYP22-YFP marker line³ was used for imaging vacuolar morphology. We used a mounting system³³, which allows the injection of new liquid medium during imaging. Images were taken before and 30min after Mock or 100nM IAA treatment and the YFP (excitation 488 nm, emission 527 nm) intensity was detected with C-Apochromat 40x/1.20 W Korr objective in an inverted Zeiss LSM 800 confocal microscope.

The GCaMP marker line⁶ was crossed into ccvTIR1 and control transgenic plants¹⁰ and used for imaging cytoslic Ca²⁺ level in vRootchip. Images were taken every 14.4s for 1h. GFP (excitation 488nm, emission 514 nm) signal was detected by Plan-Apochromat 20x/0.8 air objective in the vertical Zeiss LSM 800 confocal microscope³⁰.

Non-invasive microelectrode (MIFE) ion flux measurements

Net fluxes of H⁺, K⁺, and Ca²⁺ were measured using the non-invasive microelectrode ion flux estimation (MIFE) technique essentially as described elsewhere³⁴. In brief, microelectrodes were pulled out by PE-22 puller (Narishige), dried in an oven and silanized with tributylchlorosilane (Cat 90794, Sigma-Aldrich, Australia). The prepared electrode blanks were backfilled with respective solutions for each measured ion and electrode tips front-filled with selective liquid ion exchangers (LIX) purchased from Sigma-Aldrich) to measure ions of interest (H⁺- Cat. 95291; K⁺- Cat. 99311, Ca²⁺- Cat. 99310). A root of intact 6d-old Arabidopsis WT seedlings was immobilised in a measuring chamber using Perspex holders and basic salt media (BSM) added. The composition of the BSM solution was 0.5mM KCl and 0.1mM CaCl₂; pH 5.8, unbuffered. Measurements were recorded from root elongating epidermal cells (~450µm from the root tip). After 40min of conditioning, the microelectrodes were positioned 20µm from the root surface and moved in a slow (6s cycle, 100μm amplitude) square-wave by a computer-driven micromanipulator (MHW-4, Narishige). Net ion fluxes were calculated by the MIFEFLUX software based on the measured difference in electrochemical gradient between these two positions using the cylindrical diffusion geometry as described elsewhere³⁴. The steady fluxes were recorded for 5-10min to make sure that steady state condition was reached. Then 10nM IAA was applied to the measuring chamber, and transient H⁺, K⁺, and Ca²⁺ kinetics were measured for further 20min. At least 9 individual plants from several batches were used. The sign convention is “influx positive”.

Membrane potential measurements

Membrane potential (MP) values were measured from root epidermal elongating cells of intact Arabidopsis seedlings. Conventional microelectrodes (Harvard Apparatus) were filled with 1M KCL and connected to the MIFE electrometer via the Ag/AgCl half-cell. During MP measurement, the microelectrode with a tip diameter of 0.5µm was manually impaled into the epidermal cells of elongation (~450µm from root tip) using a 3D-micromanipulator (MHW-4, Narishige). MP values were recorded by the MIFE CHART software for at least two minutes after stabilization³⁴. Prior to measurements, a 6d-old seedling was immobilised on a Perspex block using Parafilm strips, the block then was inserted into a vertical Perspex measuring chamber and filled with basic salt media (BSM: 0.5mM KCl and 0.1mM CaCl2; unbuffered, of required pH). After 40min conditioning in BSM, the measuring chamber was mounted on a MIFE microscope stage located in a Faraday cage for MP measurements. MP measurements were conducted in two ways: under steady state conditions (at different pH values) and as transient kinetics (in response to IAA application). In the steady state experiments, MP values were recorded from roots of 5-6 individual seedlings with a new electrode being used for each measurement to ensure that the electrode tip was not blocked. At least 4 measurements were made for each seedling. In transient kinetics experiments, MP was recorded from a root in BSM (pH 5.8) for 1-2min after the initial cell penetration and then IAA prepared in BSM was added to the chamber (final concentration 10nM) followed by 5min MP recording.

Evaluating the TIR1-transcriptional response using DR5::LUC

4d-old DR5::LUC seedlings⁴ are placed on the surface of solidified ½MS medium with 200µl of 5mM D-luciferin dissolved in a 1x PBS drop on the root tips for 30min as pre-treatment. Subsequently, the samples were transferred to solidified ½MS medium supplemented with mock, 10nM IAA, 10µM FC and IAA+FC, and immediately imaged in an in-house established dark box with a Photometric Evolve^® EMCCD camera equipped with a 17mm fixed lens/0.95 and an additional 125mm lens²⁷. The multiplier EMCCD gain was set to 70s and the exposure time to 35s, and images were acquired every 2min. The resulting time-lapse video was analysed in ImageJ as described previously²⁷.

