OD750 was determined on a NannoDrop 2000c, cell number (108 mL−1) was counted with a hemocytometer under light microscopy, and dry weight (g L−1) was weighted using a pre-dried Whatman GF/C filter, according to the procedures described in our previous study [14 (link)]. Fv/Fm, the maximum quantum yield of photosystem II, were measured using dark-adapted algal cultures (15 min) in a water pulse-amplitude-modulated (PAM) fluorometer (Walz, Germany) [42 (link)].
Pam fluorometer
Manufactured by Walz
Sourced in Germany
The PAM fluorometer is an instrument used to measure the fluorescence of photosynthetic organisms. It provides information about the efficiency of the photosynthetic process by analyzing the light-induced changes in the fluorescence of chlorophyll.
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Lab products found in correlation
15 protocols using pam fluorometer
1
Microalgal Growth Characterization
2
Photosynthetic Health Evaluation of Corals
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Photosynthetic health of the corals was checked using a diving pulse amplitude modulated (PAM) fluorometer (Walz, Germany) in the tfinal Control and tfinal 31°Ctreatments. Corals were dark-adapted for 10 min before their minimum fluorescence in the dark (FO) was recorded. Maximum fluorescence (FM) was determined using a saturating pulse of light for 0.8 s. The corals were then illuminated under 616 μmol photon m-2 s-1 light for 5 min to test their ability to sustain photosynthetic function under light. Maximum Quantum Yield (FV/FM) was measured on dark-adapted samples and effective quantum yield Y(PSII), regulated non-photochemical quenching Y(NPQ), and non-regulated non-photochemical quenching Y(NO) were measured on light adapted samples. To compare the changes in the FV/FM, Y(PSII), Y(NPQ), and Y(NO) measurements in the t0, tfinal Control, and tfinal Heat Stress treatments, a 1-way analysis of variance (ANOVA) was used (treatment) to determine significant differences (P < 0.05) between these measurements. Prior to this, data was tested for normality using the Kolmogorov–Smirnov test and Levene’s test was used for homogeneity of variance.
3
Chlorophyll Fluorescence Induction Curves
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Induction curves were made with a saturating light pulse (Delay: 20 s, width: 40 s and 1500 μmol m−2 s−1) in leaves that were dark-adapted for 30 min using a JUNIOR PAM fluorometer (Walz, Germany), two leaves per individual, five individuals per species and treatment were measured. After the light pulse, the leaves were exposed to a series of pulses of saturating light to obtain the maximum fluorescence yield in a light-adapted state (Fm’). From the curves, the maximum quantum efficiency of photosystem II (Fv/Fm), non-photochemical quenching NPQ = (Fm − Fm’)/Fm’ [45 (link)], and the electron transport rate (ETR) were obtained, calculated as ETR = φPSII × PPFD × 0.5 × 0.84 [64 (link)]. Where φPSII is the current quantum yield of PSII (Fm’ − Ft /Fm’) [45 (link)], PPFD is the photosynthetic photon flux density, 0.5 corresponds to the partition of absorbed photons between the PSI and PSII, and 0.84 is the absorbance of the leaf.
4
Evaluating Photosynthetic Resilience
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Maximal photochemical efficiency of photosystem (PS) II (F v /F m ) was used as a proxy for the photosynthetic integrity of the tissue and was measured with a portable modulated PAM fluorometer (Walz, Effeltrich, Germany): PAM 2500 was used in the case study I (bryophytes) and in the protocol setup, and a Junior PAM in the case study II (tracheophytes). The maximum Chl a fluorescence yield (F m ) was induced with a saturating pulse, while minimum fluorescence (F o ) was recorded with low measuring light intensities after several hours of dark acclimation.
The maximal photochemical efficiency of PSII (F v /F m ) was then calculated as (F m -F o )/F m . The relative rate of recovery of F v /F m after desiccation-rehydration (t Rh ) with respect to the initial values (t Control ) was used as an estimator of tolerance to the reached RH, in each case similar to Hájek & Vicherová (2014) . The average F v /F m recovery of the three desiccating treatments at t Rh was used as a proxy for the DT level of each species.
The maximal photochemical efficiency of PSII (F v /F m ) was then calculated as (F m -F o )/F m . The relative rate of recovery of F v /F m after desiccation-rehydration (t Rh ) with respect to the initial values (t Control ) was used as an estimator of tolerance to the reached RH, in each case similar to Hájek & Vicherová (2014) . The average F v /F m recovery of the three desiccating treatments at t Rh was used as a proxy for the DT level of each species.
5
Chlorophyll Extraction and Quantification
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Chlorophyll from leaves, Protoplasts or thylakoid membranes was extracted by 95% ethanol at 80°C. The chlorophyll content in each unit mass or unit volume of sample was determined as described by Lichtenthaler (1987) (link), and then counting or concentration adjusting was performed, so that it would be more convenient to release the data or perform the next stage of the experiment. Integrity of PSII affected the ratio of maximum variable fluorescence to maximum yield of fluorescence (Fv/Fm), which was measured by a PAM fluorometer (Walz GmbH, Effeltrich, Germany) (Ralph et al., 2005 (link)).
