Integration of a Galdieria plasma membrane sugar transporter enables heterotrophic growth of the obligate photoautotrophic red alga Cynanidioschyzon merolae

Abstract The unicellular thermoacidophilic red alga Cyanidioschyzon merolae is an emerging model organism of photosynthetic eukaryotes. Its relatively simple genome (16.5 Mbp) with very low‐genetic redundancy and its cellular structure possessing one chloroplast, mitochondrion, peroxisome, and other organelles have facilitated studies. In addition, this alga is genetically tractable, and the nuclear and chloroplast genomes can be modified by integration of transgenes via homologous recombination. Recent studies have attempted to clarify the structure and function of the photosystems of this alga. However, it is difficult to obtain photosynthesis‐defective mutants for molecular genetic studies because this organism is an obligate autotroph. To overcome this issue in C. merolae, we expressed a plasma membrane sugar transporter, GsSPT1, from Galdieria sulphuraria, which is an evolutionary relative of C. merolae and capable of heterotrophic growth. The heterologously expressed GsSPT1 localized at the plasma membrane. GsSPT1 enabled C. merolae to grow mixotrophically and heterotrophically, in which cells grew in the dark with glucose or in the light with a photosynthetic inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) and glucose. When the GsSPT1 transgene multiplied on the C. merolae chromosome via the URA Cm‐Gs selection marker, which can multiply itself and its flanking transgene, GsSPT1 protein level increased and the heterotrophic and mixotrophic growth of the transformant accelerated. We also found that GsSPT1 overexpressing C. merolae efficiently formed colonies on solidified medium under light with glucose and DCMU. Thus, GsSPT1 overexpresser will facilitate single colony isolation and analyses of photosynthesis‐deficient mutants produced either by random or site‐directed mutagenesis. In addition, our results yielded evidence supporting that the presence or absence of plasma membrane sugar transporters is a major cause of difference in trophic properties between C. merolae and G. sulphuraria.

We have fixed the error.

Reviewer #2:
In this paper, the authors propose a novel tool for studying photosynthesis in C. merolae, by use of a sugar transporter from Galderia to enable heterotrophic or mixotrophic growth. Their approach has merit and is generally well-documented. They comprehensively characterize and demonstrate heterotrophic growth by combinations of light/dark treatments, DCMU treatments and glucose titrations in SPT1 expressors and WT. There are a few concerns which, when addressed, would improve the quality of the manuscript.
Thank you very much for your positive evaluation on our manuscript. We have revised the manuscript according to your suggestion as describe below. Thank you for your important suggestion. We have newly added an immunoblotting result showing SPT1 level (expressed by CPCC promoter) in the culture in the dark with glucose and that in the light with glucose and DCMU (Fig. 4b). A previous paper showed that CPCC mRNA level in the dark was lower than that in the light under photoautotrophic condition (Kawase et al., 2017). However, in our result in heterotrophic conditions (Fig. 4b), SPT1 protein level was not so much different between CP-SPT1 in the dark with glucose and that in the light with glucose and DCMU. Therefore, the difference in the growth will be because of reasons other than the light response of CPCC promoter. We have added the description of the result and discussion in the text (Page 22, lines 468-475).

Major Comments
2. Making C. merolae hetero or mixotrophic could indeed help advance studies of photosynthesis in this unique red algae, but there should be some comment on the altered physiology resulting from direct sugar import. For example, what is known about sugar sensing in C. merolae or related species?
Thank you very much for your comment. We have added following statements in the text (Page 26, lines 564-576).
Regarding the physiological changes resulting from direct sugar uptake in Thus, it is probably feasible to obtain photosynthesis-deficient mutants of G. sulphlaria by random mutagenesis. However, further molecular genetic analyses will be limited because G. sulphuraria is not genetically tractable. In addition, a rigid cell wall of G.
sulphlaria hampers extraction and fractionation of cellular contents in mild processing conditions. This situation contrasts with C. merolae which does not possess a rigid cell wall.  Table 1. 8. Line 327: Could the similar initial growth rates and higher final growth level be due to exhausting the glucose in the media? Could this also indicate transport limitation? It should be noted how the transformed lines will be made available to other researchers.
Thank you very much for pointing it out. We have added following statements (Page 16, lines 340-344).
The similar initial growth rate among different concentrations of exogenous glucose was probably because of limitation of glucose uptake activity of the HA-SPT1 proteins.
In addition, the difference in their final cellular concentrations was probably because of exhaustion of exogenous glucose.
We have added following statement in Acknowledgements (Page 27, lines 601-602).
The strains described in this manuscript are available from TK upon request. 9, Line 346: This is an interesting observation since this is about the same time that growth levels off in Ap-SPT1 under 25 mM glucose and no DCMU.
Yes, the growth of Ap-SPT1 cells with DCMU and glucose was suddenly accelerated from day 29 to 41 until exogenous glucose was exhausted. The final cellular concentration and the duration for reaching the concentration were almost the same between the culture in the dark with glucose and that in the light with glucose and DCMU (Fig. 2b). However, the reasons remain unclear at this point.
10. Line 469: Why is it considered a putative sugar transporter here, but seems more confirmed in other parts of the paper.
Thank you for your comment. We agree with the comment. We have removed "putative" because the sugar uptake ability of GsSPT1 was already shown in a previous study (Schilling and Oesterelt 2007).
11. Line 526: The wording in this sentence could be improved. Perhaps "kept" could be removed.
We have removed "kept" according to your suggestion.
12. Line 547 to the end of the paragraph: The wording of this section could be improved.
When a photosynthesis-deficient C. merolae mutant exhibits a similar light-sensitive phenotype, periodic light pulsing is likely effective to improve heterotrophic culture conditions according to the following finding. In C. merolae, a daily light pulse (10 min of 30 µmol m -2 s -1 ) was required for continual heterotrophic growth with 200-400 mM glycerol (Moriyama et al., 2018). Such light-activated heterotrophic growth was also reported in Synechocystis sp. PCC6803 (Anderson and Mcintosh, 1991). In Synechocystis sp., it is assumed that light pulse likely functions as an environmental signal to regulate heterotrophic metabolisms, cell division, and other cellular activities (Anderson and Mcintosh, 1991).
We believe our manuscript has been improved, thanks to the referees, and hope it is now acceptable for publication in Plant Direct. We look forward to your decision.