Identification of TMK1-interacting proteins using IP/MS-MS

Immunoprecipitation (IP) experiments were performed in 3 biological replicates as described previously³⁵ using 1g of roots of 7d-old seedlings from the pTMK1::TMK1-eGFP transgenic line and 1g of roots from WT. Interacting proteins were isolated by incubating total protein extracts with 100µL anti-GFP coupled magnetic beads (Miltenyi Biotech). 3 replicates of pTMK1::TMK1-eGFP were compared to 3 WT replicates. Tandem mass spectrometry (MS) on a Q-Exactive device (Thermo Fisher) and statistical analysis using MaxQuant and Perseus software was performed as described previously³⁶.

Identification of TIR1- and AFB1-interacting proteins using IP/MS-MS

For immunoprecipitation, ground plant material of pTIR1::TIR1-VENUS in tir1-1 and pAFB1::AFB1-VENUS in afb1-3 transgenic lines was lysed in mild lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM MgCl₂, 0.2mM EDTA, 1xCPI, 0.5mM DTT, 0.2% NP40 and 1mg/ml DNAse) and mildly sonicated using a Bioruptor (Diagenode). After lysate clearance, supernatant was submitted to enrichment using GFP-Trap agarose beads (Chromotek) for 45min at 4°C while gently rotating. Beads were subsequently washed twice in lysis buffer, twice in detergent-free lysis buffer and trice in 50mM Ammoniumbicarbonate (ABC) (Sigma) with intermediate centrifuging for 2min at 2000g at 4°C. After the final wash, bead-precipitated proteins were alkylated using 50mM Acrylamide (Sigma). Precipitated proteins were submitted to on-bead trypsin digestion using 0.35µg trypsin (Roche) per reaction. After overnight incubation at 25°C, peptides were desalted and concentrated using C18 Stagetips.

After Stagetip processing, peptides were applied to online nanoLC-MS/MS using a 60min acetonitrile gradient from 8-50%. Spectra were recorded on a LTQ-XL mass spectrometer (Thermo Scientific) and the statistical analysis using MaxQuant and Perseus software was performed as described previously³⁶.

Phospho-proteomics of auxin-treated roots

Roots from 5d-old plants were treated and immediately harvested and flash frozen in liquid nitrogen. They then were ground to fine powder in liquid nitrogen. Powder was suspended in SDS lysis buffer (100mM Tris pH 8.0, 4%SDS and 10mM DTT) and sonicated using a cooled Biorupter (Diagenode) for 10min using high power with 30s on 30s off cycle. Lysate was cleared by centrifugation at maxiumum speed for 30min. Protein concentrations were determined using the Bradford reagent (Bio-Rad).

For FASP 30kDa cut-off amicon filter units (Merck Millipore) were used. Filters were first tested by appling 50µl urea buffer UT buffer (8M Urea and 100mM Tris pH 8.5) and centrifuging for 10min at 11000rpm at 20°C. The desired amount of protein sample was next mixed with UT buffer until a volume of 200µl, applied to filter and centrifuged for 15min. All centriguge steps were at 11000rpm at 20°C. Filter was washed with UT buffer for 15 min. Retained proteins were alkylated with 50mM acrylamide (Sigma) in UT buffer for 30min at 20°C while gently shaking followed by a triple wash step with UT buffer for 15 minutes and three washes with 50mM ABC buffer. After last wash proteins were cleaved by adding trypsin (Roche) in a 1:100 trypsin to protein ratio. Digestion was completed overnight. The following day filter was changed to a new tube and peptides were eluted by centrifuging for 15min. Further elution was completed by adding two times 50mM ABC buffer and centrifuging for 10min on 11000rpm at 20°C.

For peptide desalting and concentrating 200 µl tips were fitted with 2 plugs of C18 octadecyl 47mm Disks 2215 (Empore™) material and 1mg:10µg of LiChroprep® RP-18:peptides (Merck). Tips were sequentially equilibrated with 100% methanol, 80% ACN in 0.1% formic acid and twice with 0.1% formic acid for 4min at 1500g. After equilibration peptides were loaded for 20min at 400g. Bound peptides were washed with 0.1% formic acid and eluted with 80% ACN in 0.1% formic acid for 4min at 1500g. Eluted peptides were subsequently concentrated using a vacuum concentrator for 30min at 45°C and resuspended in 50µl of 0.1% formic acid.

