1. Author Response:

    Reviewer #3:

    Maintaining the balance between stem cell proliferation and cell differentiation is an essential challenge of all stem cell niches. In the shoot apical meristem of plants, these functions are spatially separated into the central zone and peripheral zone, respectively. How these zones communicate to give rise to proper stem cell behavior has been a research focus for many years.

    In this manuscript, the authors suggest that the small secreted peptide CLE40 and the receptor kinase like protein BAM1 form a novel pathway that contributes to meristem homeostasis by stimulating the expression of the central stem cell inducer WUSCHEL primarily from the meristem periphery. Importantly, this pathway acts antagonistically to the well-studied CLV pathway, which is only active in the center of the meristem and is molecularly highly similar to the CLE40/BAM1 system. This model is experimentally supported mainly by analysis of spatial localization patterns in the meristem using transcriptional and translational reporters and by the analysis of genetic interactions.

    The findings of the authors are novel, highly relevant and would certainly be of great interest for the plant community. However, the manuscript could be substantially improved to provide better support for the conclusions laid out.

    Of major concern are the reporter genes and imaging data: Partial colocalization and exclusion from CZ and OC are one of the main arguments of the authors to claim that CLE40/BAM1 function together and antagonistically to CLV3/CLV1 in controlling WUS expression.

    Working with reporters as proxies for endogenous gene expression needs to be backed up by proper controls. Given the central importance of the reporters for the conclusions it is essential to show that the regulatory sequences used for the CLE40 reporter are sufficient to rescue a cle40 mutant.

    We show now in a new supplemental figure (1) the expression patterns of two different CLE40 reporter lines (differening in length of the promoter region) in the root, which are identical, and (2) that expression CLE40 from the CLE40 promoter rescues the cle40 mutant root phenotypes, which were described in earlier work. See Fig2-SupplFig. 1

    It is essential to show that ... the observed expression of the reporter is consistent across the majority of different T1 lines and, most importantly, that the pattern reported here is consistent with in situ data for endogenous CLE40 mRNA.

    RNA in situ analysis is difficult due to the low expression level of CLE40, and the small size of the CLE40 transcript. We show in Fig2-SupplFig2 expression data for 4 independent transgenic CLE40 reporter lines, confirming the general conclusions that we present in this manuscript.

    The authors have previously published in situs for CLE40 that do not show the exclusion from the CZ and OC (Hobe et al., 2003, Figure 2a,c), which urgently needs clarification.

    The RNA expression data from Hobe et al. are displayed at low mag and low resolution, and might have suffered from high background.

    Figures 2, 4 and 5 show imaged meristems in great detail but each focus only on a single sample. I strongly recommend to also include quantitative data on multiple samples to substantiate the claims. This could be likely be done with standard software, such as MorphographX.

    The data we showed before represented typical examples from a wide range of data that we analysed. All original data are being made publicly available for reanalysis. We have now added multiple examples from multiple samples, and also added quantitative data from fluorescence analysis. See new Supplementary Fig2-SupplFig. 2, Fig.4-SupplFig. 1, Fig.4-SupplFig. 2, Fig.4-SupplFig. 3, Fig.5-SupplFig. 1, Fig.5-SupplFig. 2, Fig.5-SupplFig. 3, Fig.5-SupplFig. 4

    Whereas the inhibitory effect of WUS on CLE40 is convincingly shown using ectopic WUS expression and the hypomorphic wus7 allele (Figure 2) the quantification of WUS positive cells in Figure 7 is problematic. Although it was done over multiple samples it heavily relies on manual scoring, which is prone to bias. The same is true for the width/height measurements of different meristems. An unbiased computational image analysis would certainly give more reliable results.

    We are grateful for this suggestion. We normally analyse samples in an anonymised manner. We have now also quantified the number of WUS positive cells using the Imaris software, as suggested, see Fig.7-SupplFig.1, and found that this analysis supported our previous conclusions. We also added a figure showing multiple samples from this experiment. See new Supplementary Fig7-SupplFig. 2

    One major point that the authors try to establish is that the CLE40 signal that eventually leads to reduction in meristem size is transduced via the BAM1 receptor. However, only genetic interactions, which are complicated by intricate feedbacks, are show to substantiate this claim. For a strong statement on CLE40/BAM1 ligand/receptor interactions, advanced imaging technologies available to the authors or biochemical experiments would be necessary.

