Peer review of: Natural variation at FLM splicing has pleiotropic effects modulating ecological strategies in (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, entitled Splicing natural variation at FLM induces 1 synergistic pleiotropy in 
Arabidopsis thaliana“, Hanemian and co-authors report the dissection of a QTL controlling the rosette 
growth difference shown by Can-0 and Col-0, two A. thaliana genotypes. The fine-mapping of a QTL in 
chromosome 1, followed by a thorough functional validation (complementation by allelic test and by 
transformation) reveals that this variation is encoded in the FLM locus. Despite the absence of coding 
variation, directed mutagenesis demonstrates that a variant affecting splicing causes the modification. 
That splicing affects function at the FLM locus had already been demonstrated, but the authors work 
further to show that this natural variant not only affects rosette growth and flowering time, it also 
alters the components of the leaf economic spectrum. A modification of the function of FLM appears to 
be sufficient to shift the leaf economic spectrum by a few percent towards increased resource-
acquisitive leaf physiology. To my knowledge, this is the first time that a flowering time allele is 
explicitly related to a phenotypic measure of central ecological relevance. The authors further show 
that the loss of function allele of Can-0 is shared by a number of other Iberian genotypes, and the 
distribution of the alleles associates with areas where temperature is relatively less homogeneous. 
The results presented in this manuscript are excellent, and the association between flowering and LES 
very novel and convincing.The elaboration on the putative adaptive relevance of the variant is an 
interesting addition that adds relevance to the work. I therefore strongly recommend this manuscript 
for publication. I think at this point that the discussion needs some work. 

Here are several points that the final version should improve. 
1) I am confused about how the authors interpret temperature effects. The QTL at FLM seems to be 
dependent on mean temperature and the polymorphism seems to associate with temperature 
heterogeneity. Why should that imply that its action is dependent on the amplitude of temperature 
fluctuations? Differences between 16 and 23 degrees are well within the realm of the daily 
temperature fluctuations one expects, no? Would it not be straightforward to validate this hypothesis? 
2) Why are Col-0 and flm-3 so far off the spectrum measured within A. thaliana and across species 
(Fig.4b)? I think there is something unrealistic there. Why should thaliana display the LES range of all 
herbaceous species (it is not a succulent)? 
3) Is it necessary to arm-wave around the role of FLM on WUE? I believe it is more relevant to discuss 
the link with LES. What does it mean? Why can one believe that this have any ecological relevance? Is 
LES related to fitness and how? 
4) Origin of the Can allele. Which of the two alleles is ancestral? Why not use the other Brassicaceae 
species to determine the ancestral state? 
5) Relict origin: while I understand that the authors are referring to this classification to describe the 
history of the Can allele, the literature on relics is unclear as to what really defines relicts. A more 
generic conclusion is that the Can-0 and the Col-0 allele have distinct admixed origins. In this context, 
it is not straightforward to determine whether the Can-0 allele is adaptive (the association with 
temperature fluctuations suggests so, but this would have to be experimentally confirmed). 
6) The last paragraph must be rewritten. I think the authors are somewhat confused about several 
concepts. Studies show that mutations with an intermediate pleiotropic effect are more likely to be 
consistently selected when seasonal conditions fluctuate. The authors show some pleiotropic effects of 
FLM, but the pattern of selection of each of these traits is unclear. Is flowering time variation 
independent of the LES, or is the LES a trait integrating variation across molecular processes, at the 
organ level? What I mean here is that the authors would not describe the effect of the mutation on 
splicing and flowering time as being pleiotropic. They are causal. As long as the causality on LES is not 
determined, it is a stretch to highlight the case studies by the authors as an example of adaptive (ie 
synergistic) pleiotropic variant. Can the two traits evolve independently as well (in which case one 
could study whether the pleiotropic mutation is more adaptive than a none pleiotropic mutation 
affecting only FT and not LES or only LES and not FT)? I understand that such follow-up work is not in 
the realm of the present manuscript, but I think the manuscript would be improved if it were 

refocused on the significance of pleiotropy, making clear that whether it is causality that links these 
traits, or a synergistic association between two traits whose genetic basis can otherwise be 
independent is not known. 
7) As a consequence, I don’t think the authors bring evidence of synergistic pleiotropy. So the author 
should either make their point more clearly or change their title. 

