LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis

  1. Mahima
  2. Sourav Chatterjee
  3. Sharmila Singh
  4. Ananda K. Sarkar

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Abstract

In higher plants, epigenetic modifications provide a stage for both transient and permanent cellular reprogramming required for vegetative to reproductive phase transition. Arabidopsis LSD1-like 1 (LDL1), a histone demethylase positively regulates floral transition, but the molecular and biochemical nature of LDL1 mediated flowering is poorly understood. Here we have shown that LDL1 mediated regulation of flowering is dependent on MADS AFFECTING FLOWERING 4 (MAF4) and MAF5 floral repressors. LDL1 binds on the chromatin of MAF4 and MAF5 and removes H3K4me2 activation marks to repress their expression. Further we show that LDL2 negatively regulates the expression of MAF4 and MAF5 redundantly with LDL1. Both LDL1 and LDL2 interact with an autonomous flowering pathway protein, FLOWERING LOCUS VE (FVE), to regulate the floral transition and thus could be a part of the FVE-corepressor complex. We show that MAF5 interacts with other floral repressors FLC and SHORT VEGETATIVE PHASE (SVP) and repress the expression of FT to delay floral transition. Thus, our results deepen the mechanistic understanding of LDL1/LDL2-FVE mediated floral transition in Arabidopsis .

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    Manuscript number: RC-2022-01480

    Corresponding author(s): Ananda, Sarkar

    1. General Statements

    We are thankful to Review commons platform that helped our manuscript critically reviewed with very constructive and valuable feedback. This gave us the opportunity to do the experiments accordingly and significantly improve the manuscript. We are hopeful that this platform will help our manuscript get published in a journal of repute.

    2. Point-by-point description of the revisions

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

    The manuscript entitled "LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis" characterized that LDL1 regulates flowering by binding on the chromatin of MAF4 and MAF5 to repress their expression. Further the authors proposed LDL1/LDL2-FVE model. Here are some comments for this manuscript.

    Major problems:

    1. This experiment is still not testing or showing/concluding that the whole complex forms on the MAF4 and MAF5

    Response: We understand reviewer’s concern regarding the complex. Previously FVE was shown to be a part of co-repressor complex including HDA6, HDA5 and FLD to regulate the expression of FLC and its clade members during floral transition1-3 We showed that LDL1 binds directly to the chromatin of MAF4 and MAF5 to suppress their expression (Figure 1 and 2). Furthermore, we discovered that LDL1 and LDL2 interact with FVE to influence floral transition (Figure 8 and 9). Hung et al., 2018 reported the interaction of LDL1 and LDL2 with HDA6 to regulate circadian rhythm4 and we found that the expression of MAF4 and MAF5 was upregulated in ldl1ldl2hda6 than ldl1ldl2 (Figure 5C and 5D). Therefore, our experimental data, together with previously reported data makes it evident that LDL1 and LDL2 are a part of co-repressor complex through their interaction with FVE and HDA6, which we concluded here. We agree with the reviewer that an additional experiment, such as complex pull-down, will be helpful, but in our opinion, it will only provide additional confirmatory evidence.

    2.It is not shown LDL1/LDL2 repress MAF4 and MAF5 by removing H3K4me2 activity. It would be useful to test whether the methylation level of MAF4 and MAF5 has been altered in ldl1/ldl2 mutant

    Response: We found altered methylation level in MAF4 and MAF5 chromatin during floral transition in ldl1 and ldl1ldl2 mutants (Figure 6 and 7). We observed that the absence of LDL1, or both LDL1 and LDL2 disturbs the shift in H3K4 methylation status on MAF4 and MAF5 during floral transition and ends up in a more active (enriched in H3K4me3 marks) chromatin state at 19 days. This result, taken together with the increased MAF4 and MAF5 expression in ldl1 and ldl1ldl2 double mutants (Figure 5C and 5D) indicates that LDL1/LDL2 repress MAF4 and MAF5 by altering H3K4 methylation.

    3.I suggest that further research is required to provide conclusive evidence concerning the physiology function of LDL1/LDL2-FVE. Such as the expression pattern of LDL1/LDL2, the methylation level of MAF4 and MAF5 before or after floral transition

    Response: Taking this suggestion into account, we performed quantification of rosette leaves and flowering time of fvec, ldlfvec and ldl2fvec along with WT, ldl1 and *ldl2 *(Figure 9). We also observed decreased expression of floral activator genes, FT and SOC1 (targets of MAF4 and MAF5) in fvec, ldlfvec and *ldl2fvec *in comparison to the WT (Supplementary Figure 10C), which corresponds to their late flowering phenotype.

