Plastoquinone measurements

The redox state of the PQ-pool of C. reinhardtii was measured with a method designed for plants (Kruk and Karpinski 2006; Mattila et al. 2020) with modifications for cyanobacteria (Khorobrykh et al. 2020). Samples were prepared by filtrating approximately 10–15 × 10⁶ cells, determined spectrophotometrically from a known relationship between OD₇₃₀ and cell density (calculated with a cell counting chamber), on a glass microfiber filter with pore size of 1.6 µm (VWR, USA, Cat. No. VWRI516-0862). All illumination treatments were done to cells on the filters. PQ was always rapidly extracted by grinding the filter in a mortar in dry ice-cold ethyl acetate under the respective treatment light or in the dark for dark treatments. The further preparation of the samples for HPLC was done as described by Khorobrykh et al. (2020). The redox state of total PQ was measured from two HPLC samples, one run without and one with addition of 5 mM of NaBH₄.

For the estimation of the size of the photochemically active PQ-pool, the redox state of the extracted PQ was measured after fully oxidizing and after fully reducing light treatments. For maximal oxidation, cells were treated with far-red light (> 700 nm) with the photon flux density (PFD) of 50 µmol m⁻² s⁻¹ for 10 min (see Fig. S1 for the spectra). PFD was calculated from measurement with an STS-VIS spectrometer (Ocean Insight, Ostfildern, Germany, D-73760). For maximal reduction, cells were illuminated for 30 s with strong white light with photosynthetic photon flux density (PPFD) 2000 µmol m⁻² s⁻¹.

To test alternative methods for PQ-pool reduction and oxidation, treatments were repeated in the presence of 5 µM 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) and 20 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), respectively. These artificial quinones were added 1 min prior to the illumination with either far-red (DCMU) or high light (DBMIB) as described above. After establishing the PQH₂/(PQH₂ + PQ) ratio at full reduction and oxidation of the photochemically active PQ-pool, the redox state of the PQ-pool of an any sample could then be calculated from Eq. (1) (Kruk and Karpinski 2006) as

where F_SAMPLE represents the ratio PQH₂/(PQ + PQH₂) in an unknown sample, obtained by measuring an aliquot of the same sample with and without addition of NaBH₄ that reduces all PQ in the sample. F_OXIDIZED and F_REDUCED refer to the same ratio in reference data obtained after full oxidation of the PQ-pool with far-red light treatment and after full reduction of the PQ-pool with a short treatment with high light, respectively.

PQ-pool redox state was examined from the growth conditions by illuminating cells on a filter with the respective growth PPFD, 100 or 50 µmol m⁻² s⁻¹, as indicated, for 5 min.

Custom-built white light sources favoring PSII or PSI were used. These types of white light were obtained by combining equal PFD of either 430, 520 and 690 nm narrow-band light (PSI light) or 470, 560 and 660 nm light (PSII light) (See Fig. S2a for spectra). LEDs equipped with 10 nm half-width at half maximum optical filters transmitting 430, 470, 520, 560, 660 or 690 nm light (Andover Corporation, Salem, New Hampshire) were used, respectively. These wavelengths were chosen because they have been shown to favor PSII or PSI in plants (Mattila et al. 2020), and the wavelength specificities of PSII and PSI are mainly determined by the combination of the Chl a/b ratios of the photosystems and the absorbance ratio of Chl a to Chl b at different wavelengths. The Chl a/b ratio of PSII is lower than that of PSI in both plants (Galka et al. 2012; Wei et al. 2016; Su et al. 2017; Caspy and Nelson 2018; Mattila et al. 2020) and C. reinhardtii, although the difference in C. reinhardtii is smaller (Drop et al. 2014a, 2014b; Shen et al. 2019).

The specific types of white light were used at low-intensity (PFD 30 µmol m⁻² s⁻¹) to illuminate the cells (grown at PPFD 100 µmol m⁻² s⁻¹) for 5 min, after which the redox state of the PQ-pool was also measured as described above.

In addition to using the two types of white light, the redox state of the PQ-pool was measured after illuminating cells (grown at PPFD 100 µmol m⁻² s⁻¹) for 5 min with the individual wavelength components of the two types of white light (see Fig. S2b for the spectra) at PFD 50 µmol m⁻² s⁻¹.

For the measurement of the PQ-pool redox state in darkness in the presence and absence of oxygen, 10 ml samples with 15 × 10⁶ cells were incubated in the dark for 2 h with continuous bubbling with either air or nitrogen, respectively. Aerobic dark incubations were conducted in 50 ml Erlenmeyer flasks placed on a horizontal shaker. Oxygen concentration was recorded directly from the cell suspension with a FireSting O₂ Fiberoptic Oxygen Meter (PyroScience GmbH, Aachen, Germany) (see Fig. S3 for the recorded oxygen concentrations). Cell filtering was done by pouring the cells on the filter directly from the flask. Anaerobic incubations were conducted in a sealed chamber to maintain anaerobic conditions in the liquid sample and in the surrounding gas phase. After flushing the chamber with nitrogen, the gas line was submerged in the sample for the duration of the incubation. Anaerobic conditions were confirmed by monitoring the oxygen levels in the gas phase inside the chamber throughout the incubation. Anaerobic incubation was also done in the presence of 5 µM DBMIB that was added to the samples prior to the sealing of the chamber. After the incubations, cells were filtered on a glass microfiber filter while keeping the cells in the dark, sealed chamber and anaerobic atmosphere.