For phosphopeptide enrichment magnetic Ti^4+-IMAC (MagResyn) were used according to manufactures protocol. Enrichments were perfromed with 1mg of peptides in biological quadruplicate.

After Stagetip processing, peptides were applied to online nanoLC-MS/MS using a 120min acetonitrile gradient from 8-50% for phospho-proteomics. Spectra recording and statistical analysis were as previously described, with the addition of phosphorylation as a variable modification³⁶. Filtering of datasets was done in Perseus in as described³⁷.

Phospho-proteomics in WT and tmk1-1 roots

4 biological replicates of WT and tmk1-1 roots were prepared and treated as indicated above. They were submitted to the phospho-proteomic pipeline^36,37 and differentially phosphorylated peptides belonging to H⁺-ATPases were specifically filtered out of the big dataset (Extended Data Table 1).

in vitro kinase assay with [g-³²P]-ATP

6xHis-MBP-TMK1^WT kinase domain (or kinase-dead TMK1^K616E) was purified from E. coli. Briefly, ca. 100ng of purified protein was added to reactions containing 5 mM HEPES, pH 7.5, 10 mM MgCl₂, 10mM MnCl₂, 1mM DTT and the assay initiated by adding 1μl of ATP solution containing 100μM (unlabeled) ATP and 33nM [g-³²P]-ATP (3000 Ci/mmol). ca. 150ng of purified GST-AHA2 C-terminal was added as indicated. Reactions were incubated at 28°C for 40min, stopped with SDS-PAGE sample buffer, run out on SDS-PAGE and phosphorylation was visualized by autoradiography. Ponceau staining was performed as loading control.

Protein extraction and Western blot analysis for co-IP and determination of AHA2 phosphorylation state

To isolate PM H⁺-ATPases and potential interactors, 5-7d-old plant roots were harvested at the indicated time points after 10 or 100nM IAA auxin treatment. 24h prior to the evaluation of auxin effects, these seedlings were sprayed with ½AM solution containing 30µM kynurenine. The root samples were flash frozen in liquid nitrogen and ground (Retsch mill, 2x 1min at 20Hz). The root powder was then resuspended in a 1:1 (w/v) ratio in protein extraction buffer (25mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 1xRoche cOmplete™ Protease Inhibitor Cocktail, 1xRoche PhosSTOP^TM, 1mM EDTA, 1mM DTT and 0.5mM PMSF). The samples were incubated on ice for 30min, followed by a centrifuging step at 10000g to discard the plant debris. The cleared supernatant containing the proteins of interest was collected and the total protein content was determined using Quick Start Bradford reagent (Bio-Rad). This could further be used for co-immunoprecipitation analysis or for SDS-PAGE analysis. In order not to lose relevant proteins, protein samples were not boiled in the presence of reducing Laemmli buffer and no harsher PM extraction or membrane enrichment was attempted.

For co-immunoprecipitation, root extracts (obtained by extraction in the Lysis buffer supplied in the Miltenyi µMACs kit, supplemented with 1xRoche cOmplete™ Protease Inhibitor Cocktail, 1mM DTT and 0.5mM PMSF), were incubated with magnetic beads from the Miltenyi anti-GFP, anti-HA or anti-FLAG µMACs kits (depending on the tags of the proteins of interest) and kept rotating for 4h at 4°C. Elution was performed with room-temperature denaturing elution buffer and the proteins were analyzed by SDS-PAGE and Western blot.

Following separation of proteins by SDS-PAGE in a 10% acrylamide gel (Protean^® TGX^TM, Bio-Rad), proteins were transferred to PVDF membranes by electroblotting (Trans-blot^®Turbo^TM, Bio-Rad). The membranes were then incubated in blocking buffer (0.05% Tween-20, 5% milk powder or 3% BSA, 20mM Tris-HCl, pH 7.5 and 150mM NaCl) for at least 60min and incubated with antibody solution against the protein of interest. All raw gel data are shown in Supplemental Information.

Antibodies

The anti-AHA2 and anti-Thr⁹⁴⁷ AHA2 antibody were shared by Toshinori Kinoshita and used as described previously³⁸ at final dilution of 1:5000 in TBST buffer+3% BSA, followed by anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (HRP) (GE Healthcare, NA934) at a dilution of 1:10000 and chemiluminescence reaction (SuperSignal West Femto, Thermo Scientific). To allow multiple antibody detections using the same PVDF membrane, mild stripping was performed using 15g/L glycine, 1g/L SDS, 10mL/L Tween-20 buffer at pH 2.2 for 2-5min.