Miscellaneous comments: 
Line 169: what is “higher” growth? Faster growth? Higher final PRA? 
Line 252-253: I don’t think the word “trigger” is appropriate. May be something like “The effect of FLM 
loss of function on LMA reaches 2-3% of the total variance….”. In addition, since LMA and Amass 
being way out of the range normally reported in plants, it is unclear whether the effect reported here 
is realistic. 
Line 283: I appreciate that the authors are being cautious on this conclusion. Their p-value is not 
massively significant, and such correlation can also arise from outliers of population structure. This 
hypothesis should be validated with ad-hoc experiment manipulating temperature fluctuations. 
Line 355-356: not clear why mentioning neo-functionalization adds to the discussion. I think this 
paragraph is not very useful. 
Line 363: P5CS1 splicing: please check this work carefully, the alternative splicing affects a transcript 
that is not necessary for function. 
Line 401: this sentence is confusing. Please rewrite. 
Line 413: reciprocal planting experiments? Where? Statement is unclear. I think the authors mean a 
common garden across environments differing in temperature fluctuations and precipitations. 
Fig. 4b: why so many points for Col-0/flm3? Are the 30 replicates shown here? Would a mean with 
error bars be more appropriate? The legend needs to be clarified. 

Reviewer #2 (Remarks to the Author): 

The middle of this paper (Results) describes a nice genetic dissection of some growth traits differences 
between 2 Arabidopsis lines. Hanemian et al. first grew RILs from the Can-0 and Col-0 cross and 
performed a QTL analysis. They then watched the major QTL segregate in the progeny of a residually 
heterozygous RIL, confirming the phenotypic effect. The QTL contained FLOWERING LOCUS M (FLM), 
so the authors went to nulls for this gene. Next, they performed a quantitative complementation assay 
in F1 hybrid plants to mutant flm-3. Finally, Hanemian et al. transformed the flm-3 mutant with the 
indicated sequence from both parental lines. In aggregate, these data show that allelic variation at 
FLM is responsible for growth and color differences between Can-0 and Col-0 owing to the QTL on 
chromosome 1. Subsequent work suggests that a SNP creates a splice site difference, causing the 
phenotype. In general, the work is a compelling demonstration of the power of forward genetics in 
Arabidopsis. 

The introduction needs a complete re-write. Despite much lingo and many broad statements, I could 
not tell what the paper was actually about until I read the results section (which is very clear). 
Regarding the motivation for the study, the fact that a mutation to a MADS-box transcription factor 
has effects on multiple phenotypes is not remotely surprising. Any mutation that affects growth or 
allocation will affect numerous plant dimensions through time. However, the fact that the Quantitative 
Trait Nucleotide (QTN) is a splice variant is notable discovery. While not unprecedented (line 359 
onward), the result bears directly on the general question of what sorts of mutations make a QTN. 
This would be a better focus for the Introduction/Discussion. 

Reviewer #3 (Remarks to the Author): 

The manuscript by Haneman et al describes how natural variation at the FLOWERING LOCUS M (FLM) 
gene contributes to pleiotropy in Arabidopsis. The authors mapped QTLs for several growth related 
traits, which turned out to be mapping to the FLM locus. When they analysed the FLM locus in strains 
that differed in the alleles, the underlying molecular mechanism hinted at changes in splicing. The 
authors go to length to try and establish that variation in splicing contributes to this variation across 
multiple growth related traits. The authors claim that Natural variation in FLM splicing mediates 
synergistic pleiotropy in Arabidopsis. The authors show that the alleles of interest have an 
interpretable geographic distribution indicative of adaptive significance. Overall the story is interesting, 
as it goes beyond flowering and it would be of interest to plant biologists and evolutionary biologists. 
However the study suffers from few issues, which I have listed below, addressing which would 
substantially improve this manuscript. 

