Nitric oxide controls shoot meristem activity via regulation of DNA methylation

Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.

Zeng et al. present a very intriguing finding about the connection of NO signaling to stem cell function and provide a mechanistic model involving the canonical stem cell maintenance factor WUS and the small RNA effector AGO4 and its RdDM activity.This work could be of broad interest to researchers interested in stem cell biology and epigenetics from the animal and plant fields.
The Authors show that the central zone of the SAM contains less NO, likely due to less activity of NO biosynthetic genes NIA1, NIA2, and NOA.Reduced NO levels result in smaller meristems with more WUS and CLV3 expression.They also used a pharmacological approach to show that NO levels can modify WUS expression.Next, they show that WUS directly represses NIA1.Using transcriptome data, they identify AGO4 as a probable target of NO signaling.Therefore they investigate the meristem of mutant plants but cannot see differences in ago4, but only in ago4ago6ago9 or drm1drm2met1 triple mutants.Next, they find that AGO4 and WUS can interact and, given the involvement of AGO4 in RdDM, investigate DNA methylation in SNP-treated meristematic material.They find a reduction of DNA methylation and conclude that SNP interrupts the AGO4-WUS repressive activity.

Major concerns:
The interaction of AGO4 with WUS and the possibility that AGO4 is recruited with or without loaded small RNAs by WUS to the promoter of genes is fascinating.But the interaction assays rely on transiently overexpressed proteins.Therefore, this interaction should be confirmed in Arabidopsis with, e.g., pAGO4:AGO4cherry and pWUS:WUS-GFP or at least with their inducible system for WUS.Alphafold could also add additional evidence for an interaction.
They should also investigate if this proposed chromatin tethering of AGO4 by WUS would depend on small RNAs (e.g., using poliv) or not.To show that AGO4 is recruited to the same sites as WUS (e.g., to promoters of NIA1 or ARR7) by chip-pcr would be affirmative.Also, the promoters of potential target genes should be analyzed if they carry transposon fragments as target sites for RdDM.They should also investigate the existence of small RNAs at the promoter regions of common AGO4/WUS targets.Less importantly, it would also be interesting to investigate a potential connection to 22nt small RNAs, as there is a strong association between the silencing of NIA1 and NIA2 and the activity of small RNAs (Wu et al., Nature 581, 2020).
Another concern is the analysis and interpretation of the DNA methylation data.First, AGO4 is almost entirely responsible for RdDM-mediated CHH methylation (Stroud et al., 2013).Still, many effects the authors claim to be connected to RdDM are visible only in ago4/ago6/ago9.Also, the choice of drm1/drm2/met1 is unfortunate, as met1 has global DNA methylation defects, not limited to RdDM.A better option would have been strict RdDM mutants e.g., drm1/drm2, rdr2, rdm1, or nrpe1.Could it be that the observed transcriptional changes are not related to RdDM but to additional functions of AGO6 and AGO9 or MET1?
The authors should at least use one more RdDM mutant to reduce this concern.It would also be excellent to see potential changes in promoter CHH methylation of NIA1 and NIA2 plus SNP.