The content was determined spectrophotometrically using the formula Chl (a + b) = 5.24 A664.2 + 22.24 A648.6, the content of Chlorophyll (%) = Chl (a + b) ×V × 10-3/m, where Chl (a + b) equaled the chlorophyll concentration in in μg ml-1, A represented the absorption, V was the volume of the 95% ethanol and m symbolized the mass of individual leaves.
The content was determined spectrophotometrically using the formula Chl (a + b) = 5.24 A664.2 + 22.24 A648.6, the content of Chlorophyll (%) = Chl (a + b) ×V × 10-3/m, where Chl (a + b) equaled the chlorophyll concentration in in μg ml-1, A represented the absorption, V was the volume of the 95% ethanol and m symbolized the mass of individual leaves.
6
Symbiotic Anemone Heat Stress Responses
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Differences in tissue coloration of symbiotic anemones indicating endosymbiont loss were recorded at the beginning and end of the heat stress experiment for H2-SSB01 and CC7-SSA01 using a Nikon Coolpix AW 130. Light-adapted photosynthetic efficiencies (ΔF/Fm’) of photosystem II (PSII) for each anemone were measured daily for the duration of the long-term heat stress experiment using a diving Pulse Amplitude Modulated (PAM) fluorometer (Walz, Germany).
7
Seaweed Photosynthetic Efficiency Analysis
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The state of health of the seaweeds was assessed prior to the experiment using a diving Pulse Amplitude Modulator (PAM) Fluorometer (Zarges 40701 Heinz Walz GMBH) and after the 4 h incubation (Chaloub et al., 2010 (link); Leedham Elvidge et al., 2015 (link); Li et al., 2014 (link)). The change in Fv∕Fm was calculated according to the following formula:
Percentage decrease in Fv∕Fm (%) The maximum quantum yield (Fv∕Fm) indicates the stress level of the seaweeds prior to and post incubation. This was done by determining the ratio of the difference between the maximum (Fm) and minimum (Fv) fluorescence level to the maximum fluorescence emitted by the seaweed fronds after dark adaptation of seaweeds for at least 15 min using dark leaf clips (Walz, Germany) (Keng et al., 2013 (link)). The correlation test was done to determine the effect of seawater pH change on Fv∕Fm as Fv∕Fm indicates photosynthetic efficiency of the seaweeds.
Percentage decrease in Fv∕Fm (%) The maximum quantum yield (Fv∕Fm) indicates the stress level of the seaweeds prior to and post incubation. This was done by determining the ratio of the difference between the maximum (Fm) and minimum (Fv) fluorescence level to the maximum fluorescence emitted by the seaweed fronds after dark adaptation of seaweeds for at least 15 min using dark leaf clips (Walz, Germany) (Keng et al., 2013 (link)). The correlation test was done to determine the effect of seawater pH change on Fv∕Fm as Fv∕Fm indicates photosynthetic efficiency of the seaweeds.
8
In vivo Chlorophyll Fluorescence Assessment
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In vivo chlorophyll a fluorescence associated with photosystem II (PSII) was determined using a portable fluorometer Junior PAM fluorometer with WinControl-3.2 software (Walz GmbH, Effeltrich, Germany). Samples of U. compressa were collected at each measurement point (6 h, 24 h, 48 h, and 6 days) and placed in 10 mL incubation chambers to obtain rapid light curves (RLC) for each treatment (three RLCs per treatment). RLCs represented the saturation characteristics of PSII electron transport and overall photosynthetic performance [26 (link),29 (link)].
9
Photosynthetic Efficiency in Symbiotic Anemones
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Light-adapted photosynthetic efficiency (ΔF/Fm′) of photosystem II (PSII) for each symbiotic anemone (N=12 per experimental condition and host–endosymbiont pairing, total of N=144) was measured daily (5 h into the light phase) for the period of the experiment using a diving Pulse Amplitude Modulated (PAM) fluorometer (Walz, Germany) (see Fig. S1B ).
10
Photosynthetic Quantum Yield Measurement
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The experiments were performed following the protocol published previously [53 (link)] with some minor modifications. Six plants for each group were dark-adapted for 20 min before taking measurements with a PAM fluorometer (Walz). All measurements were taken at the same time during the day. A saturating pulse of radiation (2700 μmol m− 2 s− 1) was applied to record the maximum fluorescence yield (Fm), and a weak modulating radiation (0.5 μmol m− 2 s − 1) was used to measure the minimum fluorescence yield (F0). The maximum photosynthetic quantum yield was then calculated as Fv (variable fluorescence yield)/Fm = (Fm-F0)/Fm.
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