Peer review of: Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Xie et al. dissected the involvement of FHY3 and FAR1 in the regulation of branching in response 
to light quality change. fhy3/far1 has reduced branching under white light but diminished the 
difference in branching under white light versus EOD-FR shown for wild type. Inside to the story, 
FHY3 and FAR1 inhibit the binding of SPL9 and SPL15 to the BRC1 promoter, and SPL9 and SPL15 
directly activates BRC1 expression. Meanwhile, three SMXL proteins also interact with SPL9 and 
SPL15 but rather repress their transactivation activity. Finally, FHY3 and FAR1 bind the first exons 
of SMAXL6 and SMAL7 and activate their expression. A model is followed to present their overall 
discoveries. 

Major comments 
1) In Figure 3d, another control, MYC plus FHY3:HA, is needed for in vivo co-IP on the right panel. 
In addition, in vivo co-IP with FAR1 may show a low affinity toward either SPL9 or SPL15. The in 
vivo results with FAR1 may partially explain the subtle phenotype of far1. In Figure 6d, FAR1 
appears to have a weak transactivation activity for SMXL6 and 7 compared to that of FHY3. 
2) In Seven-day-old seedlings grown under WL conditions with EOD-FR treatment, both FHY3 and 
FAR1 proteins accumulated much less as shown in Figure 1d and 1e. Similar results were also 
shown for adult plants of four-week old in Figure S1. In Figure 4, EOD-FR may affect the ChIP of 
SPL9 and SPL15 to the BRC1 promoter. The authors may choose either wild type or FHY3 OX 
plants and variable ages to detect the best responses. 
3) In Figure 6, the model suggests that “simulated shade treatment reduces the accumulation of 
FHY3 protein, leading to increased expression of BRC1 and reduced branching”. As EOD-FR affects 
FHY3 and FAR1 accumulation, EOD-FR may affect the expression of SMXL6 and SMXL7 in either 
wild type or FHY3 OX. Data with EOD-FR treatment added in this figure plus experiments 
suggested in 2) would strengthen the model. 

Minor points 
1) In line 705, revise as both FHY3 and FAR1 protein level rapidly declined in seedlings treated 
with EOD-FR. 
2) In line 454, fill the missing parts for “2_-pBRC1 or 2_-pBRC2”. 

Reviewer #2 (Remarks to the Author): 

This manuscript describes some potentially very interesting results regarding the integration of 
light signalling into the shoot branching pathway in Arabidopsis. The authors propose that FHY3 
and FAR1 regulate branching both by blocking SPL9:SPL15 regulation of BRC1, and by promoting 
transcription of SMXL678, which also block SPL9:SPL15 regulation of BRC1. Along the way, this 
involves showing that SPL9/SPL15 regulate BRC1 in Arabidopsis (not shown 
before, but homologous to rice), and that SMXL678 act through SPL9/SPl15 (again homologous to 
rice, not previously shown in Arabidopsis). So there is potentially a lot of interesting work here 

Each part of the manuscript uses multiple different approaches to support its conclusions, and 
superficially, the manuscript seems very convincing. However, the manuscript lacks a substantial 
amount of methodological detail regarding what was actually done, and how it was done. I have 
major concerns about some of the results, because although it is clear what the result shows it is 
not clear how the results were obtained. 

In particular, the authors claim to have expressed full length SMXL6 protein 
using a simple bacterial expression system. This is widely acknowledged to be impossible among 
labs working on strigolactone signalling. I simply don’t believe that the authors expressed the 
protein as described in the manuscript. The SMXL6 protein doesn't appear to be the correct size 

either - I'd expect a full-length His-SMXL6 from pET28a to be about 111 kDa (107.8 kDa for 
SMXL6 plus 3.4 kDa extra for the His-tag and linker residues), but the band here is under 100 kDa. 
It also isn't particularly pure (none of the "pure" proteins are), and the coomassie gel looks to me 
like it might have been altered to make the contaminant bands less prominent compared to the 
desired bands. 

To credit the authors purification work, I would want to see: 
1) the exact mass and sequence of the SMXL6 fusion protein they express 
2) more information on their expression methods ie. growth medium, extraction method, 
extraction buffer (plus any additives) 
3) raw Coomassie gels of their expression with ladders, including induced vs uninduced cells, and 
soluble vs insoluble fractions for both, plus any westerns they might have done (eg anti-His) with 
ladders 
4) raw Coomassie gels of their purification with ladders, including crude, flow through, wash and 
elution fractions, plus any westerns they might have done (eg anti-His) with ladders 
5) confirmation whether they did any secondary purifications prior to that gel, and any related 
evidence 

The same applies to some extent for the other protein work: the supporting evidence and methods 
are simply not up to scratch. 

I would also highlight that the genetic crosses the authors perform to support their protein work 
are largeky uninformative, and do not provide any evidence for genetic interactions. 
1) BRC1 vs SPL9/15 
The brc1 brc1 x rSPL9 OE is additive, and BRC1OE spl9 spl15 is epistatic - no evidence for 
interaction here. Indeed, the evidence is more in favour of non-interaction. 

2) FHY3/FAR1 vs SPL9/SPL15 
Again, all the crosses of fhy3 far and FHY3OE x spl9 spl15 demonstrate is that spl9 spl15 is 
epistatic to FHY3. That in itself isn’t good evidence for an interaction. 

3) SMXL678 vs SPL9/SPL15 
Again, the results just show that SPL9/SPL15 are epistatic to SMXL678. The authors state that 
“while SMXL6D-OE/ SPL9-OE had more rosette branches than SPL9-OE”, but that is absolutely not 
what the data in Fig 6 shows.