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 (Fv/Fm), determined after 15 min of dark adaptation, with a pulse-amplitude–modulated Chl fluorescence imaging system (MAXI-IMAGING-PAM; Heinz Walz).
Imaging pam maxi
Manufactured by Walz
Sourced in Germany
The IMAGING PAM MAXI is a non-invasive instrument for the rapid and accurate measurement of chlorophyll fluorescence. It provides comprehensive information about the photosynthetic performance of plants and algae.
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Lab products found in correlation
Fms 2, by Hansatech (2 mentions)
Multisizer 3, by Beckman Coulter (1 mentions)
Paromomycin, by Merck Group (1 mentions)
Dual pam 100, by Walz (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Uv 2550, by Shimadzu (1 mentions)
Imag k7, by Walz (1 mentions)
Plant efficiency analyser, by Hansatech (1 mentions)
21 protocols using imaging pam maxi
1
Chlamydomonas Photoinhibition Assay
2
Chlorophyll Fluorescence Analysis
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Chlorophyll fluorescence was determined using Maxi-Imaging-PAM (Walz, Germany). Five or six plants of the same line were selected. After 20 min of dark treatment, the plants were exposed to a photon density of 81 μmol m–2 s–1 and excited every 20 s. Seventeen time points were used to obtain a stable status of the following parameters: effective PSII quantum yield [Y(II)], quantum yield of nonregulated energy dissipation in PSII [Y(NO)], electron transport rate (ETR), and quantum yield of regulated energy dissipation in PSII [Y(NPQ)].
3
Chlorophyll Fluorescence and Pigment Analysis
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Chlorophyll a fluorescence parameters were analysed following the methodologies reported in Araniti et al. (2017 (link)) using a Maxi Imaging PAM fluorometer (Walz, Effeltrich, Germany). Values of F0 and Fm were measured in 40 min dark-adapted leaves before and after a saturating pulse (8.000 μmol m−2 s−1 for 0.1 s). The maximal photosystem II (PSII) photochemical efficiency [Fv/Fm = (Fm−F0)/Fm] and the operational PSII efficiency [ΦII = (Fm'−Fs)/Fm'] were calculated according to Genty et al. (1989 (link)). The proportion of open reaction centres, qL, and the quantum yield of regulated (ΦNPQ) or non-regulated (ΦNO) photochemical energy loss in PSII were determined as reported by Kramer et al. (2004 (link)) based on the lake model. Photochemical quenching (qP) was calculated according to Schreiber et al. (1986 (link)). The apparent Electron Transport Rate (ETR) was calculated as reported in Guidi et al. (2017 (link)). Analyses were conducted on six leaves in each plant belonging to a selected plot [see Sample collection].
Chlorophyll a and b and carotenoids were measured on 100 mg of liquid-nitrogen-powdered plant materials following the Wellburn’s protocol (Wellburn 1994 (link)) modified by Araniti et al. (2017 (link)). Pigments content was measured and expressed using Wellburn’s equations (1994 (link)) as µg g−1 of DW.
Chlorophyll a and b and carotenoids were measured on 100 mg of liquid-nitrogen-powdered plant materials following the Wellburn’s protocol (Wellburn 1994 (link)) modified by Araniti et al. (2017 (link)). Pigments content was measured and expressed using Wellburn’s equations (1994 (link)) as µg g−1 of DW.
4
Chlorophyll a Fluorescence Analysis
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The MAXI IMAGING-PAM (Walz, Effeltrich, Germany) was used for chlorophyll a fluorescence analysis of whole rosettes. Minimal fluorescence F0 and maximal fluorescence Fm of 30 min dark-adapted plants were derived from the measuring light only and during application of a saturating light pulse (0.8 s), respectively. The maximum quantum efficiency of PSII, Fv/Fm, was calculated as (Fm-F0)/Fm. Measurements were conducted 6 h into the light period. For each experiment, the same set of 7-10 plants for each genotype was measured repeatedly at the indicated time points (i.e., once per day).
