Antagonistic cell surface and intracellular auxin signalling regulate plasma membrane H+-fluxes for root growth

doi:10.21203/rs.3.rs-266395/v1

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Antagonistic cell surface and intracellular auxin signalling regulate plasma membrane H⁺-fluxes for root growth

https://doi.org/10.21203/rs.3.rs-266395/v1

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published 26 Oct, 2021

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Growth regulation tailors plant development to its environment. A showcase is growth adaptation to gravity, where shoots bend up and roots down. This paradox is based on different responses to the phytohormone auxin, which promotes cell expansion in shoots, while inhibiting it in roots via a yet unknown cellular mechanism. Here, by combining microfluidics, live imaging, genetic engineering and phospho-proteomics in Arabidopsis thaliana, we reveal 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, which is the direct growth-determining mechanism. Cell surface-based TRANSMEMBRANE KINASE 1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H⁺-ATPases for apoplast acidification, while intracellular TIR1/AFB-mediated signalling triggers 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 to subtle changes in the environment.

Plant Physiology and Morphology

auxin signalling

root growth

PM Hᐩ-ATPases

TMK1

Acid Growth Theory

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ExtFig1.jpg
Extended Data Figure 1. Investigation of cellular mechanisms potentially involved in auxin-induced rapid root growth inhibition. a-b, Dynamic cortical microtubule (CMT) reorientation in root cells from transversal to longitudinal in response to 100 nM IAA treatment. CMT was imaged in late elongating epidermal cells in the pEB1b::EB1-GFP marker line in vRootchip. Scans were made at 6.25 second intervals and a max Z-projection of 10 subsequent time frames was analysed using the FibrilTool macro. The average orientation of the microtubules is represented by the slope of the red line and the length of the line represents its anisotropy (a). Quantification of CMT reorientation after 100 nM IAA treatment in (a). The percentage of CMT reorientation at every time point is calculated as the difference in the angle of that time point and the initial time point divided by the difference in the angles of the initial time point and end time point at 42 minutes. Mean of 5 elongating cells, ± SD (b). c-e, Analysis of CMT reorientation in elongating epidermal root cells (c, d) and root growth (e) of 35S::MAP4-GFP in response to 10 nM IAA, 10 µM taxol and IAA+taxol co-treatment. Orientation of CMT was analysed with the Bioline script. Green-colored CMTs mark transversal oriented CMT (angle between -45° and +45°), while red-colored CMTs indicate longitudinal orientation (angle between +45° and 135°). Scale bar = 15 µm (c). Quantification of (c) as the percentage of longitudinal CMT to total CMT detected. n > 11, One-way ANOVA (d). Growth on respective treatments for 2 hours were measured. n > 10, One-way ANOVA (e). *p≤0.05, ****p≤0.0001. f, Vacuolar morphology tracked in the pSYP22::SYP22-YFP marker line (green signal) in the same elongating cells before and after 30 minutes of 100 nM IAA. Scale bar = 15 µm. Magenta signal indicates propidium-iodide stained cell walls. g, Dynamics of apoplastic pH measured in different cells across the whole EZ (p1-p8) in vRootchip. The TL and blue-yellow scale image are from the same sample shown in Fig. 1a. Scale bar= 30 µm. The upper charts depict the dynamics of apoplastic pH in the indicated cells in response to 5 nM IAA, and the lower charts represent the pH in response to washout. The left two charts show the apoplastic pH measured by the HPTS dye. The right two charts show the speed at which each cell reaches its maximum pH change calculated as the difference between the pH at a given time point and the pre-stimulus pH, divided by the end pH change in that cell. h, Dynamics of root surface pH and medium pH in vRootchip. Left graph shows the elongation zone of the root. The ROIs p1-p5 were chosen along the root, 30 µm away from the root surface indicated by the vertical white dotted line, while ROIs p6-p9 were distanced away from the root. The pH at the surface of the root (vertical positions p1-p5) increased after IAA and recovered after washout within 30 seconds. In contrast, the pH away from the root surface did not change significantly (horizontal positions p6-p9). i, PM H+-net influx measured by a non-invasive microelectrode before and after 10 nM IAA treatment in the elongating zone of WT roots. Means of 9 roots ± SEM. j-k, The medium pH (j) and apoplastic pH (k) changed rapidly after the exchange of the medium of different pH in vRootchip. Following media were used sequentially: basal medium at pH 5.8, auxin-containing medium at pH 5.8, gradually more acidic medium of pH 5.6, followed by pH 5.4 and lastly again basal medium at pH 5.8. l, Quantification of root growth rate in response to the gradual addition of KOH in the medium in the vRootchip. The greener the shade, the more KOH was added followed by washout with the initial basal pH 5.8 medium. m, Scheme of the modified vRootchip adding valve 6 and with valve routes adjusted.
ExtFig2.jpg