    To understand the role of LDL1and LDL2 during floral transition, we first analyzed the expression of LDL1 and LDL2 during floral transition (Supplementary Figure 8). We observed that the expression of LDL1 and LDL2 expression peaks at 16 days and gets stabilized till 19 days. Then we checked the enrichment of H3K4me1, H3K4me2 and H3K4me3 on MAF4 and MAF5 chromatin in ldl1 and ldl1ldl2 plants with respect to the WT at 16 days (before floral transition) and 19 days (after floral transition). We found an increase in the conversion of H3K4me1 to H3K4me3, when LDL1 and LDL2 were not present (Figure 6 and 7).

    Reviewer #1 (Significance (Required)):

    The manuscript provide some evidences how LDL1 involve in flowering through epigenetic regulation.

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

    Mahima and colleagues investigated LDL1/LDL2-MAF4/MAF5 in Arabidopsis flowering time control. The manuscript contains some interesting observations. To my point of view, however, the data need to be consolidated to support conclusions drawn in the manuscript.

    • Title: it does not correctly reflect the manuscript content. Data in relation with FVE were limited to Fig 6, where the data themselves appear preliminary.

    Response: We agree with the reviewers that our title didn’t reflect the manuscript content precisely and are happy to take this criticism into consideration. We have revised the title to, “LDL1 and LDL2 affect the dynamics of H3K4 methylation on the chromatin of MAF4 and MAF5 to allow floral transition in Arabidopsis”. Additionally, have provided the quantification data for fvec, ldlfvec and *ldl2fvec *with respect to WT, ldl1 and ldl2 plants (Figure 9)

    • Abstract: most conclusions are over-stated. The current data shown in the manuscript cannot support such strong conclusions.

    Response: We have rigorously revised the abstract and toned down the overstated conclusions

    • Introduction: It is necessary to make clear that the role of the LDL1 and LDL2 genes in flowering time control had been well established in previous studies, including their repression of transcription of FLC, MAF4 and MAF5 (Berr et al., 2015, Plant J 81:316).

    Response: We have revised the introduction to include the previously known roles of LDL1 and LDL2 in regulating flowering time.

    • Results:

    Regarding LDL1-overexpression lines, 'Relative expression' in Supplementary Fig 2B referred to normalization to WT? The phenotype of plants needs to be shown.

    Response: Yes, the level of upregulation of LDL1 expression in different T1 plants (after selection from Hygromycin) was calculated with respect to the WT.

    Regarding flowering time, have the observation and measures been performed in the same experiments for the ldl1, ldl1 flc, ldl1 maf4 and ldl1 maf5 mutants (Fig 3 and Supplementary Fig 1)? The late-flowering phenotype of ldl1 shown in Fig 3D-F is much severe than the same mutant shown in the other Figs, any explanation? What's the interpretation that ldl1 is epistatic to flc, maf4 and maf5?

    Response: We agree with the reviewer’s observation which is correct. The following quantifications were taken at various points during the study:

    flc, ldl1, and ldlflc (Supplementary Figure 1)

    WT, ldl1, and ldl1maf4 (Figure 3A, 3B and 3C)

    WT, ldl1, and ldl1maf5 (Figure 3D, 3E and 3F)

    The rosette leaf numbers and flowering time of the plants in Figure 3D-3F are more severe than the others because seeds were directly sprinkled onto the soil in this phenotyping, whereas in previous phenotypings, plants were grown on 1/2MS plates before being transferred to soil. However, all the components of a single experiment were grown in the same condition. We appreciate your observation, the present data does suggest ldl1 being epistatic to flc, maf4 and maf5.

    The in vitro test of LDL1 for its enzyme activity (Fig 4) appears preliminary and fragmented. The quantification data in Fig 4C-D need repeats. Have other histone methylation types (e.g. H3K4me3, H3K27me3, H3K36me3) been tested? The only two types (H3K4me2 and H3K9me2) shown are both down-regulated by LDL1-GST. Can H3K9 demethylation also play a role in flowering time control? In any case, the current in vitro data only are not sufficient to draw the strong conclusions as those appeared in the manuscripts.

