Water pam chlorophyll fluorometer

Whole-cell absorbance spectra (350–750 nm) were determined on a UV/Visible Ultrospec 3,100 pro (Amersham) in 1 mL of cultures previously adjusted to an OD₇₅₀ of 0.4–0.5. Pigment content absorbance maxima at 635 and 685 nm corresponded to phycocyanin and chlorophyll a, respectively (Poniedziałek et al., 2017 (link)). Data were normalized by the OD₇₅₀ of each strain at the different timepoints. To quantify the in vivo apparent PSII quantum yield, 100 μL of the previously adjusted samples (light adapted) was diluted 1:20 with 1.9 mL of H₂O and measurements were carried out in a WATER-PAM chlorophyll fluorometer (Walz GmbH) as described previously (Selim et al., 2018 (link), 2021 (link)). The maximal PSII quantum yield (F_v/F_m) was determined with the saturation pulse method. ΔF yield indicates the maximal PSII quantum yield (F_v/F_m) of three measurements of each sample.
To evaluate the oxygen evolution, 1 ml of cultures was adjusted to an OD₇₅₀ of 0.5. Measurements were taken at room temperature using a Clark-type oxygen electrode DW1 during a 300/300 s light/dark to follow the release and consumption of oxygen, respectively. The light (50 μmol photons m⁻² s⁻¹) was provided from a high-intensity white light source LS2 (Hansatech). Data were normalized by the initial measure (in light) for each strain.

To detect the photosynthetic activity, pulse–amplitude–modulation fluorometry (PAM) was used. This measures the relative quantum yield of the photosystem II, Y(II). A Heinz Walz GmbH (Effeltrich, Germany) WATER-PAM Chlorophyll Fluorometer with WinControl Software was used. For the measurements, a cell suspension with an OD₇₅₀ between 0.4 and 1 was used and diluted 20-fold. After 5 min incubation in the dark, the maximum PSII quantum yield (Fν/Fm) was determined applying the saturation pulse method [22 (link)]. For each time point, three measurements with a time constant of 30 s were taken.

For details on Symbiodinium cell culture and experimental methodology see Additional file 10. Briefly, batch cultures of Symbiodinium type B1 (culture ID Ap1; N = 6 per treatment), grown at 25°C and 40–50 μmol quanta m⁻² s⁻¹ (LI-COR Quantum light meter LI-189 with cosine sensor, LI-COR, Inc., USA) were exposed to 33°C over three days after rapid heating (1°C h⁻¹). This Symbiodinium type was chosen, because previous experiments have indicated a high degree of thermal susceptibility under the experimental setup employed here [36 ]. Samples were taken on Days 0, 1 and 3 by pelleting seven 50 mL aliquots (2000 x g, 5 min) that were flash frozen in liquid nitrogen and stored at −80°C. In addition, 5–10 mL aliquots were taken for determination of maximum quantum yield of photosystem II (F_v/F_m) via PAM fluorometry (Water-PAM chlorophyll fluorometer, Heinz Walz GmbH, Germany) and measurement of cell density via haemocytometer counts (see Additional file 10).

After 72 h exposure, oxygen evolution/consumption under growth light/dark was measured by a YSI 5100 Dissolved Oxygen Meter (YSI, Yellow Springs, OH, USA) and the concentrations of samples were kept the same at around 2 × 10⁵ cells/mL. A fluorescence induction curve was measured by a WATER-PAM Chlorophyll Fluorometer (Walz GmbH, Effeltrich, Germany) according to Ref. [55 (link)]. In brief, cultures were dark-acclimated for 15 min, and then minimal fluorescence yield (F₀) and maximal fluorescence yield (F_M) were measured respectively before and after being pulsed with a saturating light (800 ms, 3000 µmol photons m⁻²∙s⁻¹). After that, an actinic light with the same intensity as the growth light was turned on for 6.5 min, and every half minute a saturating light was applied to get the fluorescence yield F’ and F’_M before and after the saturating light pulse. Maximal PSII quantum yield (Φ_M) and operational PSII quantum yield (Φ’_M) were calculated following these equations: Φ_M = (F_M − F₀)/F_M [56 (link)] and Φ’_M = (F’_M − F’)/F’_M [57 (link)]. Non-photochemical quenching was calculated as: NPQ = (F_M − F’_M)/F’_M [58 (link)]. NPQ and Φ’_M are the average values calculated from the last three saturating light measurements.

The pulse-amplitude-modulation fluorometry (PAM) was used to detect the photosynthetic activity by measuring the relative quantum yield of photosystem (PS) II of the photosynthetic apparatus. For this, the WATER-PAM Chlorophyll Fluorometer with WinControl Software of Heinz Walz GmbH (Effeltrich) was used. In the measuring cuvette, cell suspensions between OD₇₅₀ = 0.4 – 1 were diluted 1:20. The cuvette was placed in the measuring device and the yield was measured three times with a time constant of 30 s.