Chlamydomonas Photoinhibition Assay

Chlamydomonas strains used in this study are WT 4A+ (mt+), 2pac mutant (mt+) (34 (link)), His-tagged psbB (His47) (mt+) (68 (link)), and the yellow-in-the-dark mutant chlL (mt+) (37 (link)). Cells were grown heterotrophically or mixotrophically in Tris acetate-phosphate (TAP) medium (69 ) or photoautotrophically in high salt medium (70 (link)). Cell densities were determined with a Multisizer3 (Beckman Coulter). For photoinhibition studies, dark-grown cells were concentrated to about 2 to 3 × 10⁷ cells⋅mL⁻¹ in liquid TAP and incubated either under very LL or shifted to HL (800 µmol photons⋅m⁻²⋅s⁻¹) for the indicated times in a temperature controlled (25 °C) HL chamber (Percival). TAP medium was supplemented with 100 μg/mL CAP or equivalent volume of 100% ethanol. Whole-cell samples were collected at different indicated time intervals for protein analyses. The PSII efficiency (71 (link)) was monitored by measuring the maximum quantum yield of PSII (F_v/F_m), determined after 15 min of dark adaptation, with a pulse-amplitude–modulated Chl fluorescence imaging system (MAXI-IMAGING-PAM; Heinz Walz).