PREreview of The role of LHCBM1 in non-photochemical quenching in Chlamydomonas reinhardtii

The manuscript “The role of LHCBM1 in non-photochemical quenching in Chlamydomonas reinhardtii” by Liu et al. aims to elucidate how LHCBM1 is involved in non-photochemical quenching (NPQ) in Chlamydomonas reinhardtii. The Chlamydomonas mutant lacking LHCBM1 (npq5) displays a low NPQ phenotype. The authors found that the antenna size and LHCSR3 accumulation are not responsible for the lower NPQ phenotype in npq5. They also artificially acidified the lumenal pH to protonate LHCSR3 for NPQ induction and found that npq5 NPQ is still low. They propose that absence of LHCBM1 could alter the association of LHCSR3 with the PSII supercomplex or that LHCBM1 interacts with LHCSR3 which would enhance its quenching capacity. This work enriches the knowledge about the impact of lack of LHCBM1 on antenna size, PSII function, LHCSR1 and 3 proteins accumulation and NPQ capacity during a 48-h high light treatment.

Major comments

- Fig. 1, it is stated that LHCBM1 (Type IV) does not accumulate but Fig. S1 and S2 show that Type IV accumulates between 7 and 9% of the total amount of LHCBMs. Could you comment on this accumulation, and whether it increases in high light?

- Statistical test between WT and npq5 for all the data represented in bar graphs would be required to state that differences are significant. Consider showing all data points in addition to error bars.

- Consider providing Fo and Fm values for all fluorescence measurements (and D1 accumulation) to discuss whether a possible increased Fo in npq5 in HL is due to disconnected antenna and/or loss of functional core complexes.

- Discussion, discuss why there is much less LHCSR1 in the npq5 mutant? Is this regulation at the transcriptional or post-translational level? It could be that LHCBM1 interacts with LHCSR1 preventing its degradation. In the abstract, and discussion, as no interaction data is shown, consider toning down statements regarding LHCBM1 interaction with LHCSR3.

- Discussion, discuss whether similar results were observed for the npq4 mutant (increased antenna size in HL and less functional PSII observed in npq5). Is this phenotype linked to the low NPQ capacity or specific to the lower level of LHCBM1 in the npq5 mutant?

Minor comments

- Please give a title to summarize the first result in Fig. 1. In the result section, consider making the section subtitles more informative about the main result from that section (e.g., “Photoprotection capacity after high-light acclimation” could be “NPQ induction is severely hampered in npq5 after high-light acclimation”).

- Page 3, method, actinic light of 1,500 μmol photons.m-2.s-1 (and saturating pulses of 12,000  μmol photons.m-2.s-1) are used for the fluorescence measurements. Comment on the choice of such a high light intensity (and high intensity pulses, actinic effect?). Why not use a light intensity closer to the HL treatment? Also state length of dark-acclimation prior to each type of measurement.

- Immunoblot method, state dilutions used and secondary antibody type and dilution used.

- Page 4, it is stated that “the level of the other LHCBMs is similar to that of WT (CC-425)” by referring to Fig. S1 and S2. It is difficult to see in Fig. S1 that the level of other LHCBMs is similar in npq5 and WT as the data is represented in percentage of total LHCBMs. Could you also represent the amount of the different LHCBMs in npq5 normalized to WT in a bar graph? For the Type III and Type II/I LHCBMs accumulation, a dilution series might be best for quantification to ensure that CP47 antibody signal is not saturated.

In Fig. S1, npq5 should be italic. In Fig. S2, one of the labels is likely wrong: there are two 48h time points. By biological replica, is it meant three independent batches of grown and treated cells? What is the fourth band right below the upper band detected by the LHCBM5 antibody at 48h?

- Fig. 5, please enlarge panels B and C to match panel A.

- Fig. 6, specify in the legend that 24h and 48h refer to the HL treatment.

- Discussion, “and confirmed here by analysis of the [npq5] mutant” (not npq4)

Jingfang Hao and Pierrick Bru (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Aurélie Crepin, Fadime Demirel, Jack Forsman and Domenica Farci.