PREreview of Evolution of chlorophyll degradation is associated with plant transition to land

Schumacher et al. discuss the metabolic pathways evolved by green plants to degrade chlorophyll molecules. The authors combined a large-scale comparative phylogenomic approach with biochemical characterization of putative novel phyllobilins to shed light on how the degradation pathway evolved. This manuscript points to the evolution of chlorophyll degradation, in particular the later detoxification steps, having accompanied the green lineage’s transition to land. An extensive list of orthologous genes from a diverse number of species was identified.

The manuscript stands out for the wide evolutionary view on the chlorophyll degradation pathway, which is neither an easy nor a common research subject. The in silico approach for phyllobilins identification is quite innovative and will surely give great hints for biomolecules discovery. A comprehensive bioinformatics work has been done to carefully identify and select the genes analyzed in the manuscript and the related phylogenetics figures are outstanding.

Although this work is of great interest, we have some comments that could be addressed in the next version.

Major comments

• We would suggest to tone down the title as it may be that less or no chlorophyll catabolites were detected in mosses, charophytes and chlorophytes due to the smaller number of species analyzed (Fig.5), and some of these clades may have evolved other phyllobilins exporting and modifying proteins. Please discuss these possibilities.
• Page 9, line 16: regarding the identification of 15 putatively novel phyllobilins, mass spectrometry data together with in silico produced list of diagnostic ions are presented to support these structures. Would it be possible to provide additional confirmation via a standard (an internal or a synthesized one) or alternatively to state which other molecules these m/z plus profiles could be corresponding to? How likely is it that they correspond to other unrelated compounds?
• Page 5, line 11, comment on two species lacking CAO. Are they lacking chlorophyll b?

Minor comments

• Intro, page 3, line 19, explain why it is unlikely that nitrogen remobilization be a conserved evolutionary trigger.
• Page 8, line 15, what is the carbon source used for heterotrophic growth? Line 19, instead of “etc” list all conditions tested.
• Page 9, line 24, could you discuss or introduce whether oxidations are spontaneous or enzymatic?
• Could you comment in the discussion about the significance of Chl breakdown catabolites in cellular signaling from an evolutionary point of view?
• Methods, in the paragraph “Plant material growth and chlorophyll degradation induction”, page 15, lines 1-12: several of the growth conditions are missing e.g. at which temperature S. moellendorfii was growing? At which temperature and humidity was the dark incubation of leaves performed?
• In the Sup. Figure 6 B, the structure of phyllobilin 3b is not shown. It could be nice to have that too or to explain why it is not shown. Please, also include the relevant spectra from the remaining identified compounds as supplemental data (assuming that the 4 presented spectra/structures are also of novel compounds).
• Maybe we missed it, but where can we find the identifiers of the genes that were used in the gene trees shown Figures 2, 3 and 4?
• The Sup. Table 1 is first cited in the Methods. Maybe, cite it in the introduction (page 4, line 12) in order to cite it chronologically with respect to Sup. Table 2.
• Page 9, lines 3 and 23, “SFig. 6” should read SFig. 4.
• Page 5, line 34, please indicate the gene names corresponding to the abbreviation for SGR2 and SGL.
• Minor grammatical errors and typos are present in the text, e.g. page 24:
• line 24, “loss” should be “lost”
• line 29, “one orthogroups” should be “one orthogroup”
• line 33, “do belongs” should be “does belong”
• line 35, “expect” should be “except”
• line 36, “have derived a pre-existing enzyme” should be “have derived from…”

Domenica Farci, Sam Cook (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, Aurélie Crépin, André Graça, Jack Forsman, Jingfang Hao, Pierrick Bru, Jianli Duan.