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 Xu22,23 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-TMK1K616R-HA were generated by Hong Ren and William M. Gray. The DEX::TMK1WT (or TMK1K616R/E)-HA constructs were done by cloning the cDNA of TMK1WT (or TMK1K616R/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.
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 Ti4+-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 pipeline36,37 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-TMK1WT kinase domain (or kinase-dead TMK1K616E) 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 PhosSTOPTM, 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® TGXTM, Bio-Rad), proteins were transferred to PVDF membranes by electroblotting (Trans-blot® TurboTM, 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.
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.
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.
Methods and Extended Data Figure references
1 Komaki, S. et al. Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis. J. Cell Sci 123, 451-459 (2010).
2 Marc, J. et al. A GFP–MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927-1939 (1998).
3 Robert, S. et al. Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1. PNAS 105, 8464-8469 (2008).
4 Moreno-Risueno, M.A. et al. Oscillating gene expression determines competence for periodic Arabidopsis root branching. Science 329, 1306-11 (2010)
5 Martinière, A. et al. Uncovering pH at both sides of the root plasma membrane interface using non-invasive imaging. PNAS, 115, 6488-6493 (2018).
6 Toyota, M. et al. Glutamate triggers long-distance, calcium-based plant defense signaling. Science 361, 1112-1115 (2018).
7 Parry, G. et al. Complex regulation of the TIR1/AFB family of auxin receptors. PNAS 106, 22540-22545 (2009).
8 Prigge, M. J. et al. Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions. Elife 9, e54740 (2020).
9 Dharmasiri, N. et al. Plant development is regulated by a family of auxin receptor F box proteins. Dev. Cell 9, 109-119 (2005).
10 Uchida, N. et al. Chemical hijacking of auxin signaling with an engineered auxin–TIR1 pair. Nat. Chem. Biol. 14, 299 (2018).
11 Swarup, R. et al. Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell 16, 3069-3083 (2004).
12 Shih, H.-W., DePew, C. L., Miller, N. D. & Monshausen, G. B. The cyclic nucleotide-gated channel CNGC14 regulates root gravitropism in Arabidopsis thaliana. Curr. Biol. 25, 3119-3125 (2015).
13 Wang, R. et al. HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1. Nat. Commun. 7, 1-11 (2016).
14 Rast-Somssich, M. I. et al. The Arabidopsis JAGGED LATERAL ORGANS (JLO) gene sensitizes plants to auxin. J. Exp. Bot. 68, 2741-2755 (2017).
15 Haruta, M. et al. Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity. J. Biol. Chem. 285, 17918-17929 (2010).
16 Yamauchi, S. et al. The plasma membrane H+-ATPase AHA1 plays a major role in stomatal opening in response to blue light. Plant Phys. 171, 2731-2743 (2016).
17 Carbonell, A. et al. New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Phys. 165, 15-29 (2014).
18 Karimi, M., Bleys, A., Vanderhaeghen, R. & Hilson, P. Building blocks for plant gene assembly. Plant Phys. 145, 1183-1191 (2007).
19 Marquès-Bueno, M. M. et al. A versatile multisite gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis. Plant J. 85, 320-333 (2016).
20 Zhang, Y., Xiao, G., Wang, X., Zhang, X. & Friml, J. Evolution of fast root gravitropism in seed plants. Nat. Commun. 10, 1-10 (2019).
21 Fuglsang, A. T. et al. Receptor kinase-mediated control of primary active proton pumping at the plasma membrane. Plant J. 80, 951-964 (2014).
22 Cao, M. et al. TMK1-mediated auxin signalling regulates differential growth of the apical hook. Nature 568, 240 (2019).
23 Wang, Q. et al. A phosphorylation-based switch controls TAA1-mediated auxin biosynthesis in plants. Nat. Commun. 11, 1-10 (2020).
24 Lee, J. et al. Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces. Plant Phys. 158, 1803-1818 (2012).
25 Fendrych, M. et al. Rapid and reversible root growth inhibition by TIR1 auxin signalling. Nat. Plants 4, 453 (2018).
26 Robert, S. et al. ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis. Cell 143, 111-121 (2010).
27 Li, L., Krens, S. G., Fendrych, M. & Friml, J. Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis thaliana hypocotyls. Bio Protoc. 8 (2018).
28 Gelová, Z. et al. Developmental roles of auxin binding protein 1 in Arabidopsis Thaliana. Plant Sci. 303, 110750 (2021).
29 Barbez, E., Dünser, K., Gaidora, A., Lendl, T. & Busch, W. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana. PNAS 114, E4884-E4893 (2017).
30 Von Wangenheim, D. et al. Live tracking of moving samples in confocal microscopy for vertically grown roots. Elife 6, e26792 (2017).
31 Boudaoud, A. et al. FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat. Protoc. 9, 457-463 (2014).
32 Adamowski, M., Li, L. & Friml, J. Reorientation of cortical microtubule arrays in the hypocotyl of Arabidopsis thaliana is induced by the cell growth process and independent of auxin signaling. Int. J. Mol. Sci. 20, 3337 (2019).
33 Narasimhan, M. et al. Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking. Plant Phys. 186, 1122-1142 (2021).
34 Shabala, S. N., Newman, I. A. & Morris, J. Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Phys. 113, 111-118 (1997).
35 De Rybel, B. et al. A bHLH complex controls embryonic vascular tissue establishment and indeterminate growth in Arabidopsis. Dev. Cell 24, 426-437 (2013).
36 Wendrich, J. R., Boeren, S., Möller, B. K., Weijers, D. & De Rybel, B. in Plant Hormones: 147-158 (Springer, 2017).
37 Nikonorova, N. et al. Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome reveal growth regulators. J. Exp. Bot. 69, 4591-4607 (2018).
38 Hayashi, Y. et al. Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase. Plant Cell Physiol. 51, 1186-1196 (2010).
39 Inoue, S.-i., Takahashi, K., Okumura-Noda, H. & Kinoshita, T. Auxin influx carrier AUX1 confers acid resistance for Arabidopsis root elongation through the regulation of plasma membrane H+-ATPase. Plant Cell Physiol. 57, 2194-2201 (2016).
40 Spartz, A. K. et al. SAUR inhibition of PP2C-D phosphatases activates plasma membrane H+-ATPases to promote cell expansion in Arabidopsis. Plant Cell 26, 2129-2142 (2014).
41 Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428-438 (2004).
42 Leuzinger, K. et al. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J. Vis. Exp. 77: 50521 (2013).
43 Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. & Scheible, W.-R. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Phys. 139, 5-17 (2005).
44 Serre, N. B. et al. AFB1 controls rapid auxin signalling through membrane depolarization in Arabidopsis thaliana root. Nat. Plants, 1-10 (2021).
45 Dindas, J. et al. AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling. Nat. Commun. 9, 1-10 (2018).
46 Yang, Y., Hammes, U. Z., Taylor, C. G., Schachtman, D. P. & Nielsen, E. High-affinity auxin transport by the AUX1 influx carrier protein. Curr. Biol 16, 1123-1127 (2006).
47 Dumont, J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol 136, 153-179 (1972).