PREreview of Circadian and diel regulation of photosynthesis in the bryophyte Marchantia polymorpha

The manuscript by Cuitun-Coronado et al. investigates the effects of circadian and diel cycles on the photosynthetic processes of the liverwort Marchantia polymorpha. The authors clarified three points: 1) they confirmed that M. polymorpha does display circadian regulation of several photosynthetic parameters; 2) they showed that light-dark cycles mask circadian cycles for these parameters; 3) by using a pharmacological approach, they showed that chloroplast translation is necessary for the circadian regulation of photosynthesis.

The study fills a gap of knowledge in the circadian regulation of photosynthetic activity across evolution, by providing an additional link to what was known in cyanobacteria, microalgae, and vascular plants. It is original and well-conducted. The interpretation and the discussion of the results is very convincing, and the conclusions are carefully drawn and backed up by the data.

Major comments

• Page 7, lines 173-176: it is stated that the differences in period length observed between PAM and DF measurements might be due to differences in light intensity. How about light quality, as the PAM measurements were performed under blue light, vs. mixed red/blue for DF?

• Blue light, which was used in both types of experiments, has been shown to impact circadian rhythms in plants through the action of cryptochromes (e.g. Chaves et al., Annu. Rev. Plant Biol., 2011). Also, BL is responsible for chloroplast relocation responses, which is mediated by phototropins (e.g. see Kong et al. Plant and Cell Physiology, 2013). In Arabidopsis it can also impact plastid transcription through the intermediate of the blue-responsive plastid sigma factor SIG5 (Tsunoyama et al, FEBS Lett. 2002) and it is regulated by BL photoreceptors cryptochromes (Onda 2007, Plant Journal). Can you comment on these potential effects? Has their involvement in Marchantia polymorpha been investigated? Did they motivate your use of blue light for these experiments?

• Page 13, lines 316-323, please discuss the lack of effect on circadian rhythm by rifampicin: at first it may appear odd that translation has an effect but transcription doesn’t. Could it be due to rifampicin only inhibiting PEP and transcription by NEP compensates?

• Page 14, lines 354-363:
  1. Is there a reason why you performed light curves with increasing light intensity up to 789 µmol m^-2 s^-1 if you end up using only the data with light intensity closer to growth conditions (108 µmol m^-2 s^-1)?
  2. Could the 20 minutes of dark-adaptation every two hours and increasing light intensity up to 789 µmol m-2 s-1 have affected the circadian rhythms of plants that were supposed to be at constant LL? This experimental setup could be considered as fluctuating light condition. Please discuss whether this may have an effect on the observed results.

Minor comments

• Page 2, lines 27-28, "the circadian regulation of several measures of photosynthetic biochemistry (delayed fluorescence, the rate of photosynthetic electron transport, and non-photochemical quenching of chlorophyll fluorescence)": please rephrase this sentence as it seems that the circadian regulation has an effect on the measurements themselves while they are tools to see something happening at photosynthetic levels. We suggest replacing it with "circadian regulation affects photosynthesis performances, detectable by several photosynthetic parameters such as delayed fluorescence, the rate of photosynthetic electron transport, and non-photochemical quenching of chlorophyll fluorescence". Same can be applied in Pages 4-5, lines 98-99.

• Page 4, lines 84-90, this sentence is very long and difficult to read, please rephrase and split it into at least two sentences for better readability and clarity.

• Page 5, lines 114-118, discussion might be a better place for this.

• Page 5, line 118, please replace "Features of PAM fluorescence" with "Features of chlorophyll fluorescence as measured by PAM" as the first indicates a specific method to measure an intrinsic phenomena (Chl fluo) and it is not the only one for Chl fluo measurements.

• Page 5, line 123, please replace "PAM metrics" with "prompt fluorescence" for consistency with DF, named just before.

• Page 6, lines 126-127, to be consistent with the nomenclature, "LL" should be "low light", not "constant light". Consider replacing LL with CLL ("constant low light") since the plants are actually exposed to a lower light compared to the standard growth light described in materials and methods. Also, please include in the description of the light conditions “LD” together with its full name (which is directly mentioned before in line 138 without its full name "light-dark cycles").

• Pages 6-7, lines 152-154, this sentence is more technical and it does not seem necessary to understand and interpret the results described later. Please consider moving this sentence in Material and methods to keep the results section more readable.

• Page 7, lines 167-172, please provide a reason why photosynthetic activity was measured only under LL and not also in the other two conditions described for DF.

• Page 8, lines 196-199, reading the text it seems you used only T24 and T28. Please list all the T lengths (T20, T22, T24 and T28) as reported in Figure 3 legend.

• Page 10, line 254, please add a short sentence to briefly comment the results obtained with the rifampicin treatment.

• Page 13, lines 316-318, "Perhaps inhibiting the expression of these proteins prevents or reduces PSII repair, which disrupts circadian cycles of the rate of electron transport by interfering with the electron transport process". Please rephrase this sentence, as it is written it can be interpreted as defective PSII repair disrupts circadian cycles.

• Page 13, line 335, describe conditions of its natural habitat “such as”.

• Page 14, lines 341, in the growth conditions you describe that the standard light conditions used were 100 μmol photons m^-2 s^-1 of white light. Is there a reason why for the DF experiment described in this manuscript you decided to use 60 µmol m^-2 s^-1?

• Page 18, legend of Figure 1:
  • Line 421, “thallus” should be “thalli”; “light/cycles” should read “light/dark cycles”.
  • Line 430, define FFT-NLLS.
  • Please move the definition of RAE from lines 433-434, to its first occurrence line 430.
  • Line 433, define SEM.

• Page 19, line 470, please write SEM instead of s.e.m., for consistency with the legend of Figure 1.

• Figure 1:
  • the figure is very big and can create confusion, please consider to split it in two figures, e.g. a-f Figure1 and g-k Figure 2.
  • In the legend, include the abbreviations for the light conditions and correct "ligh/cycles" with "light-dark cycles" as reported in the text.
  • Panel g. Please better describe this panel, especially the colours meaning in the clock.

• Figure 1 (a,c,e) 2 and Figure 4: the alternance of white and light grey bands to mark the 12h periods can be very easily misinterpreted as light/dark cycles. To clarify and display the constant light conditions at a glance, please consider using another means to evidence them, such as dashed lines.

• Figure 4:
  • similarly to Figure 1, this figure is very large and hard to read. Consider splitting it.
  • Please write the concentrations of lincomycin and rifampicin in the relevant panels, as the different shades are difficult to distinguish and understand in panels a-d, g-j, m-p and s-v.
  • Panels f-l and r-x: please use the same scale on the y axis, or, if not possible, bring attention of the reader to the different scale in the figure legend. At first, we were puzzled by the ‘no-treatment’ not starting from similar amplitudes.

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