Plant material and growth conditions
All Arabidopsis thaliana lines are in a Col-0 background. T-DNA insertion lines were supplied by the Arabidopsis Biological Resource Center. PCR genotyping for homozygous lines was performed using the primers listed in Sup. Table 1. To generate npf2.12 npf2.13 double mutant, npf2.12 (SALK_138987) was crossed to npf2.13 (SALK_022429) to obtain an F1 population. F3 homozygous plant was selected by PCR genotyping.
Plants were sown on vertical plates containing 0.5 × Murashige-Skoog (MS) medium, 1% sucrose, and 0.8% agar, pH 5.7, stratified for 2 days at 4°C in the dark, then transferred to growth chambers (Percival CU41L5) at 21°C, 100 µE m− 2 s− 1 light intensity under long day light (16 h light/8 h dark). All plants in suberin quantification experiments were grown on 0.5 × MS medium, 1% sucrose, and 0.8% agar, pH 5.7 for 3 days and subsequently moved to 0.5 × MS medium without sucrose supplementation due to phenotype masking by the sucrose treatment. For low-nitrate experiments, plants were sown on MS with vitamins, nitrate free (Caisson labs MSP07-50LT), which was supplemented with 0.01 mM (Low nitrate) or 10 mM (High nitrate) KNO3.
Hypocotyl cross sections
Sectioning and clearing were performed as describe in Ursache et al 44. 3-Week-old hypocotyls were fixed in 4% PFA for an hour, rinsed twice in 1xPBS embedded in 5% agarose and sectioned to 150 µM slices using a Leica VT1000S vibratome. Slices were cleared using a ClearSee solution for 5 days. Following clearing sections were counterstained with 0.1% Calcofluor White in ClearSee solution for 30 min. Next, the seedlings were washed in ClearSee for 30 min with gentle shaking. For imaging, sections were mounted directly in ClearSee and imaged using a Zeiss LSM 780 inverted microscope.
Hormone was added to the agar medium at concentrations indicated in the figure legends. Seedlings were either germinated on media or moved after germination to treatment plates. GA-Fl (5 μM) was applied in liquid MS media for 16 h prior to imaging. For ga1 experiments, both Col-0 and ga1 seeds were imbibed in sterile water containing 5 μM GA3 for 16 h to induce uniform germination. Following imbibition, seeds were washed three times in sterile water to wash away excess GA and were sown on MS plates.
Cloning of NPFs overexpression and reporter lines
NPF2.12 and NPF2.14 coding sequences were synthesized by Bio Basic Inc., cloned into pENTR/D-TOPO (Invitrogen K2400), and subsequently cloned into the pH7YWG2 destination vectors using the LR Gateway reaction (Invitrogen 11791). NPF2.12 and NPF2.14 promoters were amplified with the primers listed in Sup. Table 2 using a Phusion high-fidelity polymerase (New England Biolabs), cloned into pENTR/D-TOPO, and then cloned into pMDC7 vector for NLS-YFP reporters and pGWB3 vector for GUS reporters.
To generate pNPF2.13:GUS reporter, the promoter of NPF2.13 (1.7-kb fragment) was PCR amplified from Col-0 genomic DNA with appropriate primers listed in Sup. Table 2 and inserted into pDONR221 (Invitrogen) by Gateway cloning and recombined with pGWB633 67.
Imaging and analysis
Seedlings were stained in 10 mg/L-1 propidium iodide (PI) for 5 min, rinsed, and mounted in water. Seedlings were imaged using a laser scanning confocal microscope (Zeiss LSM 780 inverted microscope), with argon laser set at 488 nm for fluorescein, 514 nm for YFP, and 561 nm for PI excitation. Emission filters used were 493-548 nm for fluorescein derivatives, 508-570 nm for YFP, and 583-718 nm for PI emission. Image analysis and signal quantification were done with the measurement function of ZEN lite software. The number of quantified biological repeats and sampling points is indicated for each graph in figure legends.
Root length characterization
For root length measurements, seedlings were imaged using Zeiss Stemi 2000-C stereo microscope and measured using ImageJ software (http://rsbweb.nih.gov/ij/index.html).
