NBR1-mediated selective autophagy of ARF7 modulates root branching

  1. E Ebstrup
  2. J Ansbøl
  3. A Paez-Garcia
  4. H Culp
  5. J Chevalier
  6. P Clemmens
  7. NS Coll
  8. A Moreno-Risueno M
  9. E Rodriguez

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Abstract

Auxin dictates root architecture via the Auxin Response Factor (ARF) family of transcription factors, which control lateral root (LR) formation. In Arabidopsis , ARF7 regulates the specification of prebranch sites (PBS) generating LRs through gene expression oscillations and plays a pivotal role during LR initiation. Despite the importance of ARF7 in this process, there is a surprising lack of knowledge about how ARF7 turnover is regulated and how this impacts root architecture. Here, we show that ARF7 accumulates in autophagy mutants and is degraded through NBR1-dependent selective autophagy. We demonstrate that the previously reported rhythmic changes to ARF7 abundance in roots are modulated via autophagy and might occur in other tissues. Additionally, we prove that the level of co- localization between ARF7 and autophagy markers oscillates and can be modulated by auxin to trigger ARF7 turnover. Furthermore, we observed that autophagy impairment prevents ARF7 oscillation and reduces both PBS establishment and LR formation. In conclusion we report a novel role for autophagy during development, namely by enacting auxin-induced selective degradation of ARF7 to optimize periodic root branching.

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  1. Version published to 10.1101/2022.08.19.504407v4 on bioRxiv
  2. Version published to 10.1101/2022.08.19.504407v3 on bioRxiv
  3. Version published to 10.1101/2022.08.19.504407v2 on bioRxiv
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    Our reply to reviewers contain figures that has been uploaded as a word file with the rest of the files. Here, we cannot past figures into the reply box, and because we don't wish to submit an incomplete response, we cannot provide a response in this format.

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    Referee #2

    Evidence, reproducibility and clarity

    Lateral root production is a process regulated by auxin, among others. The expression of auxin-dependent genes requires the activity of transcription factors of the ARF family. In this study by Ebstrup et al., the authors suggest that selective autophagy would be involved in the degradation of the ARF7 factor involved in lateral root initiation and production in Arabidopsis thaliana, even though the accumulation of ARF7 in autophagy-deficiency mutant may not affect lateral root initiation.

    Major remarks and comments:

    1. In general, some experimental data do not facilitate appropriate comparisons due to lack of statistical analysis. This is particularly the case for Figures 1-a,b,c and 4-a,b,c,d.
    2. Confocal microscopy images are not always convincing, due to a lack of necessary controls and also qualitatively. It would be useful, for example, to clearly indicate the objects of interest that the reader can use for comparisons. It is for example difficult to understand that chlorophyll fluorescence and GFP fluorescence (from the BiFC signal) colocalize almost in the same organelles (fig. 2c). The parent lines expressing the Venus and mCherry fusions should also serve as controls for figure EV3. Another point concerning fig. 2 a, b (IP): how do the authors explain the "GFP" signal, especially the apparent size and the doublet present only in one of the "YFP" controls after IP?
    3. It would be important for the authors to clarify whether the different fluorescent fusions used are indeed functional or not. This is particularly important in the context of the proteins being studied and the possible regulatory process(es).
    4. Apparently ARF7 would be degraded by the UPS system and the selective autophagy pathway. Would autophagy-deficient mutants, including atg2-1 and atg5-1 be more or less sensitive to MG132 (relative levels of ARF7 accumulation)? This is not clear from the data and its discussion.
    5. The authors seem to insist that NBR1-mediated degradation of ARF7 by selective autophagy would be observable only preferentially in mature root tissues (probably to prevent them from forming lateral roots?). If this is the case, the title of their paper should reflect this conclusion. The authors have the tools (described in their manuscript) to unambiguously clarify this important point. Just as it would be important to demonstrate that the ARF7 proteins that accumulate would indeed be ubiquitylated.

    Minor comments:

    1. Some of the figures would benefit from qualitative improvement, especially the photographs and micrographs.
    2. The authors' attention is drawn to the existence of several typos in the text and the absence of certain references cited in the bibliography.

    Significance

    Although the biological question is of unquestionable interest and importance, the data presented in this manuscript unfortunately do not allow us to rightly assess the contribution of this work to the state of our knowledge.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript shows the involvement of both the proteasome and autophagy pathways in the turnover and therefore regulation of ARF7, an auxin-responsive factor involved in lateral root formation. The authors bring crucial information for the understanding of how autophagy is involved in auxin-signaling.

    Major comments:

    The key conclusions appear overall convincing yet this reviewer would strongly advise to take into account the following remarks for a clearer and more convincing line of inquiry. This reviewer also believes that the additional experiments could be performed relatively fast apart for the point 9) where the establishment of a homozygous line could take 6 months or more.

