A trans- acting long non-coding RNA represses flowering in Arabidopsis

  1. Yu Jin
  2. Maxim Ivanov
  3. Anna Nelson Dittrich
  4. Andrew D. L. Nelson
  5. Sebastian Marquardt

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Eukaryotic genomes give rise to thousands of long non-coding RNAs (lncRNAs), yet the purpose of lncRNAs remains largely enigmatic. Functional characterization of lncRNAs is challenging due to multiple orthogonal hypothesis for molecular activities of lncRNA loci. Here, we identified a fl owering a ssociated i ntergenic l ncRNA ( FLAIL ) that represses flowering in Arabidopsis . An allelic series of flail loss-of-function mutants generated by CRISPR/Cas9 and T-DNA mutagenesis showed an early flowering phenotype. Gene expression analyses in flail mutants revealed differentially expressed genes linked to the regulation of flowering. A genomic rescue fragment of FLAIL introduced in flail mutants complemented gene expression defects and early flowering, consistent with trans- acting effects of the FLAIL RNA. Knock-down of FLAIL RNA levels using the artificial microRNA approach revealed an early flowering phenotype shared with genomic mutations, indicating a trans- acting role of FLAIL RNA in the repression of flowering time. Genome-wide detection of FLAIL -DNA interactions by ChIRP-seq suggested that FLAIL may directly bind genomic regions. FLAIL bound to genes involved in regulation of flowering that were differentially expressed in flail , consistent with the interpretation of FLAIL as a trans- acting lncRNA directly shaping gene expression. Our findings highlight FLAIL as a trans- acting lncRNA that affects flowering in Arabidopsis , likely through mediating transcriptional regulation of genes directly bound by FLAIL .

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Note: Figures we made to respond to the referee comments appear to be not supported by the ReviewCommons system.

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

    **Summary:**

    The authors characterized a new lncRNA locus named FLAIL that controls flowering time in Arabidopsis thaliana. The functional validation of this locus is strongly supported by the use of several different tools (CRISPR-Cas9 deletions, T-DNA insertion, amiRNA gene silencing, and transgene complementation of KO lines). It is also suggested that FLAIL lncRNA works in trans but not in cis. There are strong observations supporting that FLAIL works in trans.

    Moreover, it is suggested that FLAIL regulates gene expression by interacting with distant chromatin loci. This was assessed using RNA-Seq and ChIRP-Seq. Yet, the overlap between DEGs in the flail mutant and FLAIL binding sites at the chromatin is very small, with only 12 genes. From those, only 2 flowering genes' expression was rescued by FLAIL transgene complementation. The final conclusion that FLAIL lncRNA represses flowering by direct inhibition of the 2 flowering genes expression is correlative, and lacks genetic validation.

    __#1.1 We plan to support the conclusions in the manuscript genetically as the reviewer suggests. We started these experiments yet they will require the timeframe of the full revision. __

    In addition inspection of the supplementary file shows that the ChIRP analysis was done without filtering for the FDR so that some of the positive hits have an FDR of 0,232.

    #1.2 We strengthened the manuscript by implementing and FDR filter of ChIRP-seq results. The distribution of FLAIL binding sites in Fig. S7B and Table S4, and overlapping numbers between DEGs and FLAIL-ChIRP in Fig. S8A were correspondingly updated.

    In addition, many of the peaks land in intergenic regions with is not mentioned in the text a graph with the position of the peaks in respect to nearby genes would help.

    #1.3 Thank you for the suggestion, we strengthened the manuscript with the requested analysis. We implemented the FDR filter, then we used "tssRegion" in ChIPseeker to set distance to the nearest TSS as (-1000, 1000), then most peaks were located in promoter regions (67.24%) and in intergenic regions with 16.38%. Since many papers present the position of the peaks by ChIPseeker (PMID: 32338596, PMID: 28221134, PMID: 31081251, PMID: 32012197, PMID: 31649032, PMID: 32633672) we also applied a similar method to display a distribution of FLAIL binding loci relative to distance from the nearest TSS in Fig. S7C.

    In one sentence, the authors used the right model system and methodology, including advanced techniques, to characterize a new trans-acting lncRNA important for controlling the flowering time in Arabidopsis but lack evidence supporting a mechanism of action that goes beyond the interaction with several chromatin loci.

    **minor points:**

    line#63-64 the authors say the COLDAIR and ASL work on FLC in cis in my view the original papers suggested/showed they work in trans.

    #1.4 We increased precision by changing this sentence to ‘Vernalization-induced flowering associates with several lncRNAs such as ____COOLAIR____, COLDAIR____, ANTISENSE LONG (ASL), and COLDWRAP____ that in cis or in trans locally repress gene expression of FLOWERING LOCUS C (FLC), a key flowering repressor at different stages of vernalization’____.

    Fig 1B please add some more protein-coding RNAs for the bio-info analysis for comparison

    __#1.5 ____done. __

    Order of Supplementary Fig citation is mixed with S2 coming before S1B

    #1.6 Thank you, we ordered all figures by appearance in the text. __

    __

    It would help the reader to have a schematic of the crisper deletions, T-DNA insertion, and position of primers used for the RT-qPCR.

    __#1.7 We enhanced our presentation of Fig. 1A. It shows a schematic of them as well as positions of primers. __

    In the supplementary PDF file, some of the text is missing on page 3 beginning and end of lines.

    #1.8 we ensured all text in new submission.

    Reviewer #1 (Significance (Required)):

    The use of several different tools to validate the biological function of FLAIL locus is a major strength of this work.

    The authors propose that flowering time and its gene regulation are controlled by sense FLAIL lncRNAs. However, the sense transcription of FLAIL locus is not detected in wild-type plants by TSS-Seq, TIF-Seq, or plaNET-Seq.

    #1.9.1 There appears to be some confusions. Transcription of sense* FLAIL* can be observed in chr-DRS, TSS-seq, TIF-seq in wild type and even in plaNET-seq in NRPB2-FLAG nrpb2-1 plant. We enhanced presentation of Fig. 1 and provided a more clear description in Line 81-99.

