Analysis of altered photosystem stoichiometry and antenna size in chlorina mutants.

Seeds of clo-174, clo-160, clo-165, and their control cultivar RGT-Planet were incubated at 37°C for 72 h and let germinate on wet paper at 20°C. Seedlings were sown in soil and grown under constant white light at 120 μmol photons m−2 s−1 for 16 h light/8 h dark at 22°C. All plants were analysed at 20 days-after-sow (DAS). The functional PSI/PSII reaction centre ratio and an independent estimate of PSII functional antenna size were determined from electrochromic shift (ECS) measurements using a JTS-10 spectrophotometer (Bio-Logic, France) as previously described (https://doi.org/10.1038/s41477-019-0526-5, https://doi.org/10.1111/pce.14032). The PSI/PSII reaction centre ratio was measured on dark-adapted leaves using a saturating single-turnover flash to induce charge separation. Excitation was provided by a fluorescent dye emitting at approximately 630 nm and pumped by a frequency-doubled Nd laser (Minilite II, Continuum, USA; 532 nm, 5 ns full width at half maximum). ECS signals were detected at 520 nm (10 mm thick Schott glass filter; 10 nm FWHM) after subtraction of the signal measured at 546 nm (10 mm thick Schott glass filter; 10 nm FWHM). A 3 mm Schott BG39 filter was placed in front of the detector to eliminate interference from actinic light. Functional antenna sizes were determined using continuous red LED illumination (630 nm; approximately 150 μmol photons m−2 s−1). To separate PSI- and PSII-derived ECS signals, leaves were infiltrated with a solution containing 200 μM DCMU and 1 mM hydroxylamine dissolved in HEPES–sorbitol buffer. Complete inhibition of PSII activity was verified by chlorophyll fluorescence measurements, and only samples displaying Fv/Fm values below 0.2 following DCMU–hydroxylamine treatment were used for subsequent analyses. The functional antenna size of PSII was estimated by analysing OJIP chlorophyll fluorescence transients following the procedure described by https://doi.org/10.1016/j.bbabio.2012.02.003. Measurements were performed on intact, dark-adapted leaves using a HandyPea+ fluorometer (Hansatech Instruments Ltd, UK). A 1 s red light pulse (650 nm) was applied at increasing photosynthetic photon flux densities (350, 500, 650, 900, 1200, 1500, 2000, 2500 and 3000 μmol photons m−2 s−1; PPFD) to the same area of the third fully expanded leaf. Measurements were performed on three independent plants per genotype, with a 10-min dark-adaptation period between consecutive measurements. The measured fluorescence was normalised to the photosynthetic photon flux density, and linear regression was performed to determine the slope of the fluorescence intensity as a function of the light intensity at 300 μs.