Peer review of: A mycorrhizae-like gene regulates stem cell and gametophore development in mosses (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The authors describe the functional characterisation of the PpMACRO2 gene in Physcomitrella 
patens, which bears a resemblance to those found in mycorrizae-like fungi. The authors take a 
reverse genetics approach to link PpMACRO2 function to stem cell regulation and 3D growth 
processes. Firstly, I would like to remark that the developmental phenotypes described in this 
paper are of great interest and are very significant. However, careful conclusions must be made 
with regards to links to stem cell/3D growth regulation. My comments: 

Introduction 

The introduction begins with an outline of the morphological features developed by plants during 
terrestrialisation. The paper then steers towards the 3D growth literature, particularly those 
relating to the APB TFs, DEK1 and CLAVATA. Firstly, a review is cited for DEK1 and not the primary 
literature (which comprises numerous papers in Physcomitrella patens). At this point, I have no 
clue why the authors have opted to study PpMACRO2? The link to 3D growth becomes even more 
confusing when we subsequently discover that these proteins are not encoded within the genomes 
of vascular plants (where 3D growth and indeed stem cell regulation processes are present). 

Results 

Supplementary Fig S1a – Spirogyra information is superfluous. Supplementary Fig S1b+c looks to 
be a direct repeat of Figure 1. My opinion is that Fig. 1 should comprise diagrams showing exon-
intron structure of PpMACRO2 (sequenced version) and location of domains in PpMACRO2 protein. 
In my experience, it isn’t uncommon for Phytozome gene models to be incorrect. The alignment is 
valid and should remain in Fig. 1. I feel that the phylogeny should be moved to Supplementary Fig 
1. 

Description of ko and OE lines: 
- Supplementary Figure S5 – ‘only minimal expression detected’. Do the authors mean ‘no 
expression’? It seems strange that you would detect a transcript in a full deletion mutant. 
Southern blotting results should be shown – this is standard for gene targeting experiments in 
Physcomitrella patens. 
- Supplementary Fig. 7 superfluous 
- Supplementary Fig S8 and S9: Really significant results that I feel have a place in the main 
figure. These results also demonstrate that the ko and OE of this gene has an overall effect on 
development, not just on stem cell regulation and/or 3D growth. These could be combined into a 
single figure or incorporated into the current Fig. 2 (moving out the GUS/GFP data to Fig. 3). 
- Supplementary Fig. S11 – should also appear in the main figures. 
- Figure 2/3 – GUS/GFP expression appears more widespread than the authors claim. Expression is 
not restricted to stem cells. Rather in Figure 2d, expression can be observed throughout the 
filamentous network. Nevertheless, I do agree that expression looks more enriched in stem cells 
though. Perhaps a partial quantification of a GFP signal might be needed? 
- Figure 4 – gametophore development occurs earlier in OE than WT or ko lines. Perhaps it is 
simply earlier development of the gametophore initial cells that causes the enlarged gametophore 
phenotype? It is unclear why the authors are examining detached leaves. 
- Figure 5 – it is possible that the altered expression of AP2 genes is an indirect consequence of 
having fewer or more gametophore initial cells rather PpMACRO2 acting transcriptionally upstream. 
- Throughout – within figure legends and the methods section, it is unclear what the error bars 
refer to (SEM?) and which statistical tests were performed in each case. 

For me, the link to 3D growth in particular is somewhat tenuous. I appreciate that the number of 
gametophore initial cells appears to be affected, but the gametophores have no apparent cell 

orientation defects (like the dek1 and CLAVATA mutants). A stem cell focused view seems more 
appropriate… (see Aoyama et al.). 

Methods 
- How were the gametophore assays performed? It doesn’t look like the starting material was 
normalised in anyway (OD measurements or similar). How much material was added to the 
medium? Was the same amount added? 
- Components of the GUS solution? 
- In general, the methods section lacks sufficient detail. 

Reviewer #2 (Remarks to the Author): 

In their article "Mycorrhizae-like gene regulates stem cell development and three-dimensional 
growth in mosses", Wang and colleagues present a thorough wet lan study on the molecular 
function of the PpMACRO2 protein. I generally think that the authors have done a study of high 
quality and an impressive amount work. Yet, the bioinformatic analysis should be revised. Further, 
the evolutionary interpretations have some problems and require additional thought. 

From the perspective of an evolutionary biologist, the main problem of the manuscript is that 
bryophytes--or in this case Physcomitrella in particular--are treated as living representatives of the 
earliest land plants. This is wrong. The earliest land plants were organisms that lived 500 million 
years ago. They are extinct. Physcomitrella is as divergent as any plant living today from the last 
common ancestor of land plants. Read and cite "Reconstructing trait evolution in plant evo–devo 
studies" (Delaux et al. 2019; Current Biology 29 (21), R1110-R1118). 
Furthermore, with the recent analyses of Puttick et al. 2018 (which is cited) the bryophytes are 
most likely not the earliest diverging land plants. This makes the aforementioned statement even 
more problematic. 
--> Hence, sentences like "pathways that are only retained in bryophytes represent part of the 
genetic toolkit of ancestral land plants." are misleading and should be reconsidered. Anything 
found in any plant genome--no matter if moss, spruce or barley--might have been part of the 
genetic toolkit of ancestral land plants. 

l. 44: "fluctuation, and exposure to novel pathogens." --> this sentence needs a reference. Read 
and cite "On plant defense signaling networks and early land plant evolution" (2018; 
Communicative & Integrative Biology 11 (3), 1-14) 
as well as "Embryophyte stress signaling evolved in the algal progenitors of land plants" 
(Proceedings of the National Academy of Sciences 115 (15), E3471-E3480) and "Great moments in 
evolution: the conquest of land by plants" (2018; Current Opinion in Plant Biology 42, 49-54). 