ATP hydrolysis in root samples

To deplete endogenous auxin levels in the seedlings, 14d-old plants were pre-treated with 30µM kynurenine for 24h in the dark. Then, the pretreated seedlings were incubated in presence and absence of 100nM IAA for 60min under dark condition. The roots excised from the seedlings were homogenized in homogenization buffer (50mM MOPS-KOH,pH 7.0, 100mM KNO₃, 2mM sodium molybdate, 0.1mM NaF, 2mM EGTA, 1mM PMSF and 20µM leupeptin) and the homogenates were centrifuged at 10000g for 10min. The obtained supernatant was further ultra-centrifuged at 45000 rpm for 60min. The resultant precipitate (microsomal fraction) was resuspended in the homogenization buffer. ATP hydrolytic activity in the microsomal fraction was measured by the release of inorganic phosphate from ATP in a vanadate-sensitive manner following the method published³⁹ with the following modifications. The microsomal fraction (22.5µL, 0.2mg/mL) was mixed with the equal volume of the reaction buffer (60mM MES-Tris. pH 6.5, 6mM MgSO₄, 200mM KNO₃, 1 mMammonium molybdate, 10µg/mL oligomycin, 2mM NaN₃, 0.1% Triton X-100, 1mM PMSF and 20µM leupeptin) with or without 1µL of 10mM sodium orthovanadate. The reaction was started by adding 5µL of 2mM ATP and terminated by adding 50µL of the stop solution (2.6% [w/v] SDS, 0.5% [w/v] sodium molybdate and 0.6N H₂SO₄) after incubating at 30ºC for 30min.

Bimolecular Fluorescence Complementation (BiFC)

Following the method described⁴⁰, the full-length coding sequences of AHA2 and TMK1 without stop codons were amplified by PCR (primers in Extended Data Table 4), cloned into pENTR/D-TOPO or pDONR207 and recombined in pSPYNE and pSPYCE⁴¹ to generate BiFC expression constructs. The resulting binary vectors were introduced in Agrobacterium GV3101 by electroporation and these were cultured until OD₆₀₀ 0.8. Syringe infiltration was performed in Nicotiana benthamiana leaves as described⁴². For the constructs of interest, final OD₆₀₀ of 0.2 was used and p19 was co-infiltrated at OD₆₀₀ 0.1 to avoid gene silencing. Infiltration buffer of pH 5.8 contained: 10mM MgSO₄, 10mM MES-KOH and 0.15mM acetosyringone. TMK1 overexpression, even transiently, has a strong effect on the viability of the leaves, so samples were taken daily after infiltration to determine the optimal balance between expression level and viable leaf cells. To visualize protein interactions, sections of the leaves were imaged using a Zeiss LSM 700 confocal microscope.

Quantitative RT-PCR

RNA was extracted from 5d-old light-grown root tips with the RNAeasy Plant Mini Kit (Qiagen), with three biological replicates for each genotype. 2µg of RNA was used for cDNA synthesis (Qiagen). Samples were pipetted in three technical replicates using an automated JANUS Workstation (PerkinElmer) and measured by the Real-time PCR Roche LightCycler 480 using Luna® Universal qPCR mastermix (NEB, M3003S). Primers utilized for assessing gene expression are listed in Extended Table 4. Expression levels were normalized to Elongation factor 1-alpha (At5G60390)⁴³.

Statistical analysis and reproducibility

All graphs were generated using GraphPad Prism 6 or 8. For statistical analysis of vRootchip data, Two-way ANOVA was performed for the entire time frame of the experiment, except when a specific time interval is indicated. Welch ANOVA analysis was applied for the scanner growth assay with multiple time points, and one-way ANOVA assays were used for steady state (one incubation time point) pH and scanner growth datasets. Stars indicate significant differences on all graphs with ns for p>0.05, * for p≤0.05, ** for p≤0.01, *** for p≤0.001 and **** for p≤0.0001. Experiments always included sufficient biological replicates and were repeated at least twice independently with similar results. The depicted data show the results from one representative experiment.

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ExtendedData.docx
Extended Data Figures and Table Legends
ExtendedDataTable1phosphositesAHAs.xlsx
Extended Data Table 1
ExtendedDataTable2TIR1AFB1IPMS.xlsx
Extended Data Table 2
ExtendedDataTable3TMK1IPMS.xlsx
Extended Data Table 3
ExtendedDataTable4primers.xlsx
Extended Data Table 4

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

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