    If the authors would have explored further the expression of FLAIL transcripts in different stages of development (vegetative and non-vegetative) and in response to different conditions, it would make their claims on the function of FLAIL lncRNAs more convincing. Additionally, flail mutants could have been obtained in the hen-2 background, since it's there where we can observe FLAIL transcription.

    __#1.9.2 Thank you for the suggestion. We included additional analyses in ____Fig. S2 for *FLAIL *transcription level in different tissues and different abiotic stress conditions base on 20,000 publicly available RNA-seq libraries (PMID: 32768600). Although many libraries are non-stranded, this analysis determined that sense FLAIL or total FLAIL (including sense and antisense) is broadly expressed over many tissues and induced in response to many abiotic stresses (Fig. S2A-B), therefore suggesting that FLAIL may be needed broadly in Arabidopsis. __

    FLAIL locus lays on the proximal promoter region of PORCUPINE (PCP), an important regulator of plant development. As flail mutants, pcp mutants display an early flowering phenotype. The authors show no link between FLAIL and PCP from the overlap between re-analysis of published RNA-Seq data for pcp and RNA-Seq and ChIRP-Seq from the authors. This analysis is not enough to exclude the involvement of PCP from the FLAIL function. PCP expression using RT-qPCR should be performed in flail mutants to further support that FLAIL works independently from PCP.

    __#1.10 We strengthened this conclusion by adding the requested experiment. PCP transcription level in flail3 mutant was provided by RT-qPCR and RNA-seq in Fig. S11A-B. __

    This work does not hypothesize any molecular mechanism besides the interaction of FLAIL lncRNAs with several chromatin loci. It was recently proposed in Arabidopsis that a trans-acting lncRNA interacts with distant loci via the formation of R-loops. The authors do not comment on that. This work would benefit in correlating FLAIL binding sites with R-loop-forming regions mapped in Arabidopsis, regardless of the results from this analysis. Additionally, the authors could attempt to look for a motif responsible for FLAIL binding.

    Check R-loop forming data R-loops (Santos-Pereira and Aguilera, 2015) in Arabidopsis, determined by DRIP-seq (Xu et al., 2017).

    __#1.11 Thanks very much for this excellent suggestions. __

    First, we searched for a consensus DNA motif on FLAIL binding regions by Homer. We determined four commonly enriched DNA sequence motifs among FLAIL target genes (Fig. 4G). Notably, the target genes CIR1 and LAC8 contained consensus sequences that matched to all FLAIL binding motifs (Fig. 4G). These data are consistent with a model where FLAIL binds DNA targets through a sequence complementary mechanism. Functionally important sequences are frequently conserved among evolutionarily distant species, we observed three motifs that appeared to cross-species conserved (Fig. S9), suggesting a potential evolutionarily constrained role.

    __Second, we indeed identified R-loops peaks on several of FLAIL binding sites by DRIP-seq (Xu et al., 2017). For example, we observed R-loop formation over three FLAIL binding motifs at CIR1 locus and one at *LAC8 *(Fig. R1), indicating that R-loop formation may also be a factor determining FLAIL binding. Even though R-loop peaks are present at several FLAIL targets, full elucidation if R-loop formation determines FLAIL targeting requires further experimental evidence is beyond the scope of the current manuscript. __

    Fig. R1 Representative tracks at LAC8 and CIR1 showing R-loop formation by DRIP-seq on Watson strand (w-R loops), Crick strand (c-R loops). Undetectable R-loops after RNAse-H treatment was shown as negative control. Four conserved sequence regions of FLAIL binding motifs were indicated by red arrows at LAC8 and CIR1 loci. Gene annotation was shown at the bottom.

    Most of the key conclusions are convincing, except for the flowering time control directly through CIR1 and LAC8, which should be mentioned as speculative

    __ ____#1.12____ Thank you for finding most key conclusions convincing. We plan strengthen the manuscript with additional genetic evidence to as part of the full revision.__

    The words locus and loci are latin and they should be written in italic. The word Brassicaceae, referring to the family should be in italic, and should not be "Brassicaceaes". The word analysis has the wrong spelling.

    __#1.13 We follow conventions given in Scientific Style and Format: The CBE Manual for Authors, Editors and Publishers (1994) Cambridge University Press, Cambridge, UK, 6th edn. The words locus and loci are common Latin terms and should not be italicized. However, should the format of the final prefer these words in italics we will change it later. We improved consistency of using italics. “Brassicaceaes” was changed to “Brassicaceae”. __

    "How much time do you estimate the authors will need to complete the suggested revisions: this is difficult to answer as it depends to which level the author would like to take their work. In my view, if all new experiments would have to be started from scratch it is too far away to be estimated.

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

    In this ms, the authors identified the FLAIL lncRNA that represses flowering in Arabidopsis from a locus producing sense and antisense transcripts. They use an allelic series involving T-DNA insertions, CRISPR/Cas9 and artificial miRNAs to study the role of FLAIL in flowering. A complementation series of constructs of the flail3 allele allowed them to show that the sense FLAIL lncRNA can act in trans. RNAseq revealed a small group of genes linked to the regulation of flowering whose expression is affected in the mutant and restored in the complementation line. To gain further insight into FLAIL function, the authors used a ChIRPeq approach to test whether the lncRNA can recognize potential target genes along the genome and they could show that FLAIL binds specific genomic regions. Clearly, this paper shows very nice evidence that the FLAIL lncRNA can act in trans to regulate gene expression. Nevertheless, there are certain points that need to be clarified to further support the action of the sense FLAIL transcript.

    1.According to Fig. 1 A, the antisense FLAIL is "internal" to the DNA genomic area spanning the sense FLAIL. Hence, with direct RT-qPCR is very difficult to distinguish between these molecules as a minor "RT" activity of the Taq polymerase may lead to detection of low levels of antisense, idem if RDRs may generate low antisense levels. Although I think that the plaNET seq brings strong evidence about the start and ends of these molecules, to measure them by RT-qPCR is not trivial and requires the use of strand-specific RT-PCR using a 5' extension of the oligo and amplification with one oligo of the FLAIL sequence (sense or antisense) and the added oligo.