Histochemical GUS staining
Plants were immersed in 100 mM sodium phosphate buffer (pH 7.0) containing 0.1% Triton X-100, 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt (Sigma-Aldrich), 2 mM potassium ferricyanide, and 2 mM potassium ferrocyanide. Plants subject to vacuum treatment for 10 min and then incubated at 37 °C for 16 h. Tissues were cleared with 30%, 50%, and 70% ethanol for 30 min in each concentration and imaged using an AxioZoom 16, Zeiss binocular microscope.
For cross-sectioning of GUS-stained leaf petioles, after clearing in 70% ethanol, the samples were fixed in FAA solution (3.2% formaldehyde, 5% acetic acid, 50% ethanol) for 30 min and kept overnight at 4°C. The samples were then dehydrated in an ethanol gradient ranging from 50% to 96%, and incubated in 2% eosin overnight at 4° C. After several washes in 96% ethanol, the samples were progressively rehydrated in ethanol/HISTO-CLEAR II (Electron Microscopy Sciences) solution, incubated in 50% HISTO-CLEAR II 50% PARAPLAST PLUS (McCormick Scientific) at 60°C for 2 h, and embedded in 100% PARAPLAST PLUS. Paraffin-embedded samples were cross-sectioned with LEICA RM2155 microtome and imaged using a Leica Leitz Dmrb microscope.
Nile red suberin staining, imaging and quantification.
Nile red suberin staining was performed as described by Ursache et al. 44. In short, 5-day-old seedlings were fixed in paraformaldehyde for 1 h under gentle agitation and washed twice in phosphate-buffered saline, pH 7.4. Plants were covered in filtered 0.05% Nile red (Acros Organics, 7385-67-3) solution dissolved in ClearSee for 16 h. Following staining, plants were washed three times in ClearSee for 30 min each wash. Next, plants were counterstained with 0.1% calcofluor white (Glentham Life Sciences, 4404-43-7) dissolved in ClearSee for cell wall imaging. After 30 min, plants were washed in ClearSee for 30 min. Plants were mounted directly in ClearSee on slides and imaged with a Zeiss LSM780 confocal microscope. Images were taken from the upper part of the root and under the root-hypocotyl junction with an argon laser set at 514 nm for Nile red excitation and 405 nm for calcofluor excitation. Emission filters used were 561-753 nm filter for Nile red and 410-511 nm filter for calcofluor emission. Fluorescence intensity was assessed using the Zen software from 5 endodermal cells per root.
Protein sequences for Arabidopsis thaliana NPF family members were retrieved from TAIR (https://www.arabidopsis.org). Phylogenetic relationships were defined using Phylogeny.fr (http://www.phylogeny.fr/) and visualized with FigTree software (http://tree.bio.ed.ac.uk/software/figtree/).
Transport assays in Xenopus oocytes
Coding sequences were cloned into the pNB1u vector, and complementary RNA (cRNA) was produced as described in Wulff et al. 34. Xenopus oocyte assays were performed as described previously 34. Defolliculated Xenopus laevis oocytes (stage V-VI) were purchased from Ecocyte Biosciences and were injected with 25 ng cRNA in 50.6 nl using a Drummond Nanoject II and incubated for 2-4 days at 16 °C in HEPES-based kulori (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH 7.4) before use. Oocytes were pre-incubated in MES-based kulori (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES, pH 5) for 4 min and were then transferred to phytohormone-containing MES-based kulori for 60 min. After washing three times in 25 ml HEPES-based kulori followed by one wash in 25 ml deionized water, oocytes were homogenized in 50% methanol and stored for >30 min at -20 °C. Following centrifugation (25000 g for 10 min 4 °C), the supernatant was mixed with deionized water to a final methanol concentration of 20% and filtered through a 0.22-µm filter (MSGVN2250, Merck Millipore) before analytical LC-MS/MS as described below. For nitrate assays, sodium chloride in kulori was substituted for equimolar sodium nitrate in order not to affect the membrane potential.