"plants during their transition from water to land (4, 5), but also the sequence of evolutionary 
changes" --> No. To shed light on this topic, the algal relatives of land plants, the Charophytes 
a.k.a. streptophyte algae must be considered. Read and cite "Plant evolution: landmarks on the 
path to terrestrial life" (de Vries & Archibald, 2018, New Phytologist 217 (4), 1428-1434) 

"homologous genes and processes in flowering plants (e.g., Arabidopsis thaliana). This approach of 
evolutionary development has identified some core components of key pathways conserved 
throughout land plant evolution, but, on the other hand, has provided little information on the 
toolkit specific to early land plants" --> for inferring this, ALL land plants must be considered--
again, no matter if moss, spruce or barley. 

"We suggest that PpMACRO2 is involved in histone modification through ADP-ribosylation"" --> 
better say "we speculate" since no supportive data was generated here. 

I completely miss from the introduction why PpMACRO2 was chosen as a study subject. Don't get 
me wrong--it is very interesting indeed. However, it is currently unclear / not justified sufficiently 
why exactly the PpMACRO2 protein was chosen in an evolutionary context. Therefore, the main 
motivation is lacking from the introductory part and this hampers the pursuit of a red thread, i.e. 
being properly guided through the rationale of the article. 

Why was only Intefilum and not the genome of Klebsormidium nitens (back then called K. 
flaccidum) which was pusblished by Hori et al., 2014 in Nature Communications? 
Further, the authors cite the genome paper of Cheng et al. (2019) on Mesotaenium and Spirogloea. 
Yet, these genome data are not used by the authors here in this study. This is confusing. 
Also, Mesostigma viride should be queried, which was published by Liang et al. (2019) in Advanced 
Science. 
Further, why were the many (including only recently published) phylodiverse land plant genomes 
not used?--such as that of the water fern Azolla (see Li et al., 2018; Nature Plants) 
Further, the authors say that MACRO2 is in few other eukaryotes, but it is not clear whether all of 
these appear in the phylogenies. Further the labeling should be improved. E.g., the glaucophyte 
sequence can be easily missed in the mass of bacterial sequences. 
Finally, MACRO2 is also in PFAM database found in eumetazoa. 

The authors should seriously consider using an HMM profiling like a pHMMER to screen for MACRO2 
in a more robust fashion across eukaryotic genomes / proteomes. 
Were all of the results included in the phylogenetic analyses? They should be, but currently this is 
unclear. 

Please also provide a table in the supplement for all the hits. And an additional summary table (i.e. 
how many hits per phylum). 

There are some ambiguities regarding the datasets used for the bioinformatic analyses. The M&M 
does not mention all the databases (e.g. the NCBI EST data) that are mentioned in the results 
section. 

"which only contain transcriptomic data, yielded hits from green algal species, no hit could be 
identified from any complete genome of green algae that was generated from axenic cultures." --> 
these hits must be specified. The authors should use phylogenetic analyses to try to resolve this. If 
the gene trees somewhat match the species trees, than these could be meaningful sequences and 
not just contaminations (such as viral seqs). 

Line 133: why not refer to Figure 2C as well here? 

"This evidence indicates that PpMACRO2 promotes cell reprogramming" --> Please elaborate and 
explain more why this is your conclusion. 

"PpMACRO2 regulates epigenetic modification" --> there is not enough support for regulation. 
Currently a gene expression PATTERN is observed. Please tone this down. 

l.214: "genes related to epigenetic modification" --> Please explain in the text, not only in the 
figure, what these epigenetic-associated genes are. 

Figure 5: Please provide additional, more meaningful, labels for all the genes in the heatmap. The 
Pp gene numbers are a good resource but have little informational value by themselves. 

In the discussion, many issues regarding the plant terrestrialization concepts / inferences 
regarding the early land plants that were problematic in the introduction also come to bear here. 

l.239/240: "plants and other eukaryotes" this is a bit bold, considering that this study was only 

done on P. patens. 

Lines 242 to 247 should be moved to the results section. 

The network analysis is not properly explained in the material and methods section. Please rectify. 

Lines 267 to 286: This is all very confusing and needs to be streamlined. What is the direct 
connection between MACRO2 and facing terrestrial stressors?? Please elaborate. 

The context of LGT versus differential loss is especially difficult in eukaryotes; for some stimulating 
discussion on this, read and cite doi: 10.1002/bies.201700115 and 
doi.org/10.1002/bies.201700242 

Figure 6 is too cryptic and the figure legend does not help. 

Reviewer #3 (Remarks to the Author): 

The authors report a novel macro2 domain gene in Physcomitrella patens (Pp) an early 
nonvascular land plant. Macro2 domain genes regulate epigenetic modification, stem cell function, 
cell reprogramming and other processes in different organisms including plants. This macro2 
domain gene (PpMACRO2) is likely derived from Mucoromycota, a mycorrhizae-like fungi found in 
the earliest land plants. 

Sequence comparisons leave little doubt as to the similarity between Pp and fungi. 

Fig S 4 not needed and can be briefly stated. 

Gene knockout and over-expression results of PpMACRO2 in Pp significantly change the number 
and size of gametophores, the dominant form of three-dimensional growth in mosses. The authors 
employ the expected methods to demonstrate these results, so I have no doubts as to their 
competence or suggestions about their procedures. 

Given the strong expression of PpMACRO2 in apical stem cells of both protonemata and 
gametophores support the role of PpMACRO2 in epigenetic modification, stem cell development, 
and related process 

RNA Seq data supports role in stem cell development.