Peer review of: A leap in quantum efficiency through light harvesting in photoreceptor UVR8 (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
UVR8 is a 7-bladed beta-propeller protein that functions as a UV-B photoreceptor in its 
homodimeric form. Each UVR8 monomer contains 14 tryptophans: 6 distal (Wd), 3 peripheral 
(Wp), 4 central (Wc; pyramid), and 1 in the C-terminus. The present understanding is that in 
particular W285, together with W233, (both belonging to the group of 4 central tryptophans) plays 
a key role for UV-B responsiveness of UVR8. Li et al. used ultrafast fluorescence spectroscopy and 
quantum computational calculations to describe the relative contributions of all three studied 
tryptophan groups (Wp, Wd, Wc) to UV-B absorption and energy transfer to the key Wc residues 
(W285 and W233). This is an interesting and extensive study that provides an important step 
forward towards understanding how UVR8 works as a UV-B photoreceptor. The authors provide 
evidence that light-perception quantum efficiency increases from 35% to 73% through the 
additional UV-B photon harvesting and energy transfer through the Wd and Wp tryptophans. 
 
I only have a few points that should be addressed: 
 
- The 14th Trp in the C-terminus should at least be mentioned. Otherwise the text makes the 
impression UVR8 contains only 13 Trps. 
 
- Consider to add at least 1-2 sentences in the introduction (line 56) what happens after 
monomerization of UVR8 in its signaling pathway (e.g. interaction and inhibition of the E3 ubiquitin 
ligase COP1). 
 
- In table S1 W302H is indicated – mention as is done for R286A* (mono-6Wd) 
 
- In the discussion it would be interesting to shortly mention and discuss the evolutionary 
conservation of the studied tryptophan residues in plant UVR8 proteins (e.g. including comparison 
with Chlamydomonas UVR8 that has been functionally studied in quite some detail, next to 
Arabidopsis UVR8). It seems that the architecture of tryptophan residues and thus likely also the 
UV-B activation mechanism has arisen early on in the evolution of UVR8 photoreceptors (derived 
from a RCC1-type protein). 
 
- A recent study suggested that UVR8 also responds to UV-A under natural conditions (Rai et al., 
Plant Cell Environ 2020); it may be interesting to shortly discuss. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The use of naturally-occuring amino acids such as tryptophan as UV sensors is not particularly 
novel and has been well documented for at least ten years. However, the assembly of 26 
tryptophan residues into a complex antenna system with an energy gradient, as described here, to 
increase the efficiency of UV perception is intriguing. By a combination of time resolved 
fluorescence measurements, mutational analysis, and mixed quantum-classical simulations the 
authors present a convincing analysis of energy transfer pathways to the "reaction center" pyramid 
that initiates monomerization. 
 
However,I felt the paper was written in such away that its appeal to abroad audience would be 
limited. To me, the paper was too focused on the details of the fluorescence decay analysis to the 
detriment of the bigger picture - exactly how the excited tryptophans initiate monomerization is 
left to the reader to find out. What consequencies and opportunities enhanced sensing of UV light 
brings to plants is not mentioned. Both the Introduction and the Discussion seemed very Terse and 
lacking context. I suspect this will lessen the impact of the paper. 

The energy transfer is described exclusively as localized hopping by the Forster mechanism. What 
are the couplings between the closest tryps? Are all the levels confined to a single chromophore? 
Do the spectral shifts giving the directionality of the energy flow arise only from environmental 
effects or also from tryp-tryp interactions? 
 
 
 

Reviewer #3: 
Remarks to the Author: 
A work entitled "A leap in quantum efficiency through light harvesting in photoreceptor UVR8" has 
been presented by Xiankun Li, Haisheng Ren, Mainak Kundu, Zheyun Liu, Frank W. Zhong, Lijuan 
Wang, Jiali Gao and Dongping Zhong. Using a combined experimental and theoretical approach 
they study the energy-transfer network involving 26 tryptophan residues in the UVR8 receptor. 
Quantitative agreement between time-resolved fluorescence data and QM/MM-based results has 
been found, for what concerns the total resonant-energy-transfer time constants and the related 
energy-transfer pathways. Furthermore, the tryptophan residues have been grouped into three 
differents sets, assigning a specific role to them in the energy transfer. 
As a general comment, I found the main conclusions convincing, and the applied strategy reliable 
and robust. The present work deserves to be published on Nature Communications, but, in my 
opinion, a number of changes should be applied to make the manuscript clearer and accessible: 
Results, Discussion and Methods Sections should be reshaped in a more consistent way, to provide 
a precise information when needed, as specified below. My comments are given as follows: 
 
1) The authors should enrich the Methods section with, at least, a part of the Supporting 
Information, especially that regarding the energy-transfer and population dynamics: this modeling 
is an essential part of the work, and should be included in the main text; 
 
2) In many points of Results (lines 123-126, 131-134, 142-144, 159-162, 186-190, 198-199), the 
authors report numbers of energy-transfer times, transfer efficiency etc. with no reference to the 
method and/or formulas used to compute them: formulas are in Supporting Information, but a 
reference is necessary to avoid the reader looses the focus. For this reason, I suggest, as already 
mentioned, to move part of the first ten pages of the Supporting Information into Methods and to 
explicitly refer to the applied formulas. The last four subsections of Methods ("Energy transfer...", 
"Numerical...", "Model..." and "Energy transfer efficiency...") are currently too general and do not 
provide any specific insight into the employed physical models; 
 
3) Did the authors computationally study the reason why the mono-6Wd mutant is a monomer? 
 
4) In which conditions (temperature) the spectra have been recorded? 
 
5) In Figs 2b-f, spectra of 6Wd1 and 6Wd2 mutants are reported: the two species have been not 
defined in the main text; only a short comment in the caption of Figure 2 is given, which refers 
generically to the Supporting Information; 
 
6) in order to save clarity, author should specify the meaning of all the numbers on lines 131-134; 
 
7) the authors should briefly comment the choice, as an example, of W302 instead of the other 
residues in Figure 2b; if no physical reason is behind such a choice, a sentence should be however 
added; 
 
8) Computed energy-transfer rates in Figures 2g and 3 a-d show a different probability 
distributions; are these differences due to the relative mobility of the involved pairs? Did the 
authors explore this behaviour? 
9) the "ab" subscript in the formula on line 327 is not defined. 
 
10) Can an excitonic model, as typically used for light-harvesing complexes (e.g., LH2 and FMO), 
be applied to study the tryptophan network at quantum level? 
 
Few typos: 
 
i) "were" instead of "was" on line 246; 
 
ii) "GAMESS" instead of "GAMSS" on line 324; 
 
iii) "RET" and "FRET" are used to indicate the same group of quantities.