つくば生物ジャーナルⒸ2024 筑波大学生物学類

Tsukuba Journal of Biology (2024) 23, 74

Role of chlororespiration in the high CO2 tolerance of unicellular red alga Galdieria sulphuraria
Belitz Alisa Marie（筑波大学 生物学類）

指導教員：蓑田Introduction
While CO2 is essential for photosynthesis, the growth of mostphotosynthetic organisms is inhibited when CO2 levels surpass
2-5 percent (Solovchenko and Khonzin-Goldberg 2013). A few
algal species have been reported to tolerate higher CO2 levels,
including the unicellular red alga Galdieria sulphuraria. G.
sulphuraria is isolated from acidic hot springs and has beenreported to grow in up to 100% CO2 conditions. However, themechanism of high CO2 tolerance in G. sulphuraria is unknown.

歩（筑波大学 生命環境系）

Results and discussion
1. Construction of NDA2 and PTOX knockout mutants
1.1 Isolation of haploid cells
Diploid G. sulphuraria are surrounded by a thick cell wall thatprohibits genetic modification. Cell wall-less, haploid cells of G.
sulphuraria were induced by decreasing the pH of culture media
as described by Hirooka et al (2013). Isolated cells were used fortransformation.
1.2 Genetic modificationThe coding regions of the target NDA2 or PTOX genes alongwith the approximately 1 kbp upstream and downstream
regions were amplified by PCR from genomic DNA. The coding
regions were replaced by linear DNA containing a blasticidin S(BS) selectable marker. The resulting DNA was used fortransformation by homologous recombination using apolyethylene glycol (PEG)-mediated method. As selection by BS
has not yielded positive transformants, we are examining
transformation and selection conditions. After successfultransformation, we plan to evaluate the phenotype under highCO2 conditions.

In a previous study by our group, G. sulphuraria was suggested
to optimize the light-dependent reactions of photosynthesis by anovel process that prevents the overreduction of theplastoquinone pool under high CO2 conditions (Ozeki 2021).
Transcriptome analysis indicated the upregulation of a type II
NADPH dehydrogenase (NDA2) and a plastid terminal oxidase(PTOX) under high CO2 in G. sulphuraria. In green algae,
NDA2 and PTOX have been reported to be involved inchlororespiration, the reduction of oxygen using electrons fromthe plastoquinone pool, which can serve as an alternativeelectron outlet for alleviating excess reducing power (Saroussi etal. 2023). Thus, NDA2 and PTOX were identified as candidate
enzymes for optimizing electron transport in G. sulphuraria
under high CO2 conditions. This research aims to investigate therole of NDA2 and PTOX in the tolerance of G. sulphuraria toextremely high CO2 levels.

2. Effect of PTOX inhibition
Propyl gallate (PG) is known as a PTOX inhibitor. The effective
concentration of PG for G. sulphuraria was determined due to alack of previous reports. Growth rate of cells decreased in thepresence of 1 mM PG in either air or 100% CO2 conditions. This
concentration had no immediate effect on respiration rate andwas consistent with that used in studies of PTOX using green
algae (Saroussi et al. 2023). We plan to measure the chlorophyll
fluorescence of cells with and without the addition of PG using
PAM to evaluate the effect on photosynthetic electron transport.
Results will be discussed in the oral presentation.
References
Hirooka et al. (2022), PNAS. 119(41)Ozeki (2021), University of Tsukuba Thesis
Saroussi et al. (2023), Plant Phys. 192, 789-804
Seckbach et al. (1970), Nature. 227, 744-745
Solovchenko and Khonzin-Goldberg (2013), Biotechnol Lett.
35(11), 1745-1752

Fig.1 Schematic representation of the role of NDA2 and PTOX
in the photosynthetic electron transport chain.
Materials and Methods

Culture: G. sulphuraria cells were grown at 40℃ in 2 x Allen’s
medium adjusted to pH 2.5 or pH 1 for diploid or haploid cells,
respectively. Cells were bubbled with air or 100% CO2 andexposed to continuous light (90±3 μmol photons m-2 s-1). Cell
growth was evaluated by OD750.
O2 electrode: Respiration rate of cells was measured at 40℃
using Oxytherm (Hansatech).

Acknowledgements
We would like to thank Professor Yoshihisa Hirakawa for his
assistance in isolating haploid cells and Professor Shinya
Miyagishima and Dr Shota Yamashita for their guidance on thePEG-mediated transformation method.

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