    #2.1.1 Thanks for this good suggestion. We tested both sense and antisense FLAIL transcription using oligo linked gene specific reverse primers for RT and a pair of the linked oligo and gene specific forward primer for qPCR. Primer locations were shown in Fig. 1A and new data were in Fig. 1C-D, Fig. 2B-C, and Fig. S4B-C.

    It is not clear how they could distinguish precisely sense and antisense particularly when both RNAs correlate as it is the case here in all alleles (Fig. 1 C and 1D). This should be more explicitly mentioned in the materials and methods section.

    #2.1.2 We gave a description of strand specific RT-qPCR method in detail in Line 397-402.

    2.In Fig. 2, what are the levels of antisense in the complementing lines with the sense transcript? And reciprocally sense levels in antisense constructs?

    #2.2 We added this data in Fig. 2B-C and described in Line 136-143. We indeed observed that sense FLAIL transcripts in the transformed asFLAIL construct or *asFLAIL *transcripts in the transformed sense FLAIL construct was similar to the control *35S:GUS *(Fig. 2B-C), validating that NOS terminator inhibits antisense transcripts. We also noted that the transformed 35S:GUS and sense FLAIL construct expressed higher asFLAIL compared to the flail3 mutant (Fig. 2C). This may be caused by a T-DNA insertion of the resulting transgenic plants.

    This will definitively demonstrate the assumption that the T-NOS termination will not allow any expression on the other strand. At present, only one of the lncRNAs is measured in each experiment?

    __#2.3 We appreciate the next-level reflection of this reviewer, with so many regions initiating cryptic antisense transcription it is an interesting challenge to identify a 3´- terminator that initiates no or poor antisense transcription. __

    __First, previous published data argue that the NOS terminator is largely abolishing initiation of antisense transcription (PMID: 33985972, PMID: 30385760, PMID: 27856735). All these studies address roles of antisense transcription by generating mutations abolishing antisense lncRNA transcription using the NOS terminator sequences. __

    Second, to satisfy the curiosity of this reviewer, we provide data below that from another manuscript of the lab in preparation. It’s a screenshot of plaNET-seq in fas2-4 NRPB2-FLAG nrpb2-1 mutant carrying a pROK2 construct. The pROK2 T-DNA coincidentally carries a NOS terminator. We mapped plaNET-seq reads to the pROK2 scaffold to display the reads. In pROK2, a NOS promoter activates NPTII expression (red) with NOS terminator as a terminator sequence. No antisense transcription (blue) is detectable by this sensitive method to detect nascent transcripts. Taken together, the selection of the NOS terminator as a region suppressing initiation of antisense transcription represents a valid choice.

    Fig. R2 Genome browser screenshot of plaNET-seq at NPTII locus of pROK2 T-DNA vector in fas2-4 NRBP2-FLAG nrpb2-1 mutant. This mutant carries a pROK2 construct, in which a NOS promoter activates NPTII expression with NOS terminator a terminator sequence. Sense strand was shown in red and antisense strand in blue. pROK2 annotation was shown at the bottom.

    3.In Fig. 3, it will be important to also show the FLAIL locus in the flail3 mutants (in comparison to the wt) as well as the transgene locus. Here the reads will be strand specific and furthermore this will allow to show that the transgene is not generating antisense transcripts (through RDRs for gene silencing?) and confirm that the sense FLAIL is required for the complementation.

    __#2.4 Thank you very much for this suggestion. NGS reads for endogenous FLAIL and transgenic FLAIL both map to the FLAIL locus, so we show the FLAIL locus in Fig 3B. This representation shows that sense FLAIL transcripts were significantly reduced in flail3 and rescued in complementation line comparing to wild type. These data argue against the idea of gene silencing and linked antisense production from the transgene. However, RNA-seq suggests that an isoform of asFLAIL appears to accumulate in flail3. Since we fail to identify this accumulation by strand specific RT-qPCR result in flail3 and in CRISPR-deletion lines, this may be an asFLAIL isoform resulting from the T-DNA insertion. __

    4.In Fig. S5, the expression of FLAIL is shown in the artificial miRNA lines. Is the antisense FLAIL affected "indirectly" by the cleavage of the amiRNA or remains constant? This is likely the case but should be shown.

    #2.5 We added this result in Fig. S4C and expression level of asFLAIL remains constant compared to the transformed empty vector control.

    5.The ChIRPseq data adds major novelty to the ms and brings new ideas about the way of action of FLAIL. However, are there any common epigenetic states between ChIRP targets (e.g. histone modifications, antisense RNA production, homologies "detected" in the conserved regions between Camelina and Arabidopsis and the target loci? Or others) that may highlight potential mechanisms leading to repression mediated by FLAIL of these loci? There are many databases that could be explored (even during flowering) to search for potential relationships. Although precise description of the mechanism is out of the scope of this ms, this can be discussed in more detail to further expand on the nice data obtained.

    #2.6 We searched for a consensus DNA motif on FLAIL binding regions by Homer. We determined four commonly enriched DNA sequence motifs in target genes. Notably, the target genes *CIR1 and LAC8 *contained consensus sequences that matched to all FLAIL binding motifs (Fig. 4G). These data are consistent with a model where FLAIL binds DNA targets through a sequence complementarity mechanism. Functionally important sequences are frequently conserved among evolutionarily distant species, we observed three motifs that appeared to cross-species conserved (Fig. S9), suggesting a potential evolutionarily constrained role.

    **Minor comments:**

    6.In Fig. S3, a global alignment between FLAIL and two loci in Arabidopsis and Camelina is sown. What is the extent of homology? How conserved is this sequence at nucleotide level (small or very long?) to support the conservation of this lncRNA. Are there potential structures conserved among these lncRNAs?