Minor concerns:
Paragraph "Nitric oxide promotes peripheral zone fate via repression of WUS" It is hard to see in Supplementary Fig. 1a the claim that NO accumulated to higher levels in the PZ compared to the CZ.Please indicate fluorescence intensities across the picture and show more examples of meristems.Supplementary Fig. 2: the Signal for NIA2 is challenging to see.Given the difficulties of producing clear in situs, please provide at least antisense and/or knock-out mutant controls for all three genes.
To evaluate the functionality of all three genes: Did the authors observe phenotypic differences to wt in any of the double mutant combinations?For example, do nia1/nia2/noa1 plants grow slower, or are they also producing fewer organs (do they flower with the same or a different number of leaves?)? Are they growing normally on soil, or need additional nitrogen sources?
It looks like the experiment in Supplementary Figure 1a-d has been done only with one replica.Either clarify or provide more biological replicas.It would be beneficial to show this experiment performed with 35S::ALCR;ALA::GUS to exclude the influence of ethanol on the staining.Fig1 m,n): On which data is the quantification based?Is it linear measurements of in-situ hybridizations?Please clarify.Supplementary Fig. 5 comes right after supplementary Fig. 2. Please make the order chronological to make reading easier.More importantly, Supplementary Fig. 5 is an important control experiment to exclude a nonspecific effect of NO on WUS expression.But it is not possible to judge from the images the validity.Fluorescence intensities from several meristems need to be quantified.The line "We inspected our resources on direct WUS target genes".Which resources?RNA-seq data: please describe briefly in the text how DEGs are defined (which statistical test was used, what is the TPM/padj/foldchange cutoff?Fig. 3: a: it is confusing to have both SNP/Mock down and SNP/Mock up in the same Venn diagram.Either show only the down or split them, or use upset plots.AGO4 is one of 10 AGO proteins most highly expressed in the SAM.Therefore it would be essential to show the specificity of their probe by using knock-out ago4 plants.Figure 3o: please indicate the SNP concentration.The authors report that AGO4 accumulates in the cytoplasm upon SNP treatment.Are the Snitrosylateion sites on a potential NLS or NES? Chapter "AGO4 protein connects WUS function with the RdDM pathway" Sentence: "together with its orthologues AGO6 and AGO9.The functional relevance of AGO9 for RdDM has never been demonstrated.As mentioned already above, the usage of drm1/drm2/met1 was not a good choice.MET1 is a CG maintenance methyltransferase, and loss of MET1 has all kinds of secondary effects (e.g., late flowering).It would have been better to use other RdDM mutants like drm1/drm1, rdr2, or poliv.The IP should be repeated using Arabidopsis stably transformed Arabidopsis plants.Which ago4 allel was used?Supplementary Fig. 7: please write which mutant is in which genetic background.It would also be important to show the SAM size of Ler and Col wt plants.I assume ago4wus7 plants are from an F2 population from the ago4 x wus7 cross.Are the LerXCol plants also F2? LerXCol f2 plants are problematic as they are very heterogeneous.The better experiment would have been to generate a CRIPR ago4 knockout in the wus7 background.Line " this phenotype was largely suppressed in drm1/drm2/met1 triple mutant": please refer to picture.Is it Supplementary Fig. 8 c/d?Unfortunately, Supplementary Fig. 8 is also difficult to interpret as Met1 is not involved in RdDM but DRM1/DRM2, meaning the conclusion that "the RdDM pathway is required for WUS activity and proper SAM function" is not supported by the data.They could repeat this experiment with other RdDM mutants (as suggested above).It is also unclear to me how the authors interpret Supplementary Fig. 8: Does that mean DNA methylation is required to activate CLV3?That would be in contrast to the fact that most DNA methylation mutants develop similarly to wt.
Chapter: "Nitric oxide controls expression of key meristem regulators via DNA methylation" The DNA methylation data need to be analyzed more carefully.Which treatment length of SNP was used for Bisulfite sequencing?It is critical to distinguish between CG, CHG, and CHH methylation, as only CHH methylation is affected by the loss of AGO4.What kind of DNA methylation levels do the authors describe?Weighted DNA methylation?It is unclear to me how Fig. 6 b shows a trend towards "reduced DNA methylation"?Supplementary Fig. 9: I need clarification on why the authors show total DNA methylation levels, as this is irrelevant to AGO4 function.They should distinguish CG, CHG, CHH methylation, and promoter and gene body methylation.It is also unclear to me how "total 5-mC" was calculated, as it is unclear from the description in M&M.Please use methylation levels as defined in Schultz et al., Trends Genet 2012., or describe better how the calculation was done.To ease the reproducibility of the study, please include the atg numbers plus Stockidentifiers for all genes and mutants used in the study.Discussion: I do not know about RdDM activity in animals; the papers they refer to do not show this.Methods: Please provide the C to T conversion rates (using the chloroplast genome) and genome coverage for the EM-seq data.

