The mRNA decapping machinery targets LBD3/ASL9 to mediate apical hook and lateral root development in Arabidopsis

  1. Zhangli Zuo
  2. Milena Edna Roux
  3. Jonathan Renaud Chevalier
  4. Yasin F. Dagdas
  5. Takafumi Yamashino
  6. Søren Diers Højgaard
  7. Emilie Knight
  8. Lars Østergaard
  9. Eleazar Rodriguez
  10. Morten Petersen

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Abstract

Multicellular organisms perceive and transduce multiple cues to optimize development. Key transcription factors drive developmental changes, but RNA processing also contributes to tissue development. Here, we report that multiple decapping deficient mutants share developmental defects in apical hook, primary and lateral root growth. More specifically, LATERAL ORGAN BOUNDARIES DOMAIN 3 ( LBD3 )/ ASYMMETRIC LEAVES 2-LIKE 9 ( ASL9 ) transcripts accumulate in decapping deficient plants and can be found in complexes with decapping components. Accumulation of ASL9 inhibits apical hook, primary root growth and lateral root formation. Interestingly, exogenous auxin application restores lateral roots formation in both ASL9 over-expressors and mRNA decay-deficient mutants. Likewise, mutations in the cytokinin transcription factors type-B ARABIDOPSIS RESPONSE REGULATORS (B-ARRs) ARR10 and ARR12 restore the developmental defects caused by over-accumulation of capped ASL9 transcript upon ASL9 overexpression. Most importantly, loss-of-function of asl9 partially restores apical hook and lateral root formation in decapping deficient mutants. Thus, the mRNA decay machinery directly targets ASL9 transcripts for decay, possibly to interfere with cytokinin/auxin responses, during development.

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    Reply to the reviewers

    Thank you for conducting the peer-review of our manuscript. We really appreciate the constructive criticism of the reviewers, and we are happy to note the positive appreciation of our core findings regarding the role of the decapping machinery during apical hook and lateral root formation and the identification of ASL9 as a target of the decapping machinery. However, both reviewers note we over-interpretate about the function of ASL9 in cytokinin and auxin responses which is not always supported by our data. Based on their feedback, we have toned down our claims and performed additional experiments and analyses and addressed all the comments raised by both reviewers. We hope this substantially revised and improved version of our manuscript will be better accepted.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, the authors describe the role of the mRNA decay machinery in apical hook formation during germination in darkness in A.thaliana. As reported, this machinery is predominantly described in literature in stress response processes, whereas little is known about its involvement during developmental processes. In detail, the authors demonstrated, via RNA immunoprecipitation (RIP) and genetic experiments, the direct regulation of the LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3)/ASYMMETRIC LEAVES 2-19 LIKE 9 (ASL9) mRNA stability by the mRNA decapping machinery subunits DCPs. According to the manuscript, ASL9 controls apical hooking, LR development and primary root growth is regulating cytokinin signalling and hence its regulation helps to maintain a correct balance of auxin/cytokinin. Indeed, they showed an impair apical hooking and LR defects both in mRNA decapping mutants, where they observed more capped ASL9 compared to WT, and in ASL9 over-expressor lines. Moreover, they reported a largely restoration of over-expressor lines phenotype in the arr10-5arr12-1 double mutants. This work present simple but interesting data that corroborate the authors hypothesis.

    Our response: We thank the reviewer for acknowledging the significance of our findings although we wonder what it´s meant by “simple data”. Through a combination of (complicated) genetics, phenotyping, cell imaging and molecular biology, we have provided mechanistic evidence on the function of the decapping machinery during 2 different post embryonic developmental events. Please see our detailed answers to the reviewer’s comments in the following.

    Nonetheless, I have both major comments and minor comments to improve the manuscript: MAJOR COMMENTS:

    1. I am a bit concerned by the fact that cytokinin, auxin, LBD3, ARR12 and ARR10 have been largely involved in vasculature development and that the obtained results might be due to their role in vasculature development more than in LBD3 mRNA decapping process. Authors should provide evidence that their results are independent from vasculature defects present in those backgrounds or in case discuss this possibility.