Quantification of phytohormone content by LC-MS/MS
Compounds in the diluted oocyte extracts were directly analyzed by LC-MS/MS. The analysis was performed with modifications from the method described in Tal et al. 16. In brief, chromatography was performed on an Advance UHPLC system (Bruker). Separation was achieved on a Phenomenex Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 µm, 100 Å) with 0.05% v/v formic acid in water as mobile phase A and acetonitrile with 0.05% formic acid (v/v) as mobile phase B. The gradients used for elution of GAs were 0-0.5 min, 2% B; 0.5-1.3 min, 2-30% B; 1.3-2.2 min 30-100% B, 2.2-2.8 min 100% B; 2.8-2.9 min 100-2% B; and 2.9-4.0 min 2% B. The gradients used for elution of ABA were 0-0.5 min, 2% B; 0.5-1.2 min, 2-30% B; 1.2-2.0 min, 30-100% B; 2.0-2.5 min, 100%; 2.5-2.6 min, 100-2% B; and 2.6-4.0 min, 2% B. The mobile phase flow rate was 400 µl min-1, and column temperature was maintained at 40 °C. The liquid chromatography was coupled to an EVOQ Elite triple quadrupole mass spectrometer (Bruker) equipped with an electrospray ion source operated in positive and negative ionization mode. Instrument parameters were optimized by infusion experiments with pure standards. For analysis of GAs, the ion spray voltage was maintained at +4000 V and -4000 V in positive and negative ionization mode, respectively, and the heated probe temperature was set to 200 °C with probe gas flow at 50 psi. For ABA, the ion spray voltage was maintained at -3300 V in negative ionization mode, and heated probe temperature was set to 120 °C with probe gas flow at 40 psi. Remaining settings were identical for all analytical methods with cone temperature set to 350 °C and cone gas to 20 psi. Nebulizing gas was set to 60 psi and collision gas to 1.6 mTorr. Nitrogen was used as probe and nebulizing gas, and argon as collision gas. Active exhaust was constantly on. Multiple reaction monitoring was used to monitor analyte parent ion to product ion transitions for all analytes. Multiple reaction monitoring transitions and collision energies were optimized by direct infusion experiments. Detailed values for mass transitions can be found in Supplemental Table 3. Both Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (Version 8.2.1) was used for data acquisition and processing. Linearity in ionization efficiencies were verified by analyzing dilution series of standard mixtures. Sinigrin glucosinolate was used as internal standard for normalization but not for quantification. Quantification of all compounds was achieved by external standard curves diluted with the same matrix as the actual samples. All GAs were analyzed together in a single method. GA12 suffered from severe ion suppression when combined with the other GAs in the standard curve, thus quantification was not achieved for GA12.
Root suberin monomer profiling by GC-MS
Suberin monomers were extracted from Col-0 and mutant roots according to the protocols previously described by 68,69. A sample volume of 1 µL was injected in splitless mode on a GC-MS system (Agilent 7693A Liquid Auto injector, 8860 gas chromatograph, and 5977B mass spectrometer). GC was performed (HP-5MS UI column; 30 m length, 0.250 mm diameter, and 0.25 µm film thickness; Agilent J&W GC Columns) with injection temperature of 270°C, interface set to 250°C, and the ion source to 200°C. Helium was used as the carrier gas at a constant flow rate of 1.2 mL min-1. The temperature program was 0.5 min isothermal at 70°C, followed by a 30°C min-1 oven temperature ramp to 210°C and a 5°C min-1 ramp to 330°C, then kept constant during 21 min. Mass spectra were recorded with an m/z 40 to 850 scanning range. Chromatograms and mass spectra were evaluated using the MSD ChemStation software (Agilent). Integrated peaks of mass fragments were normalized for sample dry weight and the respective C32 alkane internal standard signal. For identification, the corresponding mass spectra and retention time indices were compared with the NIST20 library as well as in-house spectral libraries.
Xenopus oocyte injection based efflux transport assays
For injection-based export assays, on the second day of gene expression, oocytes were injected with 23 nl 8.2 mM in 98 mM KCl, 1 mM CaCl2, 10 mM HEPES, pH 7.4. T1 oocytes were left 10 min to heal and were then transport was evaluated as described above. T2 oocytes were left for approximately 20 h in HEPES-based kulori at 16° C, followed by transport analysis.
