Negative regulation of conserved RSL class I bHLH transcription factors evolved independently among land plants

Essential revisions:

The reviewers have largely provided points of clarification that will help to make the manuscript more approachable.

Reviewer #1:

[…] The authors mentioned "There are three putative small open reading frames". However, only two of three were tested. If there are reasons not to test all three potential ORFs, the authors should mention about it.

We did test all the three putative small ORFs individually by over-expressing each in the wild type background (Figure 4—figure supplement 1). One of the small ORFs, which we designated MpFRH1^miRNA, contains the entire miRNA producing hairpin and therefore over-expression of this 150 bp fragment reproduces the few rhizoids phenotype (Figure 4—figure supplement 1). The other two fragments were designated MpFRH1^PSORF1 and MpFRH1^PSORF2. The over-expression of the other two putative small ORFs (MpFRH1^PSORF1 and MpFRH1^PSORF2) did not reproduce the few rhizoids phenotype. We reported this in the text:

“To verify that the PSORFs do not encode peptides, we over-expressed wild type versions of two PSORFs that do not overlap with the miRNA encoding fragment in the wild type background. Transformed plants overexpressing either MpFRH1^PSORF1 or MpFRH1^PSORF2 did not have defects in rhizoid development and were undistinguishable from wild type plants”

The authors wrote "To test if MpFRH1 suppresses MpRSL1 mRNA cleavage we performed 5'RLM-RACE PCR assay". However, only the model is shown in Figure 3B. The authors should show the data indicating that the cleavage of MpRSL1 mRNA is mediated by MpFRH1 miRNA.

The experimental data for 5’RLM RACE PCR has now been included as Supplementary file 3. This is referred to in the subsection "MpFRH1 miRNA targets RSL class I gene MpRSL1 mRNA”.

The subsection "MpRSL1 positively regulates MpFRH1 transcript level" starts without any explanation to study the hypothesis. I recommend to mention the motivation for why authors want to test if MpRSL1 regulates MpFRH1 expression.

We now include a rationale. “In biological networks equilibrium is commonly achieved through negative feedback loops, in which positive regulators promote the expression of their repressors. Therefore, we hypothesised MpRSL1 may also promote MpFRH1 expression. To test this hypothesis, we measured steady state levels of MpFRH1 transcript in the MpRSL1 loss-of-function and gain-of-function mutants”

For Figure 3—figure supplement 3, do the numbers show the three replicate of experiments in the panel A? If so, the authors need to mention about the weak signal of 3. in Basic helix-loop-helix transcription factor (Mapoly0100s0033).

Yes, the three replicate experiments are shown in panel A. Two out of three samples were similar to MpFRH1^GOF and wild type. Therefore we think Mapoly0100s0033 is an unlikely target. With the third replicate sample RNA quantification could be off (Mapoly0075s0041 also gives slightly lower amplification in this replicate), there could be a pipetting error or there could be different levels of contamination from genomic DNA between the replicates (please see below). The target site is also fairly close to the 5’ UTR of the gene and therefore differences in RNA integrity could explain the difference in signal intensities.

Note that the computationally predicted MpFRH1 miRNA target site is not present on the Mapoly0100s0033 transcript found in the Bowman et al., 2017 transcriptome (see the alignment in Supplementary file 4 C). We initially carried out the target prediction using the M. polymorpha gametophyte transcriptome published in Honkanen et al., 2016. The predicted MpFRH1 miRNA target site is on a short transcript 27676, which is identical to Mapoly0100s0033 transcript in its 5’ region. However the 3’ region of this transcript including the predicted miRNA target site is within the first intron of Mapoly0100s0033. Therefore, the 3’ region of transcript 27676 may be a partial alternatively spliced transcript of Mapoly0100s0033 or the reads may have originated from genomic contamination. The Marchantia genome browser (marchantia.info) shows very low level of reads on this region (scaffold_100:358969..360369). Therefore we do not believe the reduction seen in one of the three replicate samples is biologically significant.

To prove that MpFRH1 encodes miRNA, I think stem-loop PCR is not sufficient. Northern blot analysis is a more direct and reliable approach to detect miRNA of MpFRH1. If possible, I suggest to include these data to strengthen this point.

We do not have the Northern blot data. The miRNA cataloguing paper by Tsuzuki et al. 2016 showed that MpFRH1 miRNA is expressed and accumulates in M. polymorpha. Tsuzuki et al. identified the MpFRH1 miRNA (mpo-MIR11861) in each of the three replicate sequencing experiments (Supplementary file 1). This demonstrates that the MpFRH1 miRNA exists in Marchantia polymorpha. Therefore, Northern blots while useful, are not necessary to demonstrate the presence of the MpFRH1 miRNA in Marchantia polymorpha.

Reviewer #2:

[…] The main text states that the MpRSL1 mRNA fragment terminated at the putative cleavage site, although Figure 3B only shows the gene structure and the predicted site. The authors should provide such experimental data, or revise the main text and Materials and methods.

The experimental data for 5’RLM RACE PCR data has now been included as Supplementary file 3.

The authors clearly show antagonistic relationship between MpFRH1 and MpRSL1 during rhizoid development. Overexpression of wild-type MpRSL1 by a strong EF1 α promoter did not overcome the MpFRH1 GOF mutation, implying strong degeneration activity of MpFRH1 against MpRSL1 transcripts. This raises the question how MpRSL1 is activated to specify gemma epidermal cells as the rhizoid precursor cells, in which MpFRH1 are also expressed, as shown by the MpFRH1 promoter activity. It is possible that MpFRH1 expression is significantly reduced, or diminished, before the rhizoid precursor cells differentiate on the gemma epidermal cell layer. More information about spatio-temporal pattern of the MpFRH1 promoter activity during gemma development would be helpful for better understanding of the molecular mechanism of rhizoid precursor cell differentiation. I suppose this could be done by simply observing immature gemmae of the MpFRH1 promoter:3xYFP-NLS lines.

Rhizoid precursor cells become established very early in gemmae development when it is difficult to image their development. We have not been able to acquire these data yet. We are planning to continue to do this but it will clearly take some time.