    __Our response: __We are a bit puzzled on how vasculature development could explain the apical hook phenotype observed in the decapping mutant. Data like the rapid assembly of P-bodies upon IAA (Fig. 3C) treatments and the overall decreased DR5 signal in dcp mutants (Fig.S5&6) are all consistent with a process precluding vasculature formation. However, we still discuss the possibility that the developmental defects observed in mRNA decapping mutants and ASL9 overexpressor might be related to the vasculature development in these plants (Line 239-244).

    The interaction between the described players and auxin is not clear. From the reported experiments it is difficult to understand what authors wants to report as in S4 and S5 are reported experiments not fully described in the text (authors report about introgression of DR5::GFP in dcp1 and 2 mutants, but SD4 reports ACC treatments of DR5::GFP,dcp2 mutants and SD5 of 7 dpg root meristems of this strain ). Please describe and discuss better the experiment. Also, to this reviewer it is difficult to understand whether the absence of auxin activity in the dcp2 mutants hypocotyl is merely an effect of the lack of the hook formation in this background or a cause. Please clarify this point including new experiments (axr1 or axr3 mutants might help in understand this point).

    __Our response: __We follow the reviewer’s suggestions and trust we now describe and discuss Fig S5&6 (old Fig S4&S5) clear in Line 188-193. As axr1 has been published with apical hook and lateral root defect (old Line 42, new Line 39&169), we did not repeat it in new experiments but emphasize it in Line 169.

    Authors conclude that mRNA decapping is also involved in root growth. However, they do not report direct evidences regarding root growth but mostly regarding the mere root lenght at a precise developmental stage. Please eliminate this point or provide new experiments (e.g., root length and root meristem activity over time)

    __Our response: __We follow the reviewer’s suggestions and eliminate the data regarding to primary root growth (Fig. 3-6 &S2)

    Regarding root growth defects, these might be due to defect in the vasculature development, please analyse this point or report new experiments (e.g., vasculature analysis of dcp1,2 mutants or tissue specific expression of DCP2).

    __Our response: __We largely agree with the reviewer, all the decapping components DCP1, DCP2, DCP5 and PAT1 exhibit high expression in xylem cells and low expression in procambium cells (Brady et al., 2007) indicating functions of decapping components in vasculature development. However, we did not include this knowledge in our manuscript since we decided to eliminate the primary root growth data (Fig.3-6&S2).

    For consistency the last paragraph of result section: "ASL9 directly contributes to apical hooking, LR formation and primary root growth" should be part of the result section entitled "Accumulation of ASL9 suppresses LR formation and primary root growth". Authors should move this result in the paragraph before "Interference of a cytokinin pathway and/or exogenous auxin restores developmental defects of ASL9 over-expressor and mRNA decay deficient mutants".

    __Our response: __We agree thus we reorganize the result sections and move "ASL9 directly contributes to apical hooking and LR formation" before "Interference of a cytokinin pathway and/or exogenous auxin restores developmental defects of ASL9 over-expressor and mRNA decay deficient mutants" (Line 152).

    I suggest being consistent in the description of the statistical analysis. In particular: - I suggest reporting the meaning of ANOVA letters and the P-value in each figure as sometimes these information are missing, especially in Fig.2.

    __Our response: __We used ANOVA letters when comparing among genotypes and treatments, for example Fig 2A; and we used stars when comparing to controls, for example old Fig 2F. For consistency, we use letters for all the statistical analysis now and we report the meaning of the letters clearly in the figure legends (Fig. 1-6, S1-5&7). However, we think that putting the P-values in each figure would not be reader-friendly, and thus we have not done this.

    • in Fig.S3 please report the statistical significance on bars and the statistical analysis performed.

    __Our response: __We thank the reviewer for pointing it out, we report the statistical analysis now in new Fig. S2 (old Fig. S3).

