The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha

Serine metabolism is involved in various biological processes. Here we investigate primary functions of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH encoding 3-phosphoglycerate dehydrogenase in this pathway. Growth phenotypes indicate that serine from the phosphorylated pathway in the dark is crucial for thallus growth. Sperm development requires serine from the phosphorylated pathway, while egg formation does not. Functional MpPGDH in the maternal genome is necessary for embryo and sporophyte development. Under high CO2 where the glycolate pathway of serine biosynthesis is inhibited, suppressed thallus growth of the mutants is not fully recovered by exogenously-supplemented serine, suggesting the importance of serine homeostasis involving the phosphorylated and glycolate pathways. Metabolomic phenotypes indicate that the phosphorylated pathway mainly influences the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lipid metabolism. These results indicate the importance of the phosphorylated pathway of serine biosynthesis in the dark, in the development of sperm, embryo, and sporophyte, and metabolism in M. polymorpha.

The manuscript presented by Wang et al. describes the requirement of phosphorylated serine for Marchantia growth and development.The authors provide good evidence for the expression profile of the MpPGDH enzyme and they also provide detailed phenotypic characterization of Mppgdh mutant vegetative and reproductive development specifically when grown under long-day conditions.Transcript profiling by qRT-PCR and metabolomics/lipidomics profiling further characterizes the contribution of the phosphorylated serine pathway to liverwort growth and development.I'm supportive of the manuscript overall but there a couple of issues that need to be addressed.
1. I'm somewhat confused by the result that Mppgdh-1 does not have a defect in endogenous serine.This would argue that serine isn't the primary cause for the observed phenotype.Can the authors provide any explanation on this key issue?Is it more appropriate to be measuring metabolites specific to the phosphorylated serine pathway directly?This point should be clarified in line 147 to keep the reader on track.
Discussion lines 394-397: since serine supply is often referred to throughout the paper I think it is important to expand on this section.If it isn't serine per se that is impacted, what is it?This is critical to all interpretations of the work.
2. Since the main focus of this manuscript is the phenotypic characterization of Mppgdh mutants, having data to demonstrate genetic complementation, even if only performed for growth phenotypes, would be an appropriate/important control to include.

Minor points:
Line 98: It might be worth adding 'in vitro' here to clarify that your previous study was focused on the function of purified proteins.This may provide a clearer distinction when introducing your new study.
In fig 2d: this should be expressed as a standardized value.Sperm cells per antherdia appears to be more appropriate when comparing two genotypes.
Figure S5: This is a useful piece of data but it misses a CL control that was not moved to dark.This would be useful to show that changes are due to environmental factors and not part of the circadian rhythm.Instead of performing additional experiments, I'm wondering if the authors can take advantage of existing circadian rhythm expression datasets (perhaps this set: https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.17653)to address this potential issue.
The term 'reproduction branches' appears to be inconsistent with the field, which often refers to these as gametophores.Is this what the authors are referring to here?Also, the images in Fig S16a are fairly low resolution.
The authors present an interesting study on functions of serine from the phosphorylated pathway on growth, male gametogenesis, and metabolism in liverworts.There is a comprehensive dataset on genetics, physiology and omics.The authors prepared high quality figures and the manuscript is well written.I only have a few minor comments.1.In the title, "Functions of serine from the phosphorylated pathway" is a little vague.The authors are suggested to modify the title that will highlight the major findings of the paper.2. Keywords are missing from the manuscript.The authors may need to add some based on the requirement of the journal.8. Figure 10: The high CO2 experiment is one of the highlights of the paper.The authors should justify the use of 3000ppm of CO2 for 14 days.What is the optimal CO2 level for liverworts from an evolutionary point of view?The plants were grown on media with no nutrient limitation.However, the high CO2 + serine treatment may have a different impact if the plants are grown in pots or in the natural environment where mineral nutrients are limiting.The authors may want to discuss this as well.
We greatly appreciate the valuable comments and suggestions provided by the editor and the reviewers.We have performed additional experiments and addressed the discussion points as recommended by the reviewers, although only genetic complementation experiments have not been completed due to technical reasons (please refer to one-by-one responses below).While revising the manuscript, we discussed with an expert regarding the interpretation of the results related to the defect in sperm development observed in Mppgdh mutants.Accordingly, the main text has been slightly revised (highlighted in red in the revised manuscript).In the original version, the Abstract and the main text exceeded the character limit, leading us to condense them without altering the meaning.
The following are our one-by-one responses to the reviewers' comments.