This manuscript suffers severely from a chronological effect of experiments. In 2013/14, there was 
Nature paper published by Pose et al, which proposed a model based on an assumption that the 
primary transcripts arising from FLM are FLM-beta and FLM-delta. This assumption was incorrect and 
this mis-led the field and several researchers on a wrong track. This paper also appears to be one of 
those mis-led by this story and that is reflected in their interpretation. When I look at the data they 
present it is very clear, what is really happening. In Col-0 you have primarily FLM-beta. In Can-0, you 
have a new splice acceptor site that is strong enough to outcompete the normal splice site that would 
have resulted in FLM-beta. Therefore, with the new splicing instead of AG1 (i.e., splice acceptor that is 
in front of Exon2) the new splice site is used resulting in a novel transcript, that contains a premature 
stop codon. The authors are too influenced by the beta/delta model presumably due to historical 
reasons, and fail to see this. As far as I could see their data clearly shows that the primary effect is 
loss of the normal (FLM-beta) transcript and formation of a novel transcript with a premature stop 
codon due to a hot splice acceptor site. There is no need at all to discuss their work in the context of 
FLM-delta in this paper. This is the primary problem with this paper and it requires re-writing the 
paper with the current knowledge. The authors presumably are tip-toeing around this issue, which is 
not very helpful. 

I have given some clear suggestions below. 

Fig 1A: 

rHIF068: The legend says it is fixed for FLM. The genotype panel indicates that orange is Col and Blue 
is Can. Then what do the colours indicate in rHIF68? Does this mean that rHIF068 that is fixed for Col 
and Can alleles for FLM do not differ in their phenotypes? Is this correct? Or rHIF68 is fixed for Col 
allele? This makes not much sense. 

Fig 1B & D. 
16ºC panel figure.. Did all 21 plants flowered on day 30 in the flm mutant background? Is this what 
the figure shows? How was days to flowering measured. This lack of variation is a bit strange… I really 
have difficulties to believe that all 15 plants in Col flowered on day 54 and all 30 plants of flm flowered 
on day 47 or something. Please explain. 

Fig 1C: What is this significance for? The data discussed in lines 181 and 183 is somewhat inconsistent 
with the figure. Please double check this analysis and its interpretation. 

Lines: 194 to 195. Rephrase this.. This idea of discussing FLM splicing in the context of FLM beta and 
delta leads to fundamental confusion in the manuscript. It is better to state that FLM 

splicing/expression 

Fig 2 and Fig S3. It think both these figures can be removed. They do not really add any value. It is 
pretty clear from this work as well as from earlier works that such analysis would lead to misleading 
conclusions. The authors have done a lot of work. But this does not help either the paper or the story 
itself. I suggest the authors to directly discuss the sequencing of full-length cDNA clones and remove 
this from the manuscript. The entire section of lines 194 to 213 can be removed. I believe all these 
discussions with exon2 vs exon 3 etc are not very useful. The authors can simply state that 
temperature affects FLM splicing and then present their cloning data. I suggest to make Fig S4 as the 
main fig S2, which can easily drive the point. 

Lines 231 and 232: We have no idea whether the promotion of growth has got anything to do with 
these alleles. Should be rephrased to say that there was no differences in PRA were observed between 
plants harbouring either allele. 

Lines 249-250: Unclear … what is being shown here and what does that mean? 

Lines: 241-254: I have no idea what is meant by this. E.g., “variation observed across all plant 
species”??? 

Lines: 285-300: Unclear. I am not sure whether this data is convincing enough. 

Lines 327-329: “which is expressed at a substantial level in many Arabidpsis strains”? where is this 
data from… I do not believe any paper has shown this clearly. I suggest to remove this. 

Lines 338: Remove in exon 3 etc.. A premature stop codon is a premature stop codon. End of story. 

Lines 344-346 and the full line of argument: This is incorrect to state. Nd-1 and Ei-6 carries full 
deletion of the FLM locus. I am not sure how appropriate is the line of argument made in this 
paragraph. 

Fig. S5B: Are there 2 Col-0 lanes? 

Hanemian et al. 

Nature Communications