    MINOR COMMENTS: L31- please replace "normal" with "proper"

    __Our response: __We thank the reviewer for the suggestion, now we replace "normal" with "proper"(Line 30)

    L42-please report the acronym of axr1

    __Our response: __The acronym of axr1 is correctly reported (Line 40).

    L57, L59-please include the entire name of DCP2 and XRN

    __Our response: __The entire name of DCP2 and XRN are correctly included (Line 55 &57).

    -Please report how many plants were analysed in legend or in methods section

    __Our response: __The numbers of plants in analysis are now reported in figure legends (Fig. 1-6, S1,2&7).

    -Please report how many transgenic independent lines were obtained in methods section

    __Our response: __The numbers of transgenic independent lines are now reported in methods (Line 292)

    • Please, try to change the colours of the graph in Fig.S2A-B, as it quite difficult to distinguish light grey shades.

    __Our response: __We thank the reviewer’s suggestions, the colours of new Fig.S3&4 (old Fig.S2) are changed.

    • In Fig. 5A and S5A scale bars are missing.

    __Our response: __We thank the reviewer for pointing this out, scale bars are correctly added in new Fig 4 &S6 (old Fig 5 &S5).

    Reviewer #1 (Significance (Required)): The manuscript is interesting and analyse important and overlooked aspects of the role of mRNA decapping in development. Nonetheless experiments reported are not particularly innovative and not always sound. Also authors analysis are a bit superficial probably because they decide to utilize too many systems in their research (root development, hook development and lateral root development).

    Our response: We thank the reviewer again for acknowledging the significance of our findings and hope we satisfied the reviewer with our answers above. However, we would like to ask what is the purpose of writing “experiments are not particularly innovative”? We admit we used established and robust experiments which we found sufficient to answer the overlooked aspects of the role of mRNA decpping in apical hook and lateral root development as also noted by the reviewer, but maybe we simply don't understand the comment.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Major Comments

    1. My main concern is about the authors' conclusions on the role of mRNA decay and ASL9/LBD3 in the control over cytokinin and auxin responses. I don't think that based on the data presented the authors may do the conclusions stated on lines 184-185, see also the points below.

    __Our response: __We agree thus we tone down our conclusion in our new manuscript (Line 197-199), see answers below for detail.

    The conclusion about the role of ASL9 and its direct involvement in the apical hook formation and lateral root development/main root growth is a bit exaggerated, based on rather tiny effects mediated by the introduction of asl9-1 into the dcp5-1. Rather, the data suggest that misregulation of other transcripts in the mRNA decay-deficient lines might be responsible for the observed defects. That is also apparent from slightly different phenotypes seen in dcp5-1/pat triple compared to oxASL9 (Fig. 3A). The strong dependency of oxASL9 phenotype on the presence of functional ARR10 and ARR12 implies cytokinin signaling-dependent mechanism of ASL9/LBD3 action (see also point 3 below). Considering the aforementioned phenotype differences between the dcp5-1/pat triple and oxASL9, it would be interesting to see the possible dependence of the mRNA decay-deficient line phenotypes on the cytokinin signaling, too.

    __Our response: __We note restoration of dcp5-1 developmental defects in asl9 backgrounds is partial, indicating other ASLs or non-ASLs also contributing to apical hook and lateral root development (old Line 224-225, new Line 229-230 &234-235). We also note that partial suppression is a common phenomenon when studying discrete developmental traits. Two such examples could include the knockout of TPXL5 which partially suppressed the increase of LR density in the hy5 mutant and the introduction of a point mutation in SnRK2.6 in the gsnor1-3/ost1-3 double-mutant partially suppressed the effect of gsnor1-3 on ABA-induced stomatal closure (Qian et al., 2022 The Plant Cell doi.org/10.1093/plcell/koac358; Wang et al., 2015 PNAS 112, 613). In addition to such discrete developmental traits, more dramatic phenotypes like autoimmunity may also only be partially suppressed (Zhang et al., 2012 CH&M 11, 253). However, we agree that it’s interesting to check the dependence of cytokinin signaling of the developmental defects in mRNA decay-deficient mutants. Unfortunately, we were only able to cross arr10 arr12 into dcp5-1. This line showed similar partial restoration of *dcp5-1 *developmental defects as seen for dcp5-1asl9-1. Overall, these data indicates that contribution of mRNA decapping targeting ASL9 transcripts during apical hook and LR formation depends on ARR10 and ARR12 (Fig. 4&6, Line 180-186).