    __ #2.7 T____wo consensus regions of ____FLAIL____ sequences among eleven disparate Brassicaceae genomes were shown in Fig. S9. ____Camelina sativa____ shared 98-nucleotide____ conserved sequences with Arabidopsis thaliana. In the future, it will be interesting to explore evolutional conserved structures among Brassicaceae genomes. However, these analyses are beyond the scope of the current manuscript. __

    7.In Fig. S4B, arrows may help to understand which seeds were selected.

    __#2.8 Thanks. Arrows were included.____

    __

    Reviewer #2 (Significance (Required)):

    This paper is a very nice piece of work and demonstrate the action of a long non-coding RNA (lncRNA) in trans on specific targets involved in the regulation of a developmental process, flowering. There is growing evidences that the non-coding genome hides large number of lncRNAs and there is little detailed genetic support for the action of lncRNAs globally. In contrast to many descriptive papers in the field, this ms demonstrates genetically, through an allelic series and complementation experiments, that this lncRNA locus is involved in flowering regulation and that its sense lncRNA recognizes target loci genome-wide, bringing interesting perspectives on potential new mechanisms of transcriptional regulation mediated by non-coding RNAs.

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

    In the manuscript by Jin et al authors characterize the FLAIL DNA locus in Arabidopsis (using a wide array of publicly available datasets), which produces a set of sense and anti-sense lncRNAs.

    __While our work on the FLAIL manuscript was ongoing we published the manuscripts where we presented these novel genomics methods and related data to capture nascent transcription and cryptic isoforms. We shared most data with TAIR, so we are happy to hear that these data are considered publically available. __

    Authors determined that the sense FLAIL lncRNA (or a set of sense lncRNAs, which isn't fully clear from the way the data are presented) is involved in flowering time in Arabidopsis based on the fact that the several flail mutants lead to the early flowering phenotype and this flowering defect is complemented by transgenic FLAIL DNA, meaning that FLAIL lncRNA acts in trans.

    __A series of experiments lead us to conclude that the sense isoform of FLAIL is responsible for the effect. We improved the data representation and writing of the manuscript to enhance accessibility. __

    The T DNA flail3 - mutant results in expression changes (up or down) of 1221 genes, including twenty genes linked to flowering in various ways. Expression of a group of these flowering-related genes could be either fully (for eight genes) or partially (for five genes) rescued by transgenic FLAIL. Authors also conducted the ChIRP-seq to determine which genes are physically bound by FLAIL lncRNA genome-wide. It was found that 210 genes in the genome are bound by FLAIL lncRNA. Comparison of the dataset of differentially expressed genes in the T-DNA flail3 mutant with the ChIRP-seq dataset of genes that are bound by FLAIL lncRNA revealed the 12 overlapping genes.

    Among these twelve overlapping genes, four were found to be functionally connected to flowering with expression of these four genes being down in flail3 T-DNA mutant. Two out of these four genes were ruled out from being involved in the regulation of flowering by FLAIL. Authors conclude that the two other genes (Cir1 and Lac8) are responsible for the late flowering phenotype of flail mutants based on the three lines of evidence: (i) these genes expression is reduced in the flail mutant, (ii) FLAIL lncRNA directly interacts with these genes chromatin, (iii) the mutants of these genes were previously reported by others to display early flowering phenotypes too. While I find many of the findings reported in the manuscript very interesting, building a good foundation on which to expand the study and providing a very good leads for follow up experiments, I also have serious concerns about the manuscript in its current form.

    Most importantly, this reviewer doesn't think that the mechanism of FLAIL lncRNA action was convincingly demonstrated. The main question would be how FLAIL lncRNA works and this question wasn't fully answered. It is great that FLAIL lncRNA binds directly to the two flowering-related genes, but what does it mean? Does it change any chromatin context of these genes quantitatively or qualitatively to affect the transcription? Or does it bind any components of transcriptional machinery and thus controls the transcriptional output?

    #3.1 This manuscript addresses an important question in the field question: what is the evidence for functional elements in non-coding regions of genomes? Despite many efforts, convincing genetic support for these functions often remained limited. In addition to our strong genetic data, we provided new evidence that FLAIL recognizes targets with evolutionally conserved sequence motifs as part of the revision in Fig 4F and Fig. S9. Additionally, we plan to do ChIP-qPCR to identify histone modifications on FLAIL targets.

    Additionally, flail3 T-DNA mutant affects the expression of 1221 genes and FLAIL lncRNA physically interact with 210 genes, so how can authors be fully sure that FLAIL lncRNA has only direct effect on these two genes and doesn't also contribute to the regulation of the upstream to Cir1 and Lac8 genes or even components of the transcriptional machinery that regulate these genes?

    #3.2 We agree with this opinion. It is the reason why we felt stating this exact conclusion in our previous manuscript was justified. We improved accessibility of our manuscript in the revision, these clarify our model, that the trans-acting lncRNA sense* FLAIL *can interact with the chromatin regions of its target genes to directly or indirectly regulate gene expression changes involving flowering (Line 274).

    Theoretically, doing RNA-seq in the amiR-FLAIL sense lncRNA mutant might have a chance of reducing the number of affected DEGs, making it easier to analyze the FLAIL targets, even if the allele can't be used for complementation experiments.

    __#3.3 Thanks for this suggestion. We plan to confirm key gene expression changes using amiRNA-*FLAIL *in full revision. __

    Also, auhors totally neglect putting the Cir1 and Lac8 genes into the context of flowering regulation, but it is something that needs to be done.

    #3.4 ____We discussed roles of CIR1 and LAC8 in flowering regulation in Line 260-272. Flowering is fine-tuned to maximize reproductive success and seed production and by endogenous genetic cues and external environmental stimuli such as photoperiod. Nevertheless, many details of the flowering pathways and their integration remain to be investigated____. CIR1 is a circadian clock gene, induced by light and involved in a regulatory feedback loop that controls a subset of the circadian outputs and thus determines flowering time. Our GO analysis supports that a subset of DEGs are connected to the response to red or far red light that contains among other key flowering genes such as ____phytochrome interacting factor____* 4____ (PIF4*) and CONSTANS (CO)____. FLAIL also binds the chromatin region of LAC8. LAC8 is a laccase family member that mainly modulates phenylpropanoid pathway for lignin biosynthesis____. Similar to flail, lac8 mutants flower early. While intermediates in this pathway or dysregulation of lignin-related genes could promote flowering in plants, the molecular connections of reduced LAC8 expression to effects on flowering time will require further investigation.