Reviewer #3 (Remarks to the Author):
The manuscript by Zang et al. entitled "Nitric Oxide controls shoot meristem activity via regulation of DNA methylation" reports the roles of NO and RdRM in the SAM.AGO4 is inhibited by NO at the transcriptional level and also post-transcriptional level, which is Snitroslation dependent.AGO4 interacts with WUS at the protein level, and this interaction is disrupted by NO treatment.The roles of RdRM in the SAM have been reported.For example, rice RdRM mutant plants have prominent SAM defects.This study links NO regulation to the roles of RdRM and WUS.Together, this study provides many interesting clues but there seems to be insufficient support for the models proposed by the authors.In the model, many links are not well established either.Below are my specific comments.
2. Fig. 2a-c, 3 h treatment w/ SNP or cPTIO causes WUS signal changes.Was the pWUS::2xVenusNLS reporter used?If so, the reduction after SNP treatment seems suggest a fast degradation of 2xVenusNLS.Why Venus is so unstable?Note that the authors have shown that GFP fluorescence is not responsive to NO levels (Suppl Fig 5a-c).Venus is a fast maturing protein, but 3 h is still short for a substantial portion of Venus to mature (Balleza et al, 2018 Nat Methods doi: 10.1038/nmeth.4509).
3. The protein interaction between WUS and AGO4 is interesting.However, the biological relevance is less clear.If the interaction contributes to WUS activity as a transcription factor, differential expression of WUS-target genes should be identified in ago4 mutant plants.Alternatively, if WUS participates in the RdRM activity of AGO4, AGO4 targets should be different in wus mutants or WUS-OX lines.
4. The DAF-2DA staining results is not very clear.Is the signal enriched in incipient primordia or the boundaries between primordia? 5.The expression of both WUS and CLV3 are enhanced in the nia triple mutant.Why? 6.In Fig. 2j-l, CLV3 signal seems to be enhanced after SNP and cPTIO treatment.7. To really test whether S-nitroslation is responsible for AGO4-WUS interaction, the authors need to disrupt all possible S-nitroslation sites, which can be identified by mass spectrum analysis.

Point-by-point responses to the reviewer comments
We would like to thank all reviewers for their insightful comments, which were very helpful to improve the manuscript.Following their advice, we have carried out a large number of new experiments and have extensively revised the manuscript.Specific responses to all comments can be found below: Major comments: (1) "we continuously increased NO levels in the CZ by expressing NOA1, one of the NO biosynthesis gene, from the CLV3 promoter (Supplementary Fig. 4a-e (2) To test this directly, we examined the 26 NO-induced plant stem cell-related genes identified here and previously in our methylation data and identified nine genes (Supplementary Fig. 9)".
Are there any common responsive elements located in the promoters of these 26 NO-induced plant stem cell-related genes?RESPONSE: As suggested, we carried out an analysis of known regulatory elements the 26 NOinduced plant stem cell-related genes and found a number of elements to be represented, including light, stress, and hormone responsive elements.We have added this new analysis to Supplementary Fig. 16b.Due to the small number of genes, a de novo analysis of potential response elements is not possible.
(3) "We further tested the transcriptional response of NIA1, NIA2 and NOA1 to ectopically induced WUS activity and found that at the whole seedling level NIA1 and NOA1 showed a robust repression after 4 hours WUS of induction (Supplementary Fig. 3a-c)".
Have the authors tested the binding activity of WUS to the promoter of NOA1 by ChIP-PCR or EMSAs?RESPONSE: We have carried out a new WUS ChIP-seq experiment based on the much improved eChIP protocol.Based on this very much improved data, we find strong evidence that WUS protein binds to the promotors of NIA1 and NIA2, but not NOA1.This data is now shown in the new Supplementary Figure 7.
(4) "we took advantage of the ap1/cal mutant, which is characterized by a massive overproliferation and subsequent arrest of inflorescence and young floral meristems".
The authors performed the RNA-seq analysis on microdissected apices with and without SNP treatment for 24 hours with the ap1/cal mutant.The question is that how many typical NOresponsive genes identified in whole seedlings can be repeatedly detected in the ap1/cal mutant.
That is the key point to confirm whether the transcriptome profiling performed with the ap1/cal mutant is acceptable in relative to the WT whole plants.
RESPONSE: As suggested, we performed the RNA-seq with seedlings of WT (Col-0) and ap1/cal mutants with and without 24 h SNP treatment to test for comparability of wt and ap1/cal mutans with regards to NO response.We observed that WT and ap1/cal mutants share 88% of responsive genes after SNP treatment(Supplementary Fig. 9), suggesting that the NO response of WT and ap1/cal mutants is comparable.We added the new data to our revised manuscript in the new Supplementary Fig. 9.