    Response: Before concluding that LDL1 has H3Kme2 and H3K9me2 demethylase activity, we confirmed it several times__. __Please refer to the PDF file for “response to reviewers” for supporting data.

    We analyzed the western band intensity by calculating the area under the curve with imageJ software, which varies between experiments depending on the band intensities, therefore, rather than plotting absolute values of band intensity, we plotted the ratio of LDL1-GST/GST from three independent experiments in Figure 4B. We did perform a preliminary experiment to see if LDL1 has demethylation activity against different methylation marks, such as H3k4me1, me3, H3K9me1, and me3 (1=GST, 2=LDL1-GST), but there was no significant change in the methylation marks in the presence of LDL1. Please refer to the PDF file for “response to reviewers” for supporting data.

    H3K9 is a repressive chromatin mark, and its removal would suggest gene activation. Upregulation of FLC, MAF4, and MAF5 in ldl1 and ldl2 mutant suggests LDL1 and LDL2 removes H3k4me2 methylation marks during flowering. However, JMJ28, Jumonji C (JmjC) domain-containing histone demethylase have been shown to positively regulate flowering by removing repressive H3K9me2 marks from the chromatin marks from the chromatin of CONSTANS (CO)5.

    In the manuscript, it is saying that LDL1 binds on the chromatin of MAF4 and MAF5. However, I cannot find any data shown to support this conclusion.

    Response: We would like to refer to Figure 2A and B where we have provided this information.

    Protein-protein interactions, e.g. LDL1/LDL2-FVE in Fig 6A and LDL1-LDL2 and LDL1-HDA5 in Supplementary Fig 5, are examined in yeast two-hybrid assay. Other independent assays would be required.

    Response: We have confirmed the interaction of LDL1 and LDL2 with FVE using co-immunoprecipitation assay (Figure 8B). Since Co-IP is a confirmatory experiment, we have done it for positive interactions found through Y2H only. Moreover, in the current manuscript our focus has not been on HDA5, so we didn’t proceed with further experiments.

    The study of genetic interaction between fve and ldl1/ldl2 (Fig 6B-D) looks very preliminary. It is unclear how ldl1 fve and ldl2 fve were obtained: by crosses or by CRISPR-Cas9 using ldl1 and ldl2? The phenotypes need more investigations and some molecular data regarding flowering regulatory genes (e.g. MAF4/5) are necessary. In any case, the current title and the related conclusions drawn in the manuscript are over-stated.

    Response: We performed the quantification of the genetic interaction between fve and ldl1/ldl2. The binary vector pHSE401-FVE was transformed in ldl1 and ldl2 to produce ldl1fvec and ldl2fvec, respectively. We previously mentioned it in the material methods, but we have now updated it in the results section to avoid confusion.

    Following the suggestions, we have scored the phenotype (Figure 9) and checked the expression of flowering regulatory genes (Supplementary Figure 10C).

    Fig 7 showed data about MAF5-FLC, MAF5-SVP and MAF5-MAF5 interactions in yeast two-hybrid and about transcriptional repressor activity assay in tobacco leaves using the LUC-reporter. Again, the data need to be confirmed and reproducibility of experiments need to be shown. In addition to proFT:LUC, it is also necessary to have an internal normalization reference construct. Anyway, currently it is far away to allow a strong conclusion such as drawn in the manuscript that MAF5 interacts with FLC and SVP and repress FT to delay floral transition. Response: We have confirmed the interaction of MAF5-FLC, MAF5-SVP and MAF5-MAF5 using co-immunoprecipitation (Figure 10B). We quantified the firefly luciferase activity under proFT using renilla luciferase under pro35s as an internal control and the ratio of LUC/REN represented the promoter activity of FT promoter (Figure 10C).

    Reviewer #2 (Significance (Required)):

    Topic is interesting, but data are poor to support the conculsions drawn.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis Summary This work study the role on flowering time of LDL1 and LDL2, two Arabidopsis homologs of the histone demethylase LSD1. Although this phenotype was previously described, the authors explore if LDL1 and LDL2 regulate other genes in addition to the floral repressor FLC. In fact, mRNA expression experiments and genetic analyse suggest that LDL1 modules flowering regulating the expression of MAF4 and MAF5, two FLC-like genes that has been less characterized. The also provide some in vitro biochemical evidence of the demethylase activity of LDL1 protein and yeast-two-hybrid data showing the interaction with FVE, another chromatin regulator involved in flowering time.