    Lastly, the paper needs to be totally rewritten to be even properly evaluated. In its current state it reads like a very short draft.

    __#3.5 We reorganized the structure of manuscript, improved clarity and provided new mechanistic evidence in Fig. 4G and Fig. S9 to present a more complete manuscript. __

    The Abstract is weak, the Introduction is written in a such telegraphic style that it is barely readable, in many places there is no connections between sentences leading to the information appear to be presented as random, even if it isn't.

    #3.6- We strengthened the Abstract by providing new evidence and improved for the Introduction.

    The Results section is written rather rudimentary with information not being sufficiently provided to describe the results but rather scattered between the Results and Figure legends.

    #3.7 Thanks for your suggestions, we described each FLAIL length and all constructs in detail in Results, put a schematic of T-DNA and CRISPR mutants in Fig. 1A, moved comparative genomics data to the end of Results and ensured all figures in order.

    The Discussion is the best written part of the manuscript.

    Thanks for your appreciation of the Discussion.

    The Conclusion section carries no specific information and reads more like a little summary suitable for a review article rather than experimental paper.

    #3.8 We agree this opinion, this paragraph fits Discussion better and Conclusion was removed.

    Therefore, this reviewer thinks that regardless of how authors will choose to proceed with the current experimental version of the manuscript, it'd be in the authors' best interests to at least fully revise the paper before resubmitting anywhere. I'd also advise authors to seek professional editorial help specifically using an editor with the background in the plant sciences.

    Authors might also want to consider moving Fig.3 into the Suppl. as it doesn't carry much weight or significance and perhaps make existing figures more meaningful and comprehensive and by including a better diagram of the locus (e.g., Fig. S1), etc.

    __#3.9 We thank this helpful suggestion. Fig.3 represents the RNA-seq data. In combination with supporting data in the supplementary material, it gives an easy visual readout of the reproducibility of the findings in replicates of stranded RNA-seq. In a new submission, we moved it to Fig. S5B and highlighted 13 differentially expressed flowering genes as well as sense FLAIL in flail3 that were rescued in complementation line in Fig. 3A. Moreover, we gave screenshots of FLAIL itself and four flowering related FLAIL targets in RNA-seq with a clear schematic representation of each locus. We believe these revisions improve Fig. 3. __

    It's not practical to list all issues with the writing as the paper requires total re-writing, so I can just make a few suggestions without any specific order to help authors improve the paper:

    __We are happy to improve our manuscript with the help of the reviewers. We addressed all comments including from reviewer #3 with a constructive spirit. However, since colleagues and reviewers #1 and #2 found the manuscript comprehensible to the point where they could make expert-level comments that illustrate understanding of the manuscript, a total re-writing did not feel like the most constructive suggestion to improve the manuscript. __

    --There is no statement anywhere that states the goal of the study.

    __#3.10____ We stated the goal of the study in line 50-69 and we think this is a misunderstanding. We summarized three issues currently exist in characterization of functional lncRNA in the last sentence of the first three paragraphs in Background: 1 in Line 50, the broad range of candidate hypotheses by which lncRNA loci may play functional roles call for multiple approaches to distinguish alternative molecular mechanisms. 2 in Line 59, functional characterization of trans-acting lncRNAs remains a key knowledge gap to understand the regulatory contributions of the non-coding genome. 3 in Line 69, the contribution of trans-acting lncRNAs to the regulation of distant flowering genes is currently unclear. So in the last paragraph of the background, we claimed that our goals are to address these questions through characterization of functional FLAIL lncRNA in flowering repression using multiple genetic approaches and various genomic data. __

    --No rational is provided on why authors decided to examine this specific genomic locus.

    __#3.11 For several years, our lab studies the rules and roles of non-coding transcription. We characterized and are characterizing several loci with evidence of non-coding transcription in a range of species. Early experiments suggested that FLAIL functioned in flowering, this manuscript clarifies that the function is executed as trans-acting lncRNA of the sense FLAIL isoform. __

    --Typically, the significance is in studying the function of lncRNA or a group of lncRNAs produced from a genomic locus, I don't think I ever encountered the instances when it was exciting to study just a specific genomic locus. If the locus does indeed have any significance for initiating the study, it needs to be explained.

    #3.12 This study is remarkable in many aspects. We fully discuss key strengths in the discussion. First, we ____exhibit a trans-acting lncRNA FLAIL that represses flowering by promoting the expression of floral repressor genes as discussed in Line 247-281____; Second, in Line 284-306, we informed that this study provide a compelling model about how to apply ____series of convincing genetic data____ to functionally characterize lncRNA loci. Third, in Line 307-312, evolutionary conserved FLAIL sequences across species is key to characterize the functional ____microhomology in other ____Brassicaceae.

    --The locus can produce lncRNAs, but it can't harbor them.

    __#3.13 We clarified this confusion by enhancing ____presentation of Fig. 1 and providing a more clear description of each sequencing method and results in Line 81-99. Although we provided evidence that transcription of both sense and antisense FLAIL are more stable in hen2-2, they were clearly observed in chr-DRS in wild type and plaNET-seq in *NRBP2-FLAG nrpb2-1 *and sense FLAIL was even detected in TSS-seq and TIF-seq in wild type. __

    --No length of FLAIL lncRNAs or their range is provided in the first section of Results.

    #3.14 We gave the length of sense FLAIL in Line 82 and antisense FLAIL in Line 86.

    --On many occasions authors don't state rational for doing experiments, which leads to information often flowing as random.

    #3.15 we enhanced clarity of the rational for each experiment and made some connections between sentences to make more fluent. For example, in sentences in Line 99, Line 113, Line 126, Line 159, Line 183, Line 214, and Line 219.

    --What do authors mean by the subtitle "FLAIL characterizes a trans-acting lncRNA repressing flowering"? How can lncRNA FLAIL or FLAIL locus characterize lncRNA?