Minor Comments:
(1) Page 10, line 10, 13, the AGO4 could be modified by S-nitroslation (?) in response to NO. RESPONSE: Yes, the AGO4 protein could be modified by S-nitroslation as we observed that its protein has multiple S-nitrosylation sites by in vivo biotin-switch assay (Fig. 3p).
(2) What is the concentration of cPTIO used in experiments?RESPONSE: We thank the reviewer's point and apologize for ommiting this important information.
We already added the information in the revised manuscript.
(3) In The Authors show that the central zone of the SAM contains less NO, likely due to less activity of NO biosynthetic genes NIA1, NIA2, and NOA.Reduced NO levels result in smaller meristems with more WUS and CLV3 expression.They also used a pharmacological approach to show that NO levels can modify WUS expression.Next, they show that WUS directly represses NIA1.Using transcriptome data, they identify AGO4 as a probable target of NO signaling.Therefore they investigate the meristem of mutant plants but cannot see differences in ago4, but only in ago4ago6ago9 or drm1drm2met1 triple mutants.Next, they find that AGO4 and WUS can interact and, given the involvement of AGO4 in RdDM, investigate DNA methylation in SNP-treated meristematic material.They find a reduction of DNA methylation and conclude that SNP interrupts the AGO4-WUS repressive activity.RESPONSE: We thank the rewiever for pointin out the broad impact and novelty of our work.