    Also the hypothesis on the upregulation of cytokinin signaling in the mRNA decay mutants and Col-0/oxASL9 is very indirect and should be tested using e.g. TCSn:GFP. The type A ARRs (RRAs) are not only the negative regulators of cytokinin signaling, but also the cytokinin primary response genes. Thus, the downregulation of RRAs could mean the downregulation of the cytokinin signaling pathway in the mRNA decay mutants and/or Col-0/oxASL9. The latter seems to be the case as shown recently (Ye et al., 2021).

    __Our response: __We thank the reviewer for suggesting a different annotation of our result regarding to type-A ARRs. Ye et al reported accumulation of ASL9/LBD3 induced downregulation of cytokinin pathway based on weaker ARR5 and TCSn-GFP signal(Ye et al., 2021). However, the fact that knocking out cytokinin signaling activator genes ARR10 and ARR12 largely restored developmental defects in ASL9 over-expressors lead to the hypothesis of upregulated cytokinin signaling in ASL9 over-expressors (Fig 5). Therefore, we substitute “upregulation” with “misregulation” for cytokinin signaling to compromise in our new manuscript (Line 174).

    The hypothesis on the causal link between the observed auxin-related defects and upregulated cytokinin signaling (Discussion, lines 214-216) is more than speculation. This could be tested by introducing arr10 arr12 into the dcp2-1/DR5-GFP and/or dcp5-1/DR5-GFP.

    __Our response: __We thank the reviewer for the suggestions, due to time and funds management, we decided to check auxin related gene expression in dcp5-1arr10-5arr12-1 mutants instead of making transgenic plants in triple mutant. The repressed expression of SAUR23 and TAR2 in dcp5-1 is partially restored (Fig. S4), indicating possible repression of auxin signaling caused by upregulated cytokinin signaling. However, for consistency in cytokinin signaling description, we tone down the hypothesis on the link between auxin-related defects and cytokinin signaling (Line 218-220).

    Compared to the text/quantification of the effect of asl9-1 mutant on the hook formation (Fig. S1D), I see exaggerated hook formation both in the presence and absence of ACC in asl9-1, at least on the figures shown in Fig. S1C. Are the shown seedlings not representative?

    __Our response: __We thank the reviewer for pointing our mistakes out, the shown seedlings are representative but mislabeled and the mistakes are corrected now in our new manuscript (Fig. S1C).

    Minor Comments

    1. Syntax problem in the sentence on lines 45-46 (?).

    __Our response: __We thank the reviewer for pointing it out, syntax problem of this sentence is solved now in new manuscript (Line 41-44).

    The sentence on lines 48-49 should be rephrased. It implies the cytokinins regulate the amount of RRBs, which is not correct (cytokinins control phosphorylation of RRBs, not their abundance, RRAs are not TFs).

    __Our response: __We now rephrase the sentence in a correct way (Line 46)

    In the FL for Fig. 2F there is mentioned that MYC-YFP was used as a control compared to the main text mentioning YFP-WAVE (?).

    __Our response: __We thank the reviewer for pointing this out, the YFP-WAVE line we used is MYC-YFP transgenic plants, we now include this information in our manuscript (Line 136) and for consistency we changed MYC-YFP to YFP-WAVE in Fig. 2F.

    Naito et al. (2007) suggest ASL9 as a target of cytokinin signaling, but I don't think they imply the involvement of ASL9 in the cytokinin signaling as mentioned e.g. on line 166 (?)

    __Our response: __We largely agree with the reviewer thus we also cite Ye’s paper here in our new manuscript (Line 165)

    References Ye L, Wang X, Lyu M, Siligato R, Eswaran G, Vainio L, Blomster T, Zhang J, Mahonen AP. 2021. Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes. Curr Biol 31(15): 3365-3373 e3367.