Reviewer #1 (Remarks to the Author):
The function of the phosphorylated pathway of serine biosynthesis (PPSB) in a non-vascular plant has never been addressed.This manuscript nicely describes the characterization of PGDH, the ratelimiting enzyme of the PPSB in the model bryophyte species Marchantia polymorpha.This work confirms findings found in higher plants, such as the crucial role of PPSB in vegetative growth, or its requirement for sperm formation or sporophyte development (embryo development in higher plants), even though the species are so far apart phylogenetically.This would confirm the essentiality of PPSB in the plant kingdom.
The introduction is clear, concise, and updated and the results were rigorously carried out.The discussion could be improved.I have some comments that may help to improve the manuscript.2) The authors found that the PPSB mutants show retarded growth under light/dark conditions but not under continuous light conditions.However, to determine whether the poor growth of the mutant thalli was due to insufficient endogenous serine supply, they measured the amino acid content during the light period and did not find differences in serine content in one of the mutants.Why does one mutant show serine deficiency while the other does not show it?These points should be clarified.Furthermore, in my opinion, in order to see serine insufficiency, it would have been more convenient to measure the serine content during the dark period.In fact, the authors demonstrated that exogenously added serine could revert the retarded growth phenotype, which suggests a serine deficiency in the mutants.It is also possible that there is no serine insufficiency in the mutants at the whole plant level.It may be that the serine deficiency is restricted to specific cell types involved in growth.All these considerations should be addressed.
Thank you very much for your comments.We performed additional experiments to sample thalli during the dark period and analyze amino acid contents.The result was shown in Fig. 2 in the revised manuscript.After 4 h in the dark, the serine amount in Mppgdh mutants was reduced significantly at the whole plant level compared with wild types.After 2 h in the light period, the serine content in Mppgdh mutants was recovered to wild type-level.We added the following sentences in Results and Discussion.
L136-139: 'The serine content in Mppgdh mutants was significantly decreased after 4 h in the dark and restored to wild type-level after 2 h in the light (Fig. 2).These results suggested that reduced serine content during the dark period caused the poor growth in Mppgdh mutants under L/D conditions.' L345-348: 'Furthermore, the serine content in Mppgdh mutants was strongly reduced during the dark period and rapidly restored to wild-type level under light (Fig. 2).This indicates that the decreased serine supply during the dark period caused poor growth of Mppgdh mutants under L/D conditions.' 3) The result section "DNA -dependent processes…" is too long and difficult to read.It could be considerably shortened by eliminating all negative results.
During revising the manuscript, we discussed the qRT-PCR result with Prof. Takashi Araki in Kyoto University, whose expertise is sperm development in Marchantia polymorpha.According to your comments and Prof. Araki's advice, we deleted the section "DNA-dependent processes are impaired during gametogenesis in male Mppgdh mutants" and modified the section "MpPGDH-mediated serine synthesis is essential to sperm formation" as follows.
L162-172: 'However, in the Mppgdh mutants, spermatid mother cells were in various sizes and irregular shapes (Fig. 3f, bottom left), and subsequent diagonal cell division to generate spermatids was barely observed (Fig. 3f, bottom middle and right).The qRT-PCR analysis of sperm differentiation-related genes (Supplementary Fig. 8) revealed that the expression of MpPRM, which is expressed specifically in sperm during late spermiogenesis 37 , were significantly repressed.On the other hand, the expression level of MpDUO1, which is a key transcription factor of MpPRM and expressed in spermatid mother cells and spermatids 36 , was similar in the wild type and the mutants.