    We are currently not aware of a reliable and applicable experimental approach that would allow us to show direct interaction of the CLE40 peptide with its receptors in vivo. Biochemical experiments using purified peptides and/or receptors are, so far, contradictory: Shinohara et al. (2015) used chemically synthesized arabinosylated CLV3 peptide and photoaffinity labelling to show binding of CLV3 to a BAM1-Halo-TAG fusion protein expressed in BY-2 cells. However, using BAM1 protein purified from insect cell lines which was biotinylated in the Creoptix WAVE system, Crook et al. (2020) found no significant binding activity for synthetic CLV3 peptide. Our preliminary conclusion from these data sets is that binding of peptides to receptors should be best evaluated in vivo, since important posttransciptional and posttranslational modifications, as well as coreceptors, can strongly modify peptide-receptor interactions.

    We have here added data showing that in the root, BAM1 receptor but not CLV1 is required for CLE40 dependent regulation of root meristem development, indicating again that CLE40 and BAM1 are likely to act in the same signaling pathway throughout development. See new Supplementary Fig6-SupplFig. 1

    Similarly, the genetic studies need some clarification: The authors show that cle40 and bam1 single mutants as well as cle40/bam1 double mutants all show a comparable reduction in meristem size, suggesting epistasis. In contrast, a reduction in meristem size can not be observed if cle40 is combined with clv1, which according to the proposed model appears to be unexpected. The interpretation of the genetic experiments is complicated by the well-known fact that BAM1 expression is regulated by the CLV pathway and loss of CLV signaling leads to ectopic expression of BAM1 in the OC which can partially compensate for the loss of CLV1, due to the molecular similarity of the two receptors. The shift of BAM1 expression from the PZ towards the OC could explain why there is no significant reduction in meristem size since CLE40 induced signaling at the PZ would be inhibited by the lack of the BAM1 receptor. To clarify the specific interaction of CLE40 with BAM1 and/or CLV1 the authors could try to restore BAM1 levels in the PZ of cle40/clv1 mutants by expressing BAM1-GFP from an appropriate promoter (e.g. RPS5 or UBQ10). This experiment would allow to distinguish between the genetic interaction of CLE40 with CLV1 from the feedback between CLV1 and BAM1 expression.

    The suggested experiment, to misexpress BAM1 from the RPS5 or UBQ10 promoter, is not feasible, since this results generally in a much higher expression level, which, in our hands, is not "tolerated" by CLV-family receptors. We found that higher level expression of RLKs generally causes mislocalisation of nonfunctional proteins.

    Overall, the manuscript could be strengthened by inclusion of additional molecular data probing the directness of WUS inhibiting CLE40 and/or BAM1 expression.

    We are planning to set-up experiments for detailed studies on the transciptional regulation of genes in the stem cell control pathways, and will in the future also investigate the feedback regulation of WUS onto CLE40 and BAM1. However, such analysis goes far beyond the scope of our current manuscript.

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  2. Evaluation Summary:

    Vertical patterning of the shoot meristem is regulated by a well-characterized feedback loop involving the CLAVATA3 peptide, the CLAVATA1 receptor-like kinase and the WUSCHEL transcription factor. Cell loss from the Peripheral Zone of the meristem, due to production of lateral organs, requires a compensatory size increase of the stem cell domain, i.e. there is a need to understand how stem cell activities in the Central Zone and Organizing Center are coordinated to regulate organ initiation and cell differentiation in the Peripheral Zone. The authors identify a new signaling pathway to control shoot meristem function in Arabidopsis, suggesting that the peptide CLE40 and the receptor kinase-like protein BAM1 act from the Peripheral Zone to stimulate stem cell fate via WUSCHEL expression, and antagonistically to the CLV pathway. The model is novel and exciting and will be of interest to plant scientists as well as those interested in developmental patterning. Some additional evidence is required to fully sufficient to support all claims in the manuscript.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors carry out a series of cell biology and genetics experiments in Arabidopsis to implicate CLE40 in an additional negative feedback loop. CLE40 has an expression pattern complementary to that of CLV3, so that CLV3 and CLE40 antagonistically control shoot meristem size by controlling expression of the transcription factor WUSCHEL in a CLV1- and BAM1-dependent manner.

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  4. Reviewer #2 (Public Review):

    Loss-of-function cle40 mutants have small meristems, and promoter fusions show that the CLE40 gene is active in the peripheral meristem zone around the CLV3 expression domain. Lines expressing WUSCHEL from the CLV3 promoter have enlarged meristems that lack CLE40 expression, and the CLE40 expression domain is larger than normal in loss-of-function wuschel mutants, so WUSCHEL suppresses CLE40 expression. Protein fusions of CLAVATA1 show localization at the center of the meristem and in leaf primordia in the apical dome, and of the BAM1 receptor-like kinase in many tissues of the shoot tip, but with strong expression in a cylinder around the center of the shoot apex. Overlapping expression of CLE40/BAM1 and CLV3/CLV1 is shown and mutant combinations show that the proteins act in pairs. Analysis of WUSCHEL expression in clv1, clv3, cle40 and bam1 mutant backgrounds shows that CLE40 and BAM1 promote expression, ad both genes affect the shape of the apical dome. A model for patterning of the apical dome by CLV3/CLV1, CLE40/BAM1 and WUSCHEL is proposed.