    Major comments

    1. Lines 116-117. Please rephrase these lines and remove panels C, D and E from figure 1 (these could be supplementary material). The flowering time phenotype of MAF4 and MAF5 in Col background is very well documented and was described before, see Gu et al Nat. Comm., 2013 (10.1038/ncomms2947) and Kim et al. Plant Cell, 2013 (10.1105/tpc.112.104760)

    Response: As per the suggestion, we have modified the discussion and moved the panels 1C, 1D and 1E to the supplementary.

    Lines 128-130 and Fig Sup3. The proLDL1:LDL1-GUS cannot be described as fully functional because its flowering time and LDL1 mRNA expression levels has not been compared to the wild-type plant. The line flowers earlier that the ldl1 mutant but it may only partially complement the flowering phenotype.

    Response: We have provided additional experiment that the transgene is functional in proLDL1:LDL1-GUS (ldl1) with respect to the WT plants (Supplementary Figure 5A).

    Line 135 and Figure 2. How the Chip data was normalized? What are you comparting in your statistical significance tests? Only two regions of each gene were analysed; to assess the binding of LDL1 to MAF4 and MAF5 loci more regions must be analysed.

    Response: Normalization of the ChIP data and significance of enrichment of LDL1 was calculated with respect to the fold enrichment in the empty vector control (EV (ldl1)) plants. We only examined the promoter and exon1 of MAF4 and MAF5 for LDL1 enrichment because Hung et al,2019's6 study demonstrated that LDL1 is enriched on the promoter and exon1 of the downstream protein coding genes. However, to check for methylation marks during flowering, we have employed different primer sets on various positions between the promoter and exon1 on MAF4 and MAF5 chromatin.

    Figures 6C and 6D. The genetic analysis of ldl mutant with fve-c line is prelaminar and incomplete. The epistasis cannot be evaluated as no quantitative flowering time data is provided. A questionable picture of one lonely plant cannot sustain the conclusions of lines 207-208.

    Response: We have modified the picture and quantified the flowering time data to show genetic interaction of ldl1 and ldl2 with fvec mutant plants (Figure 9).

    METODS. Please clarify the used mutant alleles for LDL1 LDL2, MAF4, MAF5 and FLC; if they has been previously described; if they are full knock-outs; and, consequently, use the appropriated allele name across the manuscript.

    Response: As per the suggestion, we have clarified the different mutant alleles used in the study.

    Minor points:

    1. I think the title does not describe the work - the interaction with FVE is very relevant but it is not the central theme of the article.

    Response: We have changed the title of the study to “LDL1 and LDL2 affect the dynamics of H3K4 methylation on the chromatin of MAF4 and MAF5 to allow floral transition in Arabidopsis”.

    It would be very informative to have short-day flowering tome data of the genetic combinations of ldl mutants with flc, maf4 and maf5 mutations.

    Response: We absolutely agree that elaborate SD experiment may open interesting avenue for LDL1 mediated regulation of flowering, which might be good for future studies. However, ldl1ldl2 shows late flowering, while maf4 and maf5 exhibit the early flowering phenotype irrespective of the day length7,8.

    I found the Discussion section rather too long.

    Response: We have shortened the discussion to make it more focused.

    Reviewer #3 (Significance (Required)):

    Although it is clear that LDL proteins regulate MAF4 and MAF 5. I found that the manuscript lacks of a general overview of flowering time regulation. At the end, it is not clear how LDL proteins regulate flowering time because they regulate FLC, FWA, MAF4 and MAF5: What is more important? Which is the main role of each protein? Are they reductant or do they have specialized functions? In a nut shell, this study is an interesting piece of work for the flowering time field: However, in my opinion, some of the presented data are redundant with previous works and the manuscript may not be relevant for a general audience.