    1. Figure 1 & Figure EV1: The nature of the loading control should be stated as it appears to be a specific protein detected by immunoblotting. Furthermore, if the authors wish to make a stronger point as to whether ARF7 is degraded by the proteasome (considering the reserves mentioned in the Discussion section), I would recommend to perform the same assays as in Figure 1 but using an alternative proteasome inhibitor such as Bortezomib and to include a proteasome subunit KO mutant such as rpt2a-2.
    2. The statement "The experiment revealed that both NBR1 (Fig 2A) and ATG8a (Fig 2B), but not free YFP, co-immunoprecipitated with ARF7-Venus." Is false as the authors did not try to co-immunoprecipitate free YFP with ARF7-Venus, they used a free YFP expressing line as a negative control for their GFP-immunoprecipitation (IP). It should further be noted that although NBR1 is detected in their free YFP IP, ATG8 is at very low levels so it should be stated that they see an enrichment of ATG8 in their ARF7-Venus IP.
    3. Authors state "we were unable to detect ARF7-Venus in the input of both Co-IPs which can likely be explained by the fact that ARF7-Venus is under the control of its native promoter and thus lowly expressed.", yet putative degradation products (i.e. a smear) can be observed in the input of Figure 2A, similarly to the bands observed in both IP blots. It would be interesting to repeat these co-IPs with proteolysis inhibitors such as MG132 or Pepstatin & E64-d to pinpoint the proteolytic machinery at the origin of ARF7-Venus degradation in the IPs.
    4. Figure 2: The use of multicolor BiFC "mcBiFC" should be stated as such for an easier understanding of the reader. It would be helpful for the reader if the "GFP" signal resulting from the complementation would be highlights thanks to some arrows. Moreover, a western blot to verify the expression levels should be performed since every construct has an epitope tag as stated in Gehl et al. 2009.
    5. General remark: all drug/chemical treatments performed in this study use a "non-treated" negative control, yet it should be pointed out that the correct corresponding negative controls should have the solvent used to dissolve the respective drug/chemical in order to exclude any effect of the solvent or vehicle.
    6. Figure 4, Figure EV4: Considering the variability in size and staining of the Rubisco large-subunit in the 4 immunoblot panels, I would suggest blotting with another antibody such as anti-tubulin or anti-histone 3 as a loading control for a more convincing quantification. Moreover, the nature of the staining used to stain the Rubisco large-subunit should be stated. The authors also state "differences in ARF7 accumulation in atg5 compared to Col-0" yet no immunoblot is shown where both genotypes are present on the same membrane, in order to verify this statement.
    7. Figure 5: In regards to LR density measurements, I recommend reading "Quantitative Analysis of Lateral Root Development: Pitfalls and How to Avoid Them" by Dubrovsky & Forde (Plant Cell, 2012) for a more robust method of evaluating lateral root density.
    8. Discussion: The authors state that "autophagy blockage leads to increased ARF7 cytoplasmic condensates". To support this statement, I recommend crossing pARF7::gARF7-Venus into atg mutants and analysing the localization and the fluorescence intensity of ARF7-Venus in specific parts of the root, as well as performing immunoblotting in order to assess overall ARF7 accumulation in autophagy deficient genetic backgrounds.

    Minor comments

    1. The following statement: « In contrast, plants are able to tolerate disruption of autophagy activity without major penalties" holds true to A. thaliana of some other plants but it must be noted that in O. sativa, autophagy-deficiency may lead to male sterility, which should be considered a major penalty for evolutionary fitness. For review see Norizuki et al. 2020 (Front. Plant Sci.).
    2. Figure 2: The molecular weights appear to be potentially misannotated as free YFP aligns with the 35 kDa marks although it should appear around 27 kDa.
    3. Figure EV3: There are 2 merged image columns, the furthest one to the right appears to include a DIC or Trans image on top of both fluorescence channels. It would be more helpful for the reader if the DIC or Trans image was shown with the overlay of fluorescent channels in order to assess the effect of 10% 1,6-hexandiol on the plant tissue. Moreover, demonstrating the absence of tissue damage or cell-death after 1,6-hexandiol treatment would be a plus.
    4. There is a typo throughout the manuscript: ZT should be "Zeitgeber" not "zeitberg".

    Significance

    This manuscript has the quality of describing the proteolytic balance of ARF7 and thereby, the involvement of the autophagy pathway in regulating auxin-signaling components. This research adds on to the growing interest in how autophagy participates in developmental cues, and how hormonal signaling is regulated throughout the plant.

  7. Version published to 10.1101/2022.08.19.504407v1 on bioRxiv