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  5. Reviewer #3 (Public Review):

    Maintaining the balance between stem cell proliferation and cell differentiation is an essential challenge of all stem cell niches. In the shoot apical meristem of plants, these functions are spatially separated into the central zone and peripheral zone, respectively. How these zones communicate to give rise to proper stem cell behavior has been a research focus for many years.

    In this manuscript, the authors suggest that the small secreted peptide CLE40 and the receptor kinase like protein BAM1 form a novel pathway that contributes to meristem homeostasis by stimulating the expression of the central stem cell inducer WUSCHEL primarily from the meristem periphery. Importantly, this pathway acts antagonistically to the well-studied CLV pathway, which is only active in the center of the meristem and is molecularly highly similar to the CLE40/BAM1 system. This model is experimentally supported mainly by analysis of spatial localization patterns in the meristem using transcriptional and translational reporters and by the analysis of genetic interactions.

    The findings of the authors are novel, highly relevant and would certainly be of great interest for the plant community. However, the manuscript could be substantially improved to provide better support for the conclusions laid out.
    Of major concern are the reporter genes and imaging data: Partial colocalization and exclusion from CZ and OC are one of the main arguments of the authors to claim that CLE40/BAM1 function together and antagonistically to CLV3/CLV1 in controlling WUS expression.

    Working with reporters as proxies for endogenous gene expression needs to be backed up by proper controls. Given the central importance of the reporters for the conclusions it is essential to show that the regulatory sequences used for the CLE40 reporter are sufficient to rescue a cle40 mutant; the observed expression of the reporter is consistent across the majority of different T1 lines and, most importantly, that the pattern reported here is consistent with in situ data for endogenous CLE40 mRNA. The authors have previously published in situs for CLE40 that do not show the exclusion from the CZ and OC (Hobe et al., 2003, Figure 2a,c), which urgently needs clarification.

    Figures 2, 4 and 5 show imaged meristems in great detail but each focus only on a single sample. I strongly recommend to also include quantitative data on multiple samples to substantiate the claims. This could be likely be done with standard software, such as MorphographX.

    Whereas the inhibitory effect of WUS on CLE40 is convincingly shown using ectopic WUS expression and the hypomorphic wus7 allele (Figure 2) the quantification of WUS positive cells in Figure 7 is problematic. Although it was done over multiple samples it heavily relies on manual scoring, which is prone to bias. The same is true for the width/height measurements of different meristems. An unbiased computational image analysis would certainly give more reliable results.

    One major point that the authors try to establish is that the CLE40 signal that eventually leads to reduction in meristem size is transduced via the BAM1 receptor. However, only genetic interactions, which are complicated by intricate feedbacks, are show to substantiate this claim. For a strong statement on CLE40/BAM1 ligand/receptor interactions, advanced imaging technologies available to the authors or biochemical experiments would be necessary. Similarly, the genetic studies need some clarification: The authors show that cle40 and bam1 single mutants as well as cle40/bam1 double mutants all show a comparable reduction in meristem size, suggesting epistasis. In contrast, a reduction in meristem size can not be observed if cle40 is combined with clv1, which according to the proposed model appears to be unexpected. The interpretation of the genetic experiments is complicated by the well-known fact that BAM1 expression is regulated by the CLV pathway and loss of CLV signaling leads to ectopic expression of BAM1 in the OC which can partially compensate for the loss of CLV1, due to the molecular similarity of the two receptors. The shift of BAM1 expression from the PZ towards the OC could explain why there is no significant reduction in meristem size since CLE40 induced signaling at the PZ would be inhibited by the lack of the BAM1 receptor. To clarify the specific interaction of CLE40 with BAM1 and/or CLV1 the authors could try to restore BAM1 levels in the PZ of cle40/clv1 mutants by expressing BAM1-GFP from an appropriate promoter (e.g. RPS5 or UBQ10). This experiment would allow to distinguish between the genetic interaction of CLE40 with CLV1 from the feedback between CLV1 and BAM1 expression.
    Overall, the manuscript could be strengthened by inclusion of additional molecular data probing the directness of WUS inhibiting CLE40 and/or BAM1 expression.

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