Additionally, a member in MpHMGBOX gene family, MpHMGBOX4 was also strongly repressed in the Mppgdh mutants.These results support the notion that cell division of spermatid mother cells to generate spermatid was blocked in the Mppgdh mutants.'Thank you very much for giving us an interesting point of view.Because Marchantia polymorpha is a basal land plant, it is reasonable to consider that Marchantia polymorpha has optimal CO 2 level which differs from those of Arabidopsis thaliana and other angiosperm.It is reported that different plant species have different optimal CO 2 concentrations.For example, Lee et al.
(https://doi.org/10.1007/s00442-010-1572-x)showed a substantial increase of the biomass of some plants when CO 2 concentration increased from about 350 ppm to 700 ppm, but the CO 2 fertilization effect on plant growth declined or vanished beyond certain CO 2 concentrations (such as 1000 ppm).
In this study, we used the 3000 ppm CO 2 condition based on other studies (for example, Benstein et al. (2013) Plant Cell; https://academic.oup.com/plcell/article/25/12/5011/6098476).We apologize for not being able to provide the optimal CO 2 level for Marchantia without checking its growth under various CO 2 concentrations.
Previous studies demonstrated that the PPSB is associated with nitrogen and sulfur metabolism.Furthermore, in this study, abundant serine supplementation had a significant impact on entire metabolism probably by perturbing carbon, nitrogen, and sulfur metabolism.We completely agree with your view that the high CO 2 + serine treatment may have a different impact if the plants are grown in pots or in the natural environment where mineral nutrients are limiting.According to the comments from you and Reviewer 1, we modified the discussion as follows.
L352-366: 'The Mppgdh mutants and the wild type grew better and appeared darker green under high CO 2 (3000 ppm) than under ambient CO 2 (Fig. 9a, b and Supplementary Fig. 14a, b), probably due to enhanced photosynthesis under high CO 2 .This result was in contrast to a study using A. thaliana, in which elevated CO 2 (4000 ppm) improved the biomass of the wild type but not AtPGDH1 silencing lines 39 .The optimal CO 2 concentration for growth may vary depending on plant species and environmental factors such as nutrient availability and light intensity.Under ambient CO 2 , the Mppgdh mutants continued to grow very slowly, but eventually reached a size comparable to that of the wild type.On the other hand, under high CO 2 , the mutant hardly grew further and exogenous serine supplementation did not promote thalli growth in the mutants as much as in the wild type.This indicated that serine is a limiting factor for growth under high CO 2 conditions, but metabolic homeostasis is crucial.Even wild type grew slower and produced fewer sperm under elevated CO 2 than under ambient CO 2 (Supplementary Fig. 16), suggesting high CO 2 widely affects metabolism and other limiting factors than serine for growth and development in M. polymorpha also exist under this condition.' 3. Introduction: Pages 3 and 4: There are lots of text on the PGDH in Arabidopsis.The author need to add relevant studies on PGDHs in other important species such as rice (there are three OsPGDHs) and Physcomitrium patens (there are four PpPGDHs).4. Add an overarching hypothesis to the last paragraph of the Introduction 5. Can the authors please explain the significant growth phenotype of Mppgdh-2 in terms of low Fresh Weight in Figure 2? 6. Figure 5: please add the full gene names in the Figure legend.7. Figure 8: there are only small number of differentially regulated lipids in the Mppgdh mutants.The authors should add full name of those lipids shown in the Venn diagrams (c,d) in the Figure legend.7. Figures 8 and 9 should be combined into one large figure.
photosynthetic tissues (Supplementary Fig.1).•••••• In dark environments and non-photosynthetic tissues, two other pathways are responsible for serine synthesis, namely, the glycerate and phosphorylated pathways.'InResult L230-232 'In the PCA score plot, Mppgdh-1 formed the clusters separated from those of Tak-1 not only under L/D condition but also under CL condition, indicating that the PPSB also works at daytime.'

Figures 8
Figures 8 and 9 were combined into one large figure as 'Fig.8 Changes in lipidome in the Mppgdh mutants.'