PREreview of Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation

The manuscript by Seydoux et al. investigates the role of proton potassium antiporter KEA3 in diatoms. The authors first demonstrated the pH dependence on photoprotection, specifically non photochemical quenching (NPQ) and showed that NPQ can be induced in the dark by acidic pH. They found that KEA3 modulates NPQ by impacting the proton motive force (PMF); indeed generated kea3 mutants showed increased partitioning into deltapH. Importantly they showed that diatom KEA3 in contrast to plant KEA3 possesses an EF hand motif which can bind Ca2+ and proposed that it controls KEA3 activity. The role of KEA3 and pH in affecting the NPQ response has been previously shown in other photosynthetic organisms however the novelty of this study lies in the demonstration that NPQ can be induced in the dark by acidic pH and the proposed role of Ca2+ in regulating KEA3 function.

Major comments

-      Page 5, you state that pH-induced quenching in the dark was accompanied by the conversion of DD into DT. Please provide de-epoxidation state (DES) at t15 time (Fig. 1B) to substantiate this statement. Starting DES would also be informative to ensure there was no retention of DT/zeaxanthin in the dark.

-      Also to ensure there is no sustained NPQ (and/or damage or disconnected antenna) at t0, please provide Fo and Fm levels for all NPQ kinetics experiments. Assessing PSII accumulation by D1 immunoblot could be done to ensure PSII damage does not occur.

-      In Fig. 2F, it is not clear which data points represent HL or ML treatment as well as which ones come from light or dark period. Please indicate them in different colors or symbols. Also clarify whether you have averaged data from the kea3 mutant alleles.

-      To confirm that lack of complementation by deltaEF is not due to mislocalization, please show whether deltaEF accumulates at the thylakoid membrane.

Minor comments

-    Page 3, Introduction, specify qE after NPQ response; PSBS should be written PsbS

-     Page 4, DD-dependent NPQ should be DT-dependent

-   Page 4, we suggest changing “crucial” to “Given the unknown role” if pH-dependence of NPQ in diatoms hasn’t been fully established before

-   Page 8, KEA3 most likely homolog, were there other homologs than the two shown in Fig. S5? also discuss conservation of other ion channels (is Phatr J11843 thylakoid-localised?) and if they could compensate for the absence of KEA3 in KO mutant (by being upregulated for instance).

-     Fig2B, comment on the band at ~80kDa in OE, is that from cleavage of GFP?

-    Fig2G, shouldn’t you expect a lower dpH in the OE? Please comment.

-    Page 13, for the statement that only dpH can modulate NPQ, we would suggest to tone down or specify that this is the assumption made here as it could be that dpsi modulates NPQ but has yet to be shown!

-   Most of the protein analyses were performed loading samples based on protein content, when possible please provide proof that chlorophyll levels are comparable between the genotypes (at least for the native gels)

-    Abstract, extra ‘of’ between capacity and via; page 23, extra ‘being’ between likely and less important

-    Define acronyms when used for the first time

-    There is a lot of ‘peculiar’ in the text

-    Fig. 2D, star symbol instead of square symbol, check consistency of symbols

Pushan Bag, 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, Maria Paola Puggioni, Jingfang Hao, Jack Forsman, Wolfgang Schröder, Emma Cocco, Jianli Duan.