Major concerns:
The interaction of AGO4 with WUS and the possibility that AGO4 is recruited with or without loaded small RNAs by WUS to the promoter of genes is fascinating.But the interaction assays rely on transiently overexpressed proteins.Therefore, this interaction should be confirmed in Arabidopsis with, e.g., pAGO4:AGO4cherry and pWUS:WUS-GFP or at least with their inducible system for WUS.Alphafold could also add additional evidence for an interaction.RESPONSE: As suggested, we now performed the co-IP experiment in Arabidopsis using UBQ10::mCherry-GR-WUS plants to confirm the interaction of AGO4 and WUS in vivo.The results showed that endogenous AGO4 indeed interacts with WUS in seedling (Fig. 5d).In addition, we could also observe the interaction between AGO4 and WUS by Alphafold prediction, which suggest that the homeodomain, as well as the disordered C-terminus of WUS are in contact with AGO4.
They should also investigate if this proposed chromatin tethering of AGO4 by WUS would depend on small RNAs (e.g., using poliv) or not.RESPONSE: To address this comment, we now examined the response of stem cell and nice cell specific genes to SNP treatment in wt and the nrpe1 mutants.Among the 12 stem cell-and niche-specific NO responsive genes in the wild type, none of them lost their response to SNP  To show that AGO4 is recruited to the same sites as WUS (e.g., to promoters of NIA1 or ARR7) by chip-pcr would be affirmative.RESPONSE: As suggested, we performed ChIP-PCR on AGO4 and found that AGO4 directly binds to the promoter of ARR7, in one of the promoter regions also bound by WUS.These results are now shown in the new (Supplementary Fig. 20).
Also, the promoters of potential target genes should be analyzed if they carry transposon fragments as target sites for RdDM.
They should also investigate the existence of small RNAs at the promoter regions of common AGO4/WUS targets.RESPONSE: We have carefully analyzed our new WUS ChIP-Seq data for the overlap between WUS chromatin binding and transposons and found that a substantial fraction of WUS binding events are located in these elements (Supplementary Fig. 14).This observation suggests that WUS may be involved in protecting stem cells from the activity of TEs.Conversely, we did not find evidence of TE sequences enriched among in the overlap of WUS and AGO4 target genes, suggesting that the chromatin binding likely does not involve small RNAs.
Scanning for siRNAs matches in the promoter regions of AGO4 and WUS targets, we identified about 51,256 and 20,595 siRNAs , respectively.However, only 354 (0.6%) of them were represented in both list (Figure . 2 for reviewer).Less importantly, it would also be interesting to investigate a potential connection to 22nt small RNAs, as there is a strong association between the silencing of NIA1 and NIA2 and the activity of small RNAs (Wu et al., Nature 581, 2020).RESPONSE: As suggested, we performed small RNA sequencing on ap1/cal shoot apices with and without SNP treatment to test the small RNA behavior in response to NO and found that the majority of small RNAs were not significantly affected by increasing endogenous NO level (Figure .3 for reviewer), indicating that NO does not have effects on small RNAs activity in the shoot meristem.Another concern is the analysis and interpretation of the DNA methylation data.First, AGO4 is almost entirely responsible for RdDM-mediated CHH methylation (Stroud et al., 2013).Still, many effects the authors claim to be connected to RdDM are visible only in ago4/ago6/ago9.Also, the choice of drm1/drm2/met1 is unfortunate, as met1 has global DNA methylation defects, not limited to RdDM.A better option would have been strict RdDM mutants e.g., drm1/drm2, rdr2, rdm1, or nrpe1.Could it be that the observed transcriptional changes are not related to RdDM but to additional functions of AGO6 and AGO9 or MET1?The authors should at least use one more RdDM mutant to reduce this concern.
It would also be excellent to see potential changes in promoter CHH methylation of NIA1 and NIA2 plus SNP.RESPONSE: We thank the reviewer for the very constructive suggestion.We examined the effects by using additional RdDM mutants, such as drm1/drm2 and rdm1 and observed similar results found in the ago4/ago6/ago9 and drm1/drm2/met1 triple mutants.We have added this new data in a number of new supplementary figures.As suggested, we also analyzed the CHH methylation of NIA1 and NIA2 with SNP treatment, but did not observe any changes (Supplementary Table  Supplementary Fig. 2: the Signal for NIA2 is challenging to see.Given the difficulties of producing clear in situs, please provide at least antisense and/or knock-out mutant controls for all three genes.RESPONSE: We thank the reviewer's suggestion.We agree with the reviewer's comment that the in situ picture of NIA2 was not clear enough.We replaced the old image and included sense probes as control for all three NO genes in the revised manuscript. To evaluate the functionality of all three genes: Did the authors observe phenotypic differences to wt in any of the double mutant combinations?For example, do nia1/nia2/noa1 plants grow slower, or are they also producing fewer organs (do they flower with the same or a different number of leaves?)? Are they growing normally on soil, or need additional nitrogen sources?RESPONSE: Thank you for bringing up these important points.We have now carefully performed phenotypic analysis of the mutants and found that the emergence of the first true leaves is severely compromised in the mutants compared with wild type plants, indicating that the meristem activity is reduced in the mutants.We have added this new data in the revised manuscript.
It looks like the experiment in Supplementary Figure 1a-d   RESPONSE: We appreciate the reviewer's comment and apologized for the missing information.
We have now added this information in the revised manuscript.

Figure 5a :
What is the p-Value of the overlap (e.g., use a hypergeometric test).The reference for Zheng et al. should be in the text and the reference list.

Figure 5c :
Please indicate the antibody used for IP in the Figure.