    #3.16 We changed it to “FLAIL represses flowering as trans-acting lncRNA” in Line 112.

    --Check all figures. E.g., Fig. 3B-E mentions only accession numbers for the genes.

    #3.17 The systematic gene IDs are a valid way to represent data, in particular for genomics data since it facilitates cross-comparisons. To make it more accessible we also show systematic names of each gene in Fig. 3A-F, Fig. S6 and Table S3.

    --It is not clear where exactly the T-DNA insertion is located relative to sense FLAIL in flail3 mutant (Fig. S4).

    #3.18 We moved the schematic to clarify this to revised Fig. 1A and the exact T-DNA insertion site is mentioned in the legend.

    --- What is the length of the complementing sense FLAIL lncRNA?

    #3.19 We now include the length of the complementing sense and antisense FLAILs in Line 351-352.

    --Check the description of each and every construct used and provide explanation for each in the Results. E.g., the pFLAIL:gFLAIL18/88 and pasFLAIL:gasFLAIL18/39 constructs aren't explained in Results, and can only be found in Fig. 2 legends.

    #3.20 We described each construct including pFLAIL:gFLAIL18/88 and pasFLAIL:gasFLAIL18/39 constructs in Line 133, amiR-FLAIL-11 and amiR-FLAIL-11 in Line 149.

    Reviewer #3 (Significance (Required)):

    Tens of thousands of lncRNAs have been identified in various eukaryotes, but their biological roles have been shown only for a small fraction of them, and the mechanisms of their action are delineated for only a very few of them. Most of the advances on the field of lncRNAs are reported in metazoan, while the field of lncRNAs in plants is lagging far behind in terms of knowledge about lncRNAs with assigned biological functions or lncRNAs with delineated mechanisms of action. From this point of view, this reviewer is always excited to see any new functional plant lncRNAs for which either biological or mechanistic functions have been determined, and deems the information on this subject significant. The manuscript's findings are potentially very interesting and present a decent body of work that lays a very solid groundwork for future experiments. My main concern about the manuscript's significance in its current form is the fact that no real solid mechanism of action for the described lncRNA or a set of lncRNAs (?) has been demonstrated. The best mechanistically studied lncRNAs in Arabidopsis are involved in the regulation of flowering time, particularly those that function in the vernalization flowering pathway and to lesser extent in autonomous pathway. The new FLAIL lncRNA or lncRNAs (?) described in this manuscript also appear to regulate the flowering time in Arabidopsis, however more experiments would be needed to provide a definite conclusion about how direct FLAIL's effect is and how exactly it functions. That unfortunately obviously diminishes the significance of the manuscript and makes it potentially interesting only to researches studying flowering in Arabidopsis and even then the manuscript results would be incomplete to make solid conclusion.

    Lots of functional phenotype have

    Additionally, the manuscript requires complete re-writing.

    We thank this reviewer for the appreciation of __a decent body of work and a very solid groundwork for future experiments. We are confident that our revisions make the manuscript more comprehensible to highlight the qualities of our manuscript more accessibly.____

    __

  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

    In the manuscript by Jin et al authors characterize the FLAIL DNA locus in Arabidopsis (using a wide array of publicly available datasets), which produces a set of sense and anti-sense lncRNAs. Authors determined that the sense FLAIL lncRNA (or a set of sense lncRNAs, which isn't fully clear from the way the data are presented) is involved in flowering time in Arabidopsis based on the fact that the several flail mutants lead to the early flowering phenotype and this flowering defect is complemented by transgenic FLAIL DNA, meaning that FLAIL lncRNA acts in trans. The T-DNA flail3 mutant results in expression changes (up or down) of 1221 genes, including twenty genes linked to flowering in various ways. Expression of a group of these flowering-related genes could be either fully (for eight genes) or partially (for five genes) rescued by transgenic FLAIL. Authors also conducted the ChIRP-seq to determine which genes are physically bound by FLAIL lncRNA genome-wide. It was found that 210 genes in the genome are bound by FLAIL lncRNA. Comparison of the dataset of differentially expressed genes in the T-DNA flail3 mutant with the ChIRP-seq dataset of genes that are bound by FLAIL lncRNA revealed the 12 overlapping genes.

    Among these twelve overlapping genes, four were found to be functionally connected to flowering with expression of these four genes being down in flail3 T-DNA mutant. Two out of these four genes were ruled out from being involved in the regulation of flowering by FLAIL. Authors conclude that the two other genes (Cir1 and Lac8) are responsible for the late flowering phenotype of flail mutants based on the three lines of evidence: (i) these genes expression is reduced in the flail mutant, (ii) FLAIL lncRNA directly interacts with these genes chromatin, (iii) the mutants of these genes were previously reported by others to display early flowering phenotypes too.
    While I find many of the findings reported in the manuscript very interesting, building a good foundation on which to expand the study and providing a very good leads for follow up experiments, I also have serious concerns about the manuscript in its current form. Most importantly, this reviewer doesn't think that the mechanism of FLAIL lncRNA action was convincingly demonstrated. The main question would be how FLAIL lncRNA works and this question wasn't fully answered. It is great that FLAIL lncRNA binds directly to the two flowering-related genes, but what does it mean? Does it change any chromatin context of these genes quantitatively or qualitatively to affect the transcription? Or does it bind any components of transcriptional machinery and thus controls the transcriptional output? Additionally, flail3 T-DNA mutant affects the expression of 1221 genes and FLAIL lncRNA physically interact with 210 genes, so how can authors be fully sure that FLAIL lncRNA has only direct effect on these two genes and doesn't also contribute to the regulation of the upstream to Cir1 and Lac8 genes or even components of the transcriptional machinery that regulate these genes? Theoretically, doing RNA-seq in the amiR-FLAIL sense lncRNA mutant might have a chance of reducing the number of affected DEGs, making it easier to analyze the FLAIL targets, even if the allele can't be used for complementation experiments. Also, authors totally neglect putting the Cir1 and Lac8 genes into the context of flowering regulation, but it is something that needs to be done. Lastly, the paper needs to be totally rewritten to be even properly evaluated. In its current state it reads like a very short draft. The Abstract is weak, the Introduction is written in a such telegraphic style that it is barely readable, in many places there is no connections between sentences leading to the information appear to be presented as random, even if it isn't. The Results section is written rather rudimentary with information not being sufficiently provided to describe the results but rather scattered between the Results and Figure legends. The Discussion is the best written part of the manuscript. The Conclusion section carries no specific information and reads more like a little summary suitable for a review article rather than experimental paper.

