The AUX1-AFB1-CNGC14 module establishes a longitudinal root surface pH profile

  1. Nelson BC Serre
  2. Daša Wernerová
  3. Pruthvi Vittal
  4. Shiv Mani Dubey
  5. Eva Medvecká
  6. Adriana Jelínková
  7. Jan Petrášek
  8. Guido Grossmann
  9. Matyáš Fendrych

  • Curated by eLife

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    This study presents valuable findings that relate the pH pattern along the root surface of the plant Arabidopsis thaliana to the auxin response and gravitropic (changes in growth orientation) response. The evidence supporting the claims of the authors is solid, based on the observation of dynamic responses at a second-to-minute time scale and the systematic correlation between the observed changes in the longitudinal surface pH profile and changes in growth rate. The work will be of interest to a wide range of plant biologists working on plant development and responses to the environment.

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Abstract

Plant roots navigate in the soil environment following the gravity vector. Cell divisions in the meristem and rapid cell growth in the elongation zone propel the root tips through the soil. Actively elongating cells acidify their apoplast to enable cell wall extension by the activity of plasma membrane AHA H + -ATPases. The phytohormone auxin, central regulator of gravitropic response and root development, inhibits root cell growth, likely by rising the pH of the apoplast. However, the role of auxin in the regulation of the apoplastic pH gradient along the root tip is unclear. Here, we show, by using an improved method for visualization and quantification of root surface pH, that the Arabidopsis thaliana root surface pH shows distinct acidic and alkaline zones, which are not primarily determined by the activity of AHA H + -ATPases. Instead, the distinct domain of alkaline pH in the root transition zone is controlled by a rapid auxin response module, consisting of the AUX1 auxin influx carrier, the AFB1 auxin co-receptor, and the CNCG14 calcium channel. We demonstrate that the rapid auxin response pathway is required for an efficient navigation of the root tip.

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  1. Version published to 10.7554/elife.85193 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    Root growth is driven by cell elongation, and its local control allows roots to navigate the complex soil environment. Cell growth is driven by the relaxation of the cell wall, a process requiring a drop in pH. Auxin is a key regulator of root development that inhibits root growth. Auxin effects on proton dynamics are complex, it can promote both acidification and alkalinization of the extracellular space through different signaling modules, some only recently uncovered. Serre et al. report on using a new dye to monitor extracellular pH in the region surrounding the Arabidopsis thaliana root. Their manuscript aims to clarify the relationships between pH around the root, proton flux, auxin, cell elongation, and root growth with this tool. They show a typical zonation of pH values along the root: a more acidic domain corresponding to the transit-amplifying compartment, followed by a more alkaline one at the transition and early elongation zones and a more acidic one in the late elongation/root hair zone. This zonation is in agreement with previous reports obtained by other methods. A particularly puzzling aspect is the origin of the more alkaline domain. Serre et al. present evidence supporting the involvement of the AUX1-AFB1-CNGC14 module for the emergence of this more alkaline domain and how it can contribute to the ability of the root to navigate its environment.

    Serre et al. show that the more alkaline domain in the transition zone is not directly determined by the activity or localization of the AHA proton pumps but rather by the auxin influx carrier AUX1. They show that the components of the rapid auxin response pathway, in particular, the auxin co-receptor AFB1 and the calcium channel CNGC14, contribute to the emergence of this more alkaline domain. Finally, they show that mutants in these two genes, impaired in the rapid auxin response pathway, show less efficient navigation of the root tip.

    The manuscript is clear and well-written. The logic is sound, and the conclusions are supported by the data.

    The new dye appears as a promising tool for monitoring the pH in the rhizosphere with advantages over the previous ones. Yet, as pointed out by the authors in the discussion, it reports on pH at the organ scale in the region around the root, not in the apoplast or the cell wall, which can eventually complexify the elaboration of a mechanistic model joining auxin, proton efflux, cell wall properties, cell elongation, and root growth. Although several of the findings confirm previous reports, the manuscript brings novelty by demonstrating the involvement of the rapid auxin response. I am overall supportive of the manuscript. Yet, several points should be addressed:

    • The presentation of the more acidic and alkaline domains could be easier to visualize.
    • The authors refer to acidic and alkaline domains but do not report on absolute pH values; they monitor the emission ratio of the dye. They justify why to use relative pH value in the discussion and refer there to internal controls that are not clearly defined. In my opinion, the wording should be more consistent across the text and figures and refer to more acidic and more alkaline domains rather than acidic (pH<7) and alkaline (pH>7) domains.
    • The data related to the unaltered distribution of AHA using antibody staining should be backed up.
    • The way the pH profile and the statistical analyses should be improved.
    • The authors should test the effect of extracellular auxin perception (tmk, abp) mutants on pH zonation.
    • Conclusion could be strengthened by moving several pieces of data currently in supplemental material to the main text.

    We agree with the comment to the definition of ‘acidic’ and ‘alkaline’ domains; we altered the text and explained that we observe ‘relatively alkaline’ and ‘relatively acidic’ domains in comparison to the medium pH in the first part of results.

    We defined the ‘internal controls’ in the text – by this we mean mock treated or wild type plants imaged together with the treated or mutant plants.

    To address the role of the apoplastic auxin pathway in the root surface pH, we analyzed the tmk1, tmk4 and abp1 mutants. Surprisingly, all three mutants appear undistinguishable from the controls, showing the crucial importance of the cytoplasmic AFB1 auxin perception pathway. We have included the data as Fig.S4-1.