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in this manuscript by Zeng et al. have focused on roles of nitric oxide (NO) in regulation of shoot meristem activity in a manner of DNA methylation in Arabidopsis.Several lines of genetic evidence reveal that NO signaling controls plant stem cell homeostasis using a set of mutant materials.Importantly, genetic, biochemical and molecular tests suggest that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in an NOdependent manner, which has provided molecular linkage to connect two signaling systems to modify DNA methylation patterns.Notably, in combination with phenotypic analysis of specific mutants, detecting specific and dynamic changes in NO generation around shoot meristem region have provided insights into better understanding the role of NO in represses central cell fate, likely via repressing WUS expression.This study appears as a promising starting point to supply lines of strong evidence to recognize the requirement of the regulation of WUS expression by NO in shoot meristem cell fate decisions.RESPONSE: We thank the reviewer for pointing out the importance of our work.
)".Actually, no images or quantification data of DAF-2DA fluorescent intensity were shown in Supplementary Fig. 4 to specify the increase in NO levels in the indicated CLV3::NOA1 transgenic plants compared with WT.RESPONSE: We have now included new data to show the distribution of NO in CLV3::NOA1 transgenic plants, along with additional pictures of wt including careful quantification of staining levels.We have added this information in the revised Supplementary Fig 6.After DAF-2DA staining, we observed that endogenous nitric oxide level was increased in the stem cell domain in the CLV3::NOA1 transgenic plants.

Fig. 1 |
Nitric oxide is required for proper meristem activity.Quantification of SAM size (m).What is the length unit for SAM size?RESPONSE: Thank you for bringing up this important point.We have now included this information in the revised figures.--Reviewer #2: Zeng et al. present a very intriguing finding about the connection of NO signaling to stem cell function and provide a mechanistic model involving the canonical stem cell maintenance factor WUS and the small RNA effector AGO4 and its RdDM activity.This work could be of broad interest to researchers interested in stem cell biology and epigenetics from the animal and plant fields.
treatment (Figure. 1 for reviewer), indicating the NO mediated stem cell regulation via AGO4-WUS pathway does not rely on Pol V activity.

Figure. 1
Figure. 1 for reviewer.NRPD1B does not mediate NO signaling in the shoot meristem.a, b, RT-qPCR detected gene expression level of stem cell-and niche-specific genes in the wild type (a) and nrpd1b-11 (b) mutants with and without SNP treatment.The data are shown as mean ± s.d.; n = 3 biological replicates, two-tailed Student's t-tests, **p < 0.01, ***p<0.001.

Figure. 2
Figure. 2 for reviewer.Distribution pattern of siRNAs in the promoter region of AGO4/WUS common targets.Venn diagram showing the overlap of siRNAs in the promoter region of AGO4 and WUS targets.

Figure. 3
Figure. 3 for reviewer.Profiling of small RNAs in ap1/cal with and without SNP treatment.Analysis of small RNAs in ap1/cal shoot apices with and without SNP treatment using small RNA sequencing.Up indicates up-regulated upon SNP treatment, Down indicates down-regulated after SNP treatment, Normal indicates no significant changes. .
oxide promotes peripheral zone fate via repression of WUS" It is hard to see in Supplementary Fig. 1a the claim that NO accumulated to higher levels in the PZ compared to the CZ.Please indicate fluorescence intensities across the picture and show more examples of meristems.RESPONSE: We have now examined more samples and carefully quantified the DAF-2DA florescent intensity (Supplementary Fig. 1).
has been done only with one replica.Either clarify or provide more biological replicas.It would be beneficial to show this experiment performed with 35S::ALCR;ALA::GUS to exclude the influence of ethanol on the staining.RESPONSE: Thank you for the suggestion, we have examined more samples as well as including a control by using 35S::AlcR；AlcA::GUS control plants.We included this new data in the new Supllemntary Fig 1.

Fig1
Fig1 m,n): On which data is the quantification based?Is it linear measurements of in-situ hybridizations?Please clarify.