    Therefore, this reviewer thinks that regardless of how authors will choose to proceed with the current experimental version of the manuscript, it'd be in the authors' best interests to at least fully revise the paper before resubmitting anywhere. I'd also advise authors to seek professional editorial help specifically using an editor with the background in the plant sciences. Authors might also want to consider moving Fig.3 into the Suppl. as it doesn't carry much weight or significance and perhaps make existing figures more meaningful and comprehensive and by including a better diagram of the locus (e.g., Fig. S1), etc.

    It's not practical to list all issues with the writing as the paper requires total re-writing, so I can just make a few suggestions without any specific order to help authors improve the paper:

    --There is no statement anywhere that states the goal of the study.

    --No rational is provided on why authors decided to examine this specific genomic locus.

    --Typically, the significance is in studying the function of lncRNA or a group of lncRNAs produced from a genomic locus, I don't think I ever encountered the instances when it was exciting to study just a specific genomic locus. If the locus does indeed have any significance for initiating the study, it needs to be explained.

    --The locus can produce lncRNAs, but it can't harbor them.

    --No length of FLAIL lncRNAs or their range is provided in the first section of Results.

    --On many occasions authors don't state rational for doing experiments, which leads to information often flowing as random.

    --What do authors mean by the subtitle "FLAIL characterizes a trans-acting lncRNA repressing flowering"? How can lncRNA FLAIL or FLAIL locus characterize lncRNA?

    --Check all figures. E.g., Fig. 3B-E mentions only accession numbers for the genes.

    --It is not clear where exactly the T-DNA insertion is located relative to sense FLAIL in flail3 mutant (Fig. S4). --- What is the length of the complementing sense FLAIL lncRNA?

    --Check the description of each and every construct used and provide explanation for each in the Results. E.g., the pFLAIL:gFLAIL18/88 and pasFLAIL:gasFLAIL18/39 constructs aren't explained in Results, and can only be found in Fig. 2 legends.

    Significance

    Tens of thousands of lncRNAs have been identified in various eukaryotes, but their biological roles have been shown only for a small fraction of them, and the mechanisms of their action are delineated for only a very few of them. Most of the advances on the field of lncRNAs are reported in metazoan, while the field of lncRNAs in plants is lagging far behind in terms of knowledge about lncRNAs with assigned biological functions or lncRNAs with delineated mechanisms of action. From this point of view, this reviewer is always excited to see any new functional plant lncRNAs for which either biological or mechanistic functions have been determined, and deems the information on this subject significant. The manuscript's findings are potentially very interesting and present a decent body of work that lays a very solid groundwork for future experiments. My main concern about the manuscript's significance in its current form is the fact that no real solid mechanism of action for the described lncRNA or a set of lncRNAs (?) has been demonstrated. The best mechanistically studied lncRNAs in Arabidopsis are involved in the regulation of flowering time, particularly those that function in the vernalization flowering pathway and to lesser extent in autonomous pathway. The new FLAIL lncRNA or lncRNAs (?) described in this manuscript also appear to regulate the flowering time in Arabidopsis, however more experiments would be needed to provide a definite conclusion about how direct FLAIL's effect is and how exactly it functions. That unfortunately obviously diminishes the significance of the manuscript and makes it potentially interesting only to researches studying flowering in Arabidopsis and even then the manuscript results would be incomplete to make solid conclusion. Additionally, the manuscript requires complete re-writing.

  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

    In this ms, the authors identified the FLAIL lncRNA that represses flowering in Arabidopsis from a locus producing sense and antisense transcripts. They use an allelic series involving T-DNA insertions, CRISPR/Cas9 and artificial miRNAs to study the role of FLAIL in flowering. A complementation series of constructs of the flail3 allele allowed them to show that the sense FLAIL lncRNA can act in trans. RNAseq revealed a small group of genes linked to the regulation of flowering whose expression is affected in the mutant and restored in the complementation line. To gain further insight into FLAIL function, the authors used a ChIRPeq approach to test whether the lncRNA can recognize potential target genes along the genome and they could show that FLAIL binds specific genomic regions. Clearly, this paper shows very nice evidence that the FLAIL lncRNA can act in trans to regulate gene expression. Nevertheless, there are certain points that need to be clarified to further support the action of the sense FLAIL transcript.

    1.According to Fig. 1 A, the antisense FLAIL is "internal" to the DNA genomic area spanning the sense FLAIL. Hence, with direct RT-qPCR is very difficult to distinguish between these molecules as a minor "RT" activity of the Taq polymerase may lead to detection of low levels of antisense, idem if RDRs may generate low antisense levels. Although I think that the plaNET seq brings strong evidence about the start and ends of these molecules, to measure them by RT-qPCR is not trivial and requires the use of strand-specific RT-PCR using a 5' extension of the oligo and amplification with one oligo of the FLAIL sequence (sense or antisense) and the added oligo. It is not clear how they could distinguish precisely sense and antisense particularly when both RNAs correlate as it is the case here in all alleles (Fig. 1 C and 1D). This should be more explicitly mentioned in the materials and methods section.

    2.In Fig. 2, what are the levels of antisense in the complementing lines with the sense transcript? And reciprocally sense levels in antisense constructs? This will definitively demonstrate the assumption that the T-NOS termination will not allow any expression on the other strand. At present, only one of the lncRNAs is measured in each experiment?