Quantification of nitrate from oocytes by HPLC
Nitrate concentration in the oocyte extracts was quantified using a Dionex ICS-2100 anion exchange chromatography system (Thermo Scientific). The separation was done on a Dionex IonPac AG11-HC analytical column coupled to the AS11-HC guard column (Thermo Scientific). The columns were connected to a Dionex AERS 500 anion suppressor (Thermo Scientific). The analyses were performed under the following conditions: sample injection volume 4.8 µl, column temperature 30 °C, flow rate of 0.38 ml/min, isocratic eluent gradient using 30 mM KOH solution in QH2O, suppressor current of 29 mA, and runtime of 15 min. The nitrate detection was done at 220 nm using a Dionex UltiMate 3000 (Thermo Scientific). QH2O water dilutions of Dionex Combined Seven Anion Standard (Thermo Scientific) were used to create a standard calibration curve. Accuracy and precision of the quantification was checked by including samples of potassium nitrate throughout the sequence.
pH measurements of oocyte lumen
The pH stabilization was performed as described previously 34. pH-electrodes were pulled from borosilicate glass capillaries (KWIK-FIL TW F120-3 with filament) on a vertical puller (Narishige Scientific Instrument Lab), baked for 120 min at 220 °C and silanized for 60 min with dimethyldichlorosilane (Silanization Solution I, Sigma Aldrich). Electrodes were backfilled with a buffer containing 40 mM KH2PO4, 23 mM NaOH and 150 mM NaCl (pH 7.5). The electrode tip was filled with a proton-selective ionophore cocktail (hydrogen ionophore I cocktail A, Sigma-Aldrich) by dipping the tip into the cocktail. Oocytes, as described above, were placed in freshly made HEPES-based ekulori (2 mM LaCl3, 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES pH 7.4) for at least 30 min prior to three-electrode voltage clamp experiments. Before each oocyte a pH calibration curve was made for each oocyte using 100 mM KCl pH 5.5, 100 mM KCl pH 6.5 and 100 mM KCl pH 7.5. Oocytes were clamped at 0 mV and perfused with HEPES-based ekulori pH 7.4, followed by MES-based ekulori (2 mM LaCl3, 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES pH 5) and internal pH response was measured continuously as a function of external pH change.
Membrane potential measurements
Membrane potentials of oocytes were measured using the automated two-electrode voltage clamp system, Roboocyte2 (Multi channel systems), in ekulori (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES, 2 mM LaCl3, pH 5) with electrodes backfilled with 1 M KCl and 1.5 M potassium acetate. All oocytes were measured using the same electrodes with a resistance of 280-350 kΩ. The experiment was terminated when the resistance of one of the electrodes shifted to approximately 600 kΩ.
Two-electrode voltage clamp electrophysiology
The electric signal elicited by GA treatment of oocytes was measured using the automated two-electrode voltage clamp system Roboocyte2 (Multi channel systems), in ekulori (90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM MES, 2 mM LaCl3 pH 4.5 or pH 5) with electrodes (resistance 280-1000 kΩ) backfilled with 1 M KCl and 1.5 M potassium acetate. Oocytes were clamped at – 60 mV, and IV curves were obtained before and after substrate addition. Substrate dependent currents were calculated by subtracting currents before addition of substrate from currents after addition of substrate.
Root hormone quantification
Hormone extraction and analysis was performed as described in Zhang et al., 2021. Standards (both labelled and non-labelled) were obtained from Olchemim Ltd. (Olomouc, Czech Republic) and National Research Council (NRC-CNRC, Canada). Standard grade solvents were used for sample preparation, Methanol, Acetic acid (LiChrosolv, Sigma-Aldrich, USA), Acetonitrile (J.T.Baker, Avantor, PA, USA), Formic acid (Honeywell Fluka, Thermo Fisher Scientific, MA, USA) and de-ionized water (Milli-Q, Synergy-UV millipore system, USA). Briefly, root tissue frozen in liquid nitrogen was grounded using motor and pestle. Around 200 mg of root sample was measured from ground powder and extracted with ice cold methanol/water/formic acid (15/4/1 v/v/v) added with deuterium labelled internal standards (IS). Similar concentrations of IS of abscisic acid and gibberellin (GA4) were added into samples and calibration standards. The samples were purified using Oasis MCX SPE cartridges (Waters, USA) according to manufacturer’s protocol. The samples were injected on Acquity UPLC BEH C18 column (1.7 µm, 2.1x100 mm, Waters; with gradients of 0.1% acetic acid in water or acetonitrile), connected to Acquity UPLC H class system (with Waters Acquity QSM, FNR sample manager and PDA) coupled with UPLC-ESI-MS/MS triple quadrupole mass spectrometer (Xevo TQ-S, Waters, equipped with ESI probe) for identification and quantification of hormones. The hormones were measured using MS detector, both in positive and negative mode, with two MRM transitions for each compound. External calibration curves were constructed with hormone standards added with IS, used for quantification, and calculated through Target Lynx (v4.1; Waters) software by comparing the ratios of MRM peak areas of analyte to that of internal standard.