    1. Yu, C.-W. et al. HISTONE DEACETYLASE6 Interacts with FLOWERING LOCUS D and Regulates Flowering in Arabidopsis. Plant Physiology 156, 173-184 (2011).
    2. Luo, M. et al. Regulation of flowering time by the histone deacetylase HDA 5 in A rabidopsis. The Plant Journal 82, 925-936 (2015).
    3. Yu, C.-W., Chang, K.-Y. & Wu, K. Genome-wide analysis of gene regulatory networks of the FVE-HDA6-FLD complex in Arabidopsis. Frontiers in plant science 7, 555 (2016).
    4. Hung, F.-Y. et al. The Arabidopsis LDL1/2-HDA6 histone modification complex is functionally associated with CCA1/LHY in regulation of circadian clock genes. Nucleic acids research 46, 10669-10681 (2018).
    5. Hung, F.-Y. et al. The Arabidopsis histone demethylase JMJ28 regulates CONSTANS by interacting with FBH transcription factors. The Plant Cell 33, 1196-1211 (2021).
    6. Hung, F.-Y. et al. The expression of long non-coding RNAs is associated with H3Ac and H3K4me2 changes regulated by the HDA6-LDL1/2 histone modification complex in Arabidopsis. NAR Genomics and Bioinformatics 2 (2020). 7 Berr, A. et al. The trx G family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway. The Plant Journal 81, 316-328 (2015).

    8 Kim, D.-H. and Sibum, S. Coordination of the vernalization response through a VIN3 and

            FLC gene family regulatory network in Arabidopsis. *The Plant Cell *__25, __454-469 (2013)
  2. 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 #3

    Evidence, reproducibility and clarity

    LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis

    Summary

    This work study the role on flowering time of LDL1 and LDL2, two Arabidopsis homologs of the histone demethylase LSD1. Although this phenotype was previously described, the authors explore if LDL1 and LDL2 regulate other genes in addition to the floral repressor FLC. In fact, mRNA expression experiments and genetic analyse suggest that LDL1 modules flowering regulating the expression of MAF4 and MAF5, two FLC-like genes that has been less characterized. The also provide some in vitro biochemical evidence of the demethylase activity of LDL1 protein and yeast-two-hybrid data showing the interaction with FVE, another chromatin regulator involved in flowering time.

    Major comments

    1. Lines 116-117. Please rephrase these lines and remove panels C, D and E from figure 1 (these could be supplementary material). The flowering time phenotype of MAF4 and MAF5 in Col background is very well documented and was described before, see Gu et al Nat. Comm., 2013 (10.1038/ncomms2947) and Kim et al. Plant Cell, 2013 (10.1105/tpc.112.104760)
    2. Lines 128-130 and Fig Sup3. The proLDL1:LDL1-GUS cannot be described as fully functional because its flowering time and LDL1 mRNA expression levels has not been compared to the wild-type plant. The line flowers earlier that the ldl1 mutant but it may only partially complement the flowering phenotype.
    3. Line 135 and Figure 2. How the Chip data was normalized? What are you comparting in your statistical significance tests? Only two regions of each gene were analysed; to assess the binding of LDL1 to MAF4 and MAF5 loci more regions must be analysed.
    4. Figures 6C and 6D. The genetic analysis of ldl mutant with fve-c line is prelaminar and incomplete. The epistasis cannot be evaluated as no quantitative flowering time data is provided. A questionable picture of one lonely plant cannot sustain the conclusions of lines 207-208.
    5. METODS. Please clarify the used mutant alleles for LDL1 LDL2, MAF4, MAF5 and FLC; if they has been previously described; if they are full knock-outs; and, consequently, use the appropriated allele name across the manuscript.

    Minor points:

    1. I think the tittle does not describe the work - the interaction with FVE is very relevant but it is not the central theme of the article.
    2. It would be very informative to have short-day flowering tome data of the genetic combinations of ldl mutants with flc, maf4 and maf5 mutations.
    3. I found the Discussion section rather too long.

    Significance

    Although it is clear that LDL proteins regulate MAF4 and MAF 5. I found that the manuscript lacks of a general overview of flowering time regulation. At the end, it is not clear how LDL proteins regulate flowering time because they regulate FLC, FWA, MAF4 and MAF5: What is more important? Which is the main role of each protein? Are they reductant or do they have specialized functions?

    In a nut shell, this study is an interesting piece of work for the flowering time field: However, in my opinion, some of the presented data are redundant with previous works and the manuscript may not be relevant for a general audience.