    Reviewer #2 (Significance (Required)):

    The authors provide interesting data suggesting possible role of mRNA decay machinery in the hook and lateral root formation and main root growth via decapping-mediated control over ASL9/LBD3 transcript abundance. Based on the observed interaction of the observed phenotypes with hormonal regulations, the authors' conclude mechanistic link between the mRNA decay/ASL9 and cytokinin and auxin responses.

    Our response: We thank the reviewer for acknowledging the significance of our findings.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Major Comments

    1. My main concern is about the authors' conclusions on the role of mRNA decay and ASL9/LBD3 in the control over cytokinin and auxin responses. I don't think that based on the data presented the authors may do the conclusions stated on lines 184-185, see also the points below.
    2. The conclusion about the role of ASL9 and its direct involvement in the apical hook formation and lateral root development/main root growth is a bit exaggerated, based on rather tiny effects mediated by the introduction of asl9-1 into the dcp5-1. Rather, the data suggest that misregulation of other transcripts in the mRNA decay-deficient lines might be responsible for the observed defects. That is also apparent from slightly different phenotypes seen in dcp5-1/pat triple compared to oxASL9 (Fig. 3A). The strong dependency of oxASL9 phenotype on the presence of functional ARR10 and ARR12 implies cytokinin signaling-dependent mechanism of ASL9/LBD3 action (see also point 3 below). Considering the aforementioned phenotype differences between the dcp5-1/pat triple and oxASL9, it would be interesting to see the possible dependence of the mRNA decay-deficient line phenotypes on the cytokinin signaling, too.
    3. Also the hypothesis on the upregulation of cytokinin signaling in the mRNA decay mutants and Col-0/oxASL9 is very indirect and should be tested using e.g. TCSn:GFP. The type A ARRs (RRAs) are not only the negative regulators of cytokinin signaling, but also the cytokinin primary response genes. Thus, the downregulation of RRAs could mean the downregulation of the cytokinin signaling pathway in the mRNA decay mutants and/or Col-0/oxASL9. The latter seems to be the case as shown recently (Ye et al., 2021).
    4. The hypothesis on the causal link between the observed auxin-related defects and upregulated cytokinin signaling (Discussion, lines 214-216) is more than speculation. This could be tested by introducing arr10 arr12 into the dcp2-1/DR5-GFP and/or dcp5-1/DR5-GFP.
    5. Compared to the text/quantification of the effect of asl9-1 mutant on the hook formation (Fig. S1D), I see exaggerated hook formation both in the presence and absence of ACC in asl9-1, at least on the figures shown in Fig. S1C. Are the shown seedlings not representative?

    Minor Comments

    1. Syntax problem in the sentence on lines 45-46 (?).
    2. The sentence on lines 48-49 should be rephrased. It implies the cytokinins regulate the amount of RRBs, which is not correct (cytokinins control phosphorylation of RRBs, not their abundance, RRAs are not TFs).
    3. In the FL for Fig. 2F there is mentioned that MYC-YFP was used as a control compared to the main text mentioning YFP-WAVE (?).
    4. Naito et al. (2007) suggest ASL9 as a target of cytokinin signaling, but I don't think they imply the involvement of ASL9 in the cytokinin signaling as mentioned e.g. on line 166 (?)

    References

    Ye L, Wang X, Lyu M, Siligato R, Eswaran G, Vainio L, Blomster T, Zhang J, Mahonen AP. 2021. Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes. Curr Biol 31(15): 3365-3373 e3367.