Phloem extract and hormone quantification
Rosette leaves of 5-week-old Col-0 and npf2-12 npf2-13 mutant plants (before bolting) were cut with a razor blade at the base of the petiole, and each leaf was dipped in a tube containing 80 µL of exudation buffer (50 mM potassium phosphate buffer, pH7.6, 10 mM EDTA). Exudation was carried out for 3h in dark in high humidity to limit transpiration. Exudation of 75 leaves was regrouped and concentrated under vacuum centrifugation. Hormone contents in phloem exudates were determined by UPLC system-MS/MS (Waters Quattro Premier XE). Concentrated residue of phloem sap was resuspended with 80% methanol-1% acetic acid including 17-2H2-labeled GA internal standards (Olchemim), mixed and passed through an Oasis HLB column. The dried eluate was dissolved in 5% acetonitrile-1% acetic acid, and the GAs were separated by UPHL chromatography (Accucore RP-MS column 2.6 µm, 100 x 2.1 mm; ThermoFisher Scientific) with a 5 to 50% acetonitrile gradient containing 0.05% acetic acid, at 400 µL/min over 22 min. The concentrations of GAs in the extracts were analyzed with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific) by targeted SIM using embedded calibration curves and the Xcalibur 2.2 SP1 build 48 and TraceFinder programs.
Grafting was performed without collars on water imbibed 0.45 µM MCE membrane (Millipore) between hypocotyls of rootstocks and scions of 6-day-old seedlings grown on 1x MS agar plate. Grafted seedlings were then kept vertically to recover, for 5 days under constant humidity. Successful grafts were transferred onto ½x MS agar plates and grown under a 16h photoperiod at 22°C. Root growth was measured every day for 4 days.
DELLA degradation assays.
12-day-old seedlings were transferred to 1x MS agar modified medium without nitrogen (bioWORLD plant media) supplemented with 0.5 mM KNO3 and 1 µM paclobutrazol (Sigma). 4 days after transfer, a drop of GA12 (5 µl at 1 µM) was placed on one of the first two leaved formed. Roots were collected 6, 12 and 24h after adding GA12. Total proteins were extracted in 2x SDS-PAGE sample buffer and separated on 10% SDS-PAGE gel. After transfer onto membrane, immunoblots were performed using a 2000-fold dilution anti-RGA (Agrisera) and a 10000-fold dilution of peroxidase-conjugated goat anti-rabbit (Thermo Fisher Scientific). Signals were detected with Fusion FX (Vilber) using Immobilon Forte Western HRP Substrate (Millipore). The blot was subsequently stained with Coomassie blue. Quantification of the signals was determined using ImageJ package.
Root templates were segmented from an experimental image using the CellSeT image analysis tool 70 (Sup. Fig. 13). We used CellSeT to manually assign a cell type to each cell and then read the geometrical and cell-type data into a tissue database (based on the OpenAlea tissue structure 71), extending the data structure to incorporate vacuolar compartments within each cell. The geometrical, topological and transporter-distribution data were used to form a system of ordinary differential equations (ODEs) to describe the GA transport, synthesis and degradation within the multicellular root cross-section. Parameters associated with the passive and transporter-mediated transport components were estimated using the oocyte data (Fig. 1A, Fig. 3B) and the remaining parameter values were obtained from the literature (Sup. Table 4). These ODEs were simulated using the solve_ivp package in python 3.6.5. Full details of the model equations and assumptions are provided as Supplementary text.