  3. 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

    Mahima and colleagues investigated LDL1/LDL2-MAF4/MAF5 in Arabidopsis flowering time control. The manuscript contains some interesting observations. To my point of view, however, the data need to be consolidated to support conclusions drawn in the manuscript.

    • Title: it does not correctly reflect the manuscript content. Data in relation with FVE were limited to Fig 6, where the data themselves appear preliminary.
    • Abstract: most conclusions are over-stated. The current data shown in the manuscript cannot support such strong conclusions.
    • Introduction: It is necessary to make clear that the role of the LDL1 and LDL2 genes in flowering time control had been well established in previous studies, including their repression of transcription of FLC, MAF4 and MAF5 (Berr et al., 2015, Plant J 81:316).
    • Results:

    Regarding LDL1-overexpression lines, 'Relative expression' in Supplementary Fig 2B referred to normalization to WT? The phenotype of plants needs to be shown.

    Regarding flowering time, have the observation and measures been performed in the same experiments for the ldl1, ldl1 flc, ldl1 maf4 and ldl1 maf5 mutants (Fig 3 and Supplementary Fig 1)? The late-flowering phenotype of ldl1 shown in Fig 3D-F is much severe than the same mutant shown in the other Figs, any explanation? What's the interpretation that ldl1 is epistatic to flc, maf4 and maf5?

    The in vitro test of LDL1 for its enzyme activity (Fig 4) appears preliminary and fragmented. The quantification data in Fig 4C-D need repeats. Have other histone methylation types (e.g. H3K4me3, H3K27me3, H3K36me3) been tested? The only two types (H3K4me2 and H3K9me2) shown are both down-regulated by LDL1-GST. Can H3K9 demethylation also play a role in flowering time control? In any case, the current in vitro data only are not sufficient to draw the strong conclusions as those appeared in the manuscripts.

    In the manuscript, it is saying that LDL1 binds on the chromatin of MAF4 and MAF5. However, I cannot find any data shown to support this conclusion.

    Protein-protein interactions, e.g. LDL1/LDL2-FVE in Fig 6A and LDL1-LDL2 and LDL1-HDA5 in Supplementary Fig 5, are examined in yeast two-hybrid assay. Other independent assays would be required.

    The study of genetic interaction between fve and ldl1/ldl2 (Fig 6B-D) looks very preliminary. It is unclear how ldl1 fve and ldl2 fve were obtained: by crosses or by CRISPR-Cas9 using ldl1 and ldl2? The phenotypes need more investigations and some molecular data regarding flowering regulatory genes (e.g. MAF4/5) are necessary. In any case, the current title and the related conclusions drawn in the manuscript are over-stated.

    Fig 7 showed data about MAF5-FLC, MAF5-SVP and MAF5-MAF5 interactions in yeast two-hybrid and about transcriptional repressor activity assay in tobacco leaves using the LUC-reporter. Again, the data need to be confirmed and reproducibility of experiments need to be shown. In addition to proFT:LUC, it is also necessary to have an internal normalization reference construct. Anyway, currently it is far away to allow a strong conclusion such as drawn in the manuscript that MAF5 interacts with FLC and SVP and repress FT to delay floral transition.

    Significance

    Topic is interesting, but data are poor to support the conculsions drawn.

  4. 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

    The manuscript entitled "LDL1 and LDL2 histone demethylases interact with FVE to regulate flowering in Arabidopsis" characterized that LDL1 regulates flowering by binding on the chromatin of MAF4 and MAF5 to repress their expression. Further the authors proposed LDL1/LDL2-FVE model. Here are some comments for this manuscript.

    Major problems:

    1. This experiment is still not testing or showing/concluding that the whole complex forms on the MAF4 and MAF5
    2. It is not shown LDL1/LDL2 repress MAF4 and MAF5 by removing H3K4me2 activity. It would be useful to test whether the methylation level of MAF4 and MAF5 has been altered in ldl1/ldl2 mutant
    3. I suggest that further research is required to provide conclusive evidence concerning the physiology function of LDL1/LDL2-FVE. Such as the expression pattern of LDL1/LDL2, the methylation level of MAF4 and MAF5 before or after floral transition

    Significance

    The manuscript provide some evidences how LDL1 involve in flowering through epigenetic regulation.

  5. Version published to 10.1101/2022.05.12.491658v1 on bioRxiv