Extended Data Figure 2. H+-ATPase activation counteracts auxin-mediated apoplast alkalinisation and growth inhibition a, Measurement of luminescence intensity on the root tip of DR5::LUC line after 10 µM FC, 10 nM IAA and IAA+FC co-treatment. n > 3. IAA and IAA+FC are significantly different from the mock (p ≤0.0001). No significant difference between IAA and IAA+FC (p > 0.05). Two-way ANOVA. b-g, FC and IAA counteract each other. Addition of IAA still increased apoplastic pH (b) and inhibited root growth (c) in presence of FC in vRootchip. n = 4 (b, c). Addition of FC still decreased the apoplastic pH (d) and promoted root growth (e) in presence of IAA in vRootchip. n = 4, the shaded area represents the duration time of the indicated treatments (d-e). Root growth after FC, IAA and co-treatment for 1 hour in steady state by scanner. 1 µM FC and 10 nM IAA were used in (f) while 10 µM FC and 2 nM IAA were used in (g). n > 9 for both graphs, ns p>0.05, *p≤0.05, **p≤0.01, ****p≤0.0001, One-way ANOVA (f-g). h, Dose-response of auxin-induced root growth inhibition of aha single mutants. n > 22. Relative GR is ratio between auxin-affected growth in the mutant to mock growth for the same genotype. ns p>0.05, **p≤0.01, ***p≤0.001, Welch ANOVA. i, Quantitative Real-time PCR on the AHA1,2,7,11 expression in root tips of AtTAS1c-AHA#2 and #4. The expression level was normalized to EF1α as housekeeping gene. Means of 3 biological replicates + SD.
ExtFig3.jpg
Extended Data Figure 3. TMK1 directly mediates auxin-induced H+-ATPase activation. a, IP-MS/MS on pTIR1::TIR1-VENUS in tir1-1 and pAFB1::AFB1-VENUS in afb1-3 lines under normal condition (upper graphs) compared to 1 hour 50 µM MG132 pre-incubation and 100nM IAA treatment for 2 minutes (lower graphs). Volcano plots show fold changes (in log2 scale, x-axis) and significance (p-value, y-axis). Proteins passing the threshold of FDR 0.05 and specific fold change are marked. Green depicts the respective bait protein and red depicts known members of the SCF E3 ubiquitin ligase complex. Pulldowns were performed in triplicate, LFQ analysis. b, IP-MS/MS on 35S::TMK1-GFP line. Volcano plot shows fold change (in log10 scale, x-axis) and significance (p-value, y-axis). Red dots are in the range of ratio > 10 and -LOG p-value > 2. P-values are calculated based on the three replicates of 35S::TMK1-GFP vs WT using a Two-sided t-test. c, co-IP of pTMK1::TMK1-GFP roots, followed by Western blot detection of AHA2 and Thr947-phosphorylated AHA2. The interaction was not regulated by 30 minutes of 100 nM IAA treatment, but the phosphorylation state of the interacting AHA2 was increased. d, Bimolecular Fluorescent Complementation (BiFC) in Nicotiana benthamiana leaves transiently transformed with YFPN-TMK1, AHA2-YFPC or both. Scale bar = 10µm e, Representation of wilting leaves of Nicotiana benthamiana that transiently express the WT TMK1 and ATP-site mutated forms: p35S::TMK1-HA K616E or K616R. f, Western blot analysis of the AHA2 levels and the Thr947 phosphorylation in roots of DEX::TMK1K616R-HA and DEX::TMK1-HA lines treated +/- DEX (30 µM for 24 hours) and +/- IAA (100 nM for 1 hour). g-h, Western blot detection of the AHA2 levels and the Thr947 phosphorylation in tmk1,3 roots treated with 100 nM IAA for 1 hour (g). Quantification of the auxin effect on AHA2 phosphorylation in (g) by normalising the intensity ratio of pThr947 to AHA2 detected in auxin-treated roots to the same ratio in mock-treated samples of the same genotype (h).
ExtFig4.jpg
Extended Data Figure 4. TIR1/AFB and TMK1 signalling converge antagonistically on apoplastic pH and growth regulation. a-b, Apoplastic alkalinisation (a) and root growth inhibition (b) in response to IAA measured in aux1-100 mutant compared to WT roots in vRootchip. Apoplastic pH is measured by the HPTS dye. n = 3; p ≤ 0.0001, Two-way ANOVA for both graphs. c-d, Apoplastic alkalinisation (c) and root growth inhibition (d) in response to the synthetic auxin analogue 2,4-D in aux1-100 mutant compared to WT roots. The steady state pH with the HPTS dye was measured 30 minutes after mock or 100 nM 2,4-D treatment. n > 6, One-way ANOVA (c). The growth obtained in 2 hours was captured by scanner. n > 4, One-way ANOVA (d). ns p>0.05, **p≤0.01, ***p≤0.001. e, Root growth of tir triple mutant (in red) compared to WT (in black) in response to 5 nM IAA in the vRootchip. n = 3, 2. p≤0.0001, two-way ANOVA. f-g, apoplastic pH (f) and root growth (g) after 10 µM PEO-IAA and 5 nM IAA. The steady state pH was measured 30 minutes after the treatments using the HPTS dye, while the root growth obtained in 1 hour was recorded by scanning the plates. n > 7, ns p>0.05, **p≤0.01, ****p≤0.0001, One-way ANOVA. h, Dose-response of auxin-induced root growth inhibition of pUBQ10::TMK1-3HA compared to WT and tmk1-1. Relative GR is the ratio between auxin-affected growth in a mutant to the mock growth for the same genotype. n > 7. ns p>0.05, *p≤0.05, Welch ANOVA. i-j, Raw data for Figure 4d and e, respectively. ns p>0.05, **p≤0.01, ****p≤0.0001, One-way ANOVA.
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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Antagonistic cell surface and intracellular auxin signalling regulate plasma membrane H⁺-fluxes for root growth

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