  3. eLife

    eLife assessment

    This study presents valuable findings that relate the pH pattern along the root surface of the plant Arabidopsis thaliana to the auxin response and gravitropic (changes in growth orientation) response. The evidence supporting the claims of the authors is solid, based on the observation of dynamic responses at a second-to-minute time scale and the systematic correlation between the observed changes in the longitudinal surface pH profile and changes in growth rate. The work will be of interest to a wide range of plant biologists working on plant development and responses to the environment.

  4. eLife

    Reviewer #1 (Public Review):

    This is a very elegant study of the dynamics of the longitudinal surface pH profile in growing Arabidopsis roots. The authors first present a new powerful method for the visualization of the surface pH profiles using the pH-sensitive fluorescent dye fluoresceine-5 (or 6)-sulfonic acid. This is an interesting new tool for studying surface pH in plants and perhaps other organisms. The main findings are that the presence of an alkaline band at the transition zone does not depend on AHA abundance (shown by immunolocalization) or activity shown by pharmacology (FC treatment) or by using plants expressing hyperactive, or PP2CD1- inhibited AHA2 or by using KO mutants aha2 or pp2c-d respectively. This band depends on auxin and AUX1-mediated auxin influx and rapid auxin response components AFB1 and CNGC14. The latter has a distribution along the root fitting the longitudinal surface pH zonation and are both required for it. Canonical auxin signaling (TIR) has more quantitative effects on the extent of the auxin-induced alkalinization. They also observe that the rapid auxin response module is constantly activated and inactivated as shown by the time-dependent variations in surface pH within the alkaline zone on both sides of the root and the rapid AUX1, AFB1, and CNGC14-dependent acidification of the upper surface and alkalinisation of the lower surface during gravitropic responses. Finally, they provide some evidence for the role of the rapid auxin responses in avoiding physical obstacles in the environment of the root.

    The data look very sound. The originality of the approach used is the observation of dynamic responses at a second-to-minute time scale and to systematically correlate between the observed changes in the longitudinal surface pH profile with changes in growth rate. The manuscript is well-written with clear figures.

  5. eLife

    Reviewer #2 (Public Review):

    Root growth is driven by cell elongation, and its local control allows roots to navigate the complex soil environment. Cell growth is driven by the relaxation of the cell wall, a process requiring a drop in pH. Auxin is a key regulator of root development that inhibits root growth. Auxin effects on proton dynamics are complex, it can promote both acidification and alkalinization of the extracellular space through different signaling modules, some only recently uncovered. Serre et al. report on using a new dye to monitor extracellular pH in the region surrounding the Arabidopsis thaliana root. Their manuscript aims to clarify the relationships between pH around the root, proton flux, auxin, cell elongation, and root growth with this tool. They show a typical zonation of pH values along the root: a more acidic domain corresponding to the transit-amplifying compartment, followed by a more alkaline one at the transition and early elongation zones and a more acidic one in the late elongation/root hair zone. This zonation is in agreement with previous reports obtained by other methods. A particularly puzzling aspect is the origin of the more alkaline domain. Serre et al. present evidence supporting the involvement of the AUX1-AFB1-CNGC14 module for the emergence of this more alkaline domain and how it can contribute to the ability of the root to navigate its environment.

    Serre et al. show that the more alkaline domain in the transition zone is not directly determined by the activity or localization of the AHA proton pumps but rather by the auxin influx carrier AUX1. They show that the components of the rapid auxin response pathway, in particular, the auxin co-receptor AFB1 and the calcium channel CNGC14, contribute to the emergence of this more alkaline domain. Finally, they show that mutants in these two genes, impaired in the rapid auxin response pathway, show less efficient navigation of the root tip.

    The manuscript is clear and well-written. The logic is sound, and the conclusions are supported by the data.

    The new dye appears as a promising tool for monitoring the pH in the rhizosphere with advantages over the previous ones. Yet, as pointed out by the authors in the discussion, it reports on pH at the organ scale in the region around the root, not in the apoplast or the cell wall, which can eventually complexify the elaboration of a mechanistic model joining auxin, proton efflux, cell wall properties, cell elongation, and root growth. Although several of the findings confirm previous reports, the manuscript brings novelty by demonstrating the involvement of the rapid auxin response. I am overall supportive of the manuscript. Yet, several points should be addressed:

    - The presentation of the more acidic and alkaline domains could be easier to visualize.
    - The authors refer to acidic and alkaline domains but do not report on absolute pH values; they monitor the emission ratio of the dye. They justify why to use relative pH value in the discussion and refer there to internal controls that are not clearly defined. In my opinion, the wording should be more consistent across the text and figures and refer to *more* acidic and *more* alkaline domains rather than acidic (pH<7) and alkaline (pH>7) domains.
    - The data related to the unaltered distribution of AHA using antibody staining should be backed up.
    - The way the pH profile and the statistical analyses should be improved.
    - The authors should test the effect of extracellular auxin perception (tmk, abp) mutants on pH zonation.
    - Conclusion could be strengthened by moving several pieces of data currently in supplemental material to the main text.

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript provides a high amount of data supporting the author's hypothesis. Serre et al aimed to address the root surface pH and the molecular factors regulating the establishment of the root surface pH pattern important for root growth and gravitropic response. The authors are able to provide solid data on the role of AUX1, AFB1, and CNGC14 in establishing an alkalic patch in the transition zone on the root surface. A weak point in the manuscript is the absence of cellular resolution. The authors mention the technical problems to assess apoplastic pH with previously published tools. They offer Fluorescein106 5-(and-6)-Sulfonic Acid, Trisodium Salt (FS) as an alternative. Even though they were able to generate valuable data with FS, bringing in cellular resolution would increase the quality of the paper even more. Overall, Serre et al provide a solid manuscript with novel data which is of high importance for the field of root and auxin biology.

  7. Version published to 10.1101/2022.11.23.517700v1 on bioRxiv