    3.In Fig. 3, it will be important to also show the FLAIL locus in the flail3 mutants (in comparison to the wt) as well as the transgene locus. Here the reads will be strand specific and furthermore this will allow to show that the transgene is not generating antisense transcripts (through RDRs for gene silencing?) and confirm that the sense FLAIL is required for the complementation.

    4.In Fig. S5, the expression of FLAIL is shown in the artificial miRNA lines. Is the antisense FLAIL affected "indirectly" by the cleavage of the amiRNA or remains constant? This is likely the case but should be shown.

    5.The ChIRPseq data adds major novelty to the ms and brings new ideas about the way of action of FLAIL. However, are there any common epigenetic states between ChIRP targets (e.g. histone modifications, antisense RNA production, homologies "detected" in the conserved regions between Camelina and Arabidopsis and the target loci? Or others) that may highlight potential mechanisms leading to repression mediated by FLAIL of these loci? There are many databases that could be explored (even during flowering) to search for potential relationships. Although precise description of the mechanism is out of the scope of this ms, this can be discussed in more detail to further expand on the nice data obtained.

    Minor comments:

    6.In Fig. S3, a global alignment between FLAIL and two loci in Arabidopsis and Camelina is sown. What is the extent of homology? How conserved is this sequence at nucleotide level (small or very long?) to support the conservation of this lncRNA. Are there potential structures conserved among these lncRNAs?

    7.In Fig. S4B, arrows may help to understand which seeds were selected.

    Significance

    This paper is a very nice piece of work and demonstrate the action of a long non-coding RNA (lncRNA) in trans on specific targets involved in the regulation of a developmental process, flowering. There is growing evidences that the non-coding genome hides large number of lncRNAs and there is little detailed genetic support for the action of lncRNAs globally. In contrast to many descriptive papers in the field, this ms demonstrates genetically, through an allelic series and complementation experiments, that this lncRNA locus is involved in flowering regulation and that its sense lncRNA recognizes target loci genome-wide, bringing interesting perspectives on potential new mechanisms of transcriptional regulation mediated by non-coding RNAs.

  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

    Summary:

    The authors characterized a new lncRNA locus named FLAIL that controls flowering time in Arabidopsis thaliana. The functional validation of this locus is strongly supported by the use of several different tools (CRISPR-Cas9 deletions, T-DNA insertion, amiRNA gene silencing, and transgene complementation of KO lines). It is also suggested that FLAIL lncRNA works in trans but not in cis. There are strong observations supporting that FLAIL works in trans.

    Moreover, it is suggested that FLAIL regulates gene expression by interacting with distant chromatin loci. This was assessed using RNA-Seq and ChIRP-Seq. Yet, the overlap between DEGs in the flail mutant and FLAIL binding sites at the chromatin is very small, with only 12 genes. From those, only 2 flowering genes' expression was rescued by FLAIL transgene complementation. The final conclusion that FLAIL lncRNA represses flowering by direct inhibition of the 2 flowering genes expression is correlative, and lacks genetic validation. In addition inspection of the supplementary file shows that the ChIRP analysis was done without filtering for the FDR so that some of the positive hits have an FDR of 0,232. In addition, many of the peaks land in intergenic regions with is not mentioned in the text a graph with the position of the peaks in respect to nearby genes would help.

    In one sentence, the authors used the right model system and methodology, including advanced techniques, to characterize a new trans-acting lncRNA important for controlling the flowering time in Arabidopsis but lack evidence supporting a mechanism of action that goes beyond the interaction with several chromatin loci.

    minor points:

    line#63-64 the authors say the COLDAIR and ASL work on FLC in cis in my view the original papers suggested/showed they work in trans.

    Fig 1B please add some more protein-coding RNAs for the bio-info analysis for comparison

    Order of Supplementary Fig citation is mixed with S2 coming before S1B

    It would help the reader to have a schematic of the crisper deletions, T-DNA insertion, and position of primers used for the RT-qPCR.

    In the supplementary PDF file, some of the text is missing on page 3 beginning and end of lines.

    Significance

    The use of several different tools to validate the biological function of FLAIL locus is a major strength of this work.

    The authors propose that flowering time and its gene regulation are controlled by sense FLAIL lncRNAs. However, the sense transcription of FLAIL locus is not detected in wild-type plants by TSS-Seq, TIF-Seq, or plaNET-Seq. If the authors would have explored further the expression of FLAIL transcripts in different stages of development (vegetative and non-vegetative) and in response to different conditions, it would make their claims on the function of FLAIL lncRNAs more convincing. Additionally, flail mutants could have been obtained in the hen-2 background, since it's there where we can observe FLAIL transcription.

    FLAIL locus lays on the proximal promoter region of PORCUPINE (PCP), an important regulator of plant development. As flail mutants, pcp mutants display an early flowering phenotype. The authors show no link between FLAIL and PCP from the overlap between re-analysis of published RNA-Seq data for pcp and RNA-Seq and ChIRP-Seq from the authors. This analysis is not enough to exclude the involvement of PCP from the FLAIL function. PCP expression using RT-qPCR should be performed in flail mutants to further support that FLAIL works independently from PCP.

    This work does not hypothesize any molecular mechanism besides the interaction of FLAIL lncRNAs with several chromatin loci. It was recently proposed in Arabidopsis that a trans-acting lncRNA interacts with distant loci via the formation of R-loops. The authors do not comment on that. This work would benefit in correlating FLAIL binding sites with R-loop-forming regions mapped in Arabidopsis, regardless of the results from this analysis. Additionally, the authors could attempt to look for a motif responsible for FLAIL binding.

    Most of the key conclusions are convincing, except for the flowering time control directly through CIR1 and LAC8, which should be mentioned as speculative

    The words locus and loci are latin and they should be written in italic. The word Brassicaceae, referring to the family should be in italic, and should not be "Brassicaceaes". The word analysis has the wrong spelling.

    I was asked "How much time do you estimate the authors will need to complete the suggested revisions: this is difficult to answer as it depends to which level the author would like to take their work. In my view, if all new experiments would have to be started from scratch it is too far away to be estimated.

  5. Version published to 10.1101/2021.11.15.468639v1 on bioRxiv