    Significance

    The authors provide interesting data suggesting possible role of mRNA decay machinery in the hook and lateral root formation and main root growth via decapping-mediated control over ASL9/LBD3 transcript abundance. Based on the observed interaction of the observed phenotypes with hormonal regulations, the authors' conclude mechanistic link between the mRNA decay/ASL9 and cytokinin and auxin responses.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, the authors describe the role of the mRNA decay machinery in apical hook formation during germination in darkness in A.thaliana. As reported, this machinery is predominantly described in literature in stress response processes, whereas little is known about its involvement during developmental processes. In detail, the authors demonstrated, via RNA immunoprecipitation (RIP) and genetic experiments, the direct regulation of the LATERAL ORGAN BOUNDARIES DOMAIN 3 (LBD3)/ASYMMETRIC LEAVES 2-19 LIKE 9 (ASL9) mRNA stability by the mRNA decapping machinery subunits DCPs. According to the manuscript, ASL9 controls apical hooking, LR development and primary root growthis regulating cytokinin signalling and hence its regulation helps to maintain a correct balance of auxin/cytokinin. Indeed, they showed an impair apical hooking and LR defects both in mRNA decapping mutants, where they observed more capped ASL9 compared to WT, and in ASL9 over-expressor lines. Moreover, they reported a largely restoration of over-expressor lines phenotype in the arr10-5arr12-1 double mutants.

    This work present simple but interesting data that corroborate the authors hypothesis. Nonetheless, I have both major comments and minor comments to improve the manuscript:

    Major comments

    1. I am a bit concerned by the fact that cytokinin, auxin, LBD3,ARR12 and ARR10 have been largely involved in vasculature development and that the obtained results might be due to their role in vasculature development more than in LBD3 mRNA decapping process. Authors should provide evidence that their results are independent from vasculature defects present in those backgrounds or in case discuss this possibility.
    2. The interaction between the described players and auxin is not clear. From the reported experiments it is difficult to understand what authors wants to report as in S4 and S5 are reported experiments not fully described in the text (authors report about introgression of DR5::GFP in dcp1 and 2 mutants, but SD4 reports ACC treatments of DR5::GFP,dcp2 mutants and SD5 of 7 dpg root meristems of this strain ). Please describe and discuss better the experiment. Also, to this reviewer it is difficult to understand whether the absence of auxin activity in the dcp2 mutants hypocotyl is merely an effect of the lack of the hook formation in this background or a cause. Please clarify this point including new experiments (axr1 or axr3 mutants might help in understand this point).
    3. Authors conclude that mRNA decapping is also involved in root growth. However, they do not report direct evidences regarding root growth but mostly regarding the mere root lenght at a precise developmental stage. Please eliminate this point or provide new experiments (e.g., root length and root meristem activity over time)
    4. Regarding root growth defects, these might be due to defect in the vasculature development, please analyse this point or report new experiments (e.g., vasculature analysis of dcp1,2 mutants or tissue specific expression of DCP2).
    5. For consistency the last paragraph of result section: "ASL9 directly contributes to apical hooking, LR formation and primary root growth" should be part of the result section entitled "Accumulation of ASL9 suppresses LR formation and primary root growth". Authors should move this result in the paragraph before "Interference of a cytokinin pathway and/or exogenous auxin restores developmental defects of ASL9 over-expressor and mRNA decay deficient mutants".
    6. I suggest being consistent in the description of the statistical analysis. In particular:
      • I suggest reporting the meaning of ANOVA letters and the P-value in each figure as sometimes these information are missing, especially in Fig.2.
      • in Fig.S3 please report the statistical significance on bars and the statistical analysis performed.

    Minor comments

    L31- please replace "normal" with "proper"

    L42-please report the acronym of axr1

    L57, L59-please include the entire name of DCP2 and XRN

    • Please report how many plants were analysed in legend or in methods section
    • Please report how many transgenic independent lines were obtained in methods section
    • Please, try to change the colours of the graph in Fig.S2A-B, as it quite difficult to distinguish light grey shades.
    • In Fig. 5A and S5A scale bars are missing.

    Significance

    The manuscript is interesting and analyse important and overlooked aspects of the role of mRNA decapping in development. Nonetheless experiments reported are not particularly innovative and not always sound. Also authors analysis are a bit superficial probably because they decide to utilize too many systems in their research (root development, hook development and lateral root development).

  7. Version published to 10.1101/2022.07.06.499076 on bioRxiv