5
Measuring Leaf Physiological Traits in B. hygrometrica
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The RWC of B. hygrometrica leaves was determined according to Lin et al. [78 (link)] with minor modifications, where all of the leaves cut from each plant were weighed immediately to record fresh weight (FW). Subsequently, the detached leaves were immersed in distilled water at room temperature for 6 h, weighed to record turgid weight (TW), and subjected to fast oven-drying at 105°C for 1 h followed by 65°C for several hours to a stable weight to record dry weight (DW). The RWC was calculated as follows: RWC (%) = (FW − DW)/(TW − DW) × 100. The chlorophyll fluorescence parameter Fv/Fm was monitored in intact leaves after dark adaptation for 1 h using the Maxi-Imaging-PAM (Walz, Germany), with a saturating light intensity of approximately 800 mmol m-2 s-1 and duration of 4.5 s. Leaf REC was measured using an EC 125 Conductivity Meter (Hanna Instruments, Padova, Italy), following the method described previously [79 ]. Six individual plants from each biological replicate were used to measure the physiological indexes.
6
Photosynthetic Mutants Screening in Chlamydomonas
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The insertion mutant library construction and photosynthetic mutants screen were described (Zhao et al., 2017 (link)). Briefly, the wild-type strain (CC400) was used and the mutant library was constructed by transforming this strain via the glass bead method with KpnI linearized plasmid pSI103 containing the aphVIII gene conferring paromomycin resistance (Kindle, 1990 (link)). Transformants that grew on TAP plates with 10 μg ml-1 paromomycin (Sigma) were isolated for photosynthetic mutants screening. Mutant screening was based on chlorophyll a fluorescence measurements. Sample preparation was done as previously (Zhao et al., 2013 (link)) and the measurements were taken with a chlorophyll fluorometer (Maxi-Imaging PAM; Walz, Effeltrich, Germany) by following the manufacturer’s instructions. Among the mutants with both the lowest Fv/Fm and Y(II) values, x32 was chosen for subsequent characterization.
7
Chlorophyll Fluorescence Imaging for Quantum Yield
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Maximal quantum efficiency (Fv/Fm) was calculated from chlorophyll a fluorescence signals obtained from attached fronds using a modulated fluorometer (FMS2, Hansatech, UK) as previously described in [17 (link)]. Briefly, attached fronds were carefully cover and dark acclimated for 30 min using FMS2 leaf clips. After dark acclimation the modulated light was turned on to obtain F0, then a saturating pulse (800 mS at ~ 3000 μmol m− 2 s− 1) was applied to obtain the maximum fluorescence (Fm). Variable fluorescence (Fv) and Fv/Fm ratio were calculated according to [54 (link)] at ambient temperature (17 C° approx.). Data was analyzed after check normality assumptions under an ANOVA test with P value ≤0.05. When data did not meet the normality assumptions, we used the non-parametric test of Kruskal-Wallis. Additionally, fluorescence images of Fv/Fm were obtained using detached fronds under each hydration condition FH, DH and RH using a Maxi-Imaging PAM (Walz, Effeltrich, Germany) to observe the rate of recovery of the quantum yield of fluorescence (YII) at the whole frond level of both species (Fig. 2 b).
8
Mutagenesis and Chlorophyll Fluorescence Screening
9
Chlorophyll Fluorescence Measurements for NPQ Analysis
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Chlorophyll fluorescence was measured at room temperature using a pulse-amplitude-modulated fluorometer (FMS2; Hansatech Instruments) or an Imaging-PAM Maxi (Walz). Cells were dark acclimated for 30 min prior to measurement, unless stated otherwise. Maximum fluorescence levels after dark acclimation (Fm) and maximum fluorescence levels in the light (Fm′) were recorded after applying a saturating pulse of light. NPQ was calculated as (Fm − Fm′)/Fm′. For the FMS2 measurements, 3 to 5 × 107 cells were filtered onto a glass-fiber filter that was placed in the instrument’s leaf clip. This was followed by an additional 5-min dark phase with far-red light of 0.6 µmol photons m−2 s−1. Fm and Fm′ were recorded after applying a saturating pulse of 1,800 µmol photons m−2 s−1. NPQ was induced for 9.5 min with 600 µmol photons m−2 s−1, followed by recovery in the dark with far-red light. On the Imaging-PAM, NPQ was induced for 6 to 10 min with 550 µmol photons m−2 s−1 and relaxed for 2.5 to 8 min in the dark. False-colored NPQ images were generated by the Walz software.
10
Photosynthetic Pigment and Chlorophyll Fluorescence Analysis
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Fresh leaf disks of 0.1 g cut from the youngest fully expanded leaves were soaked in 5 mL of N,N-dimethylformamide (Sigma chemical Co.) in the dark for 48 h at 4 C before measuring the absorptions at 647, 664, and 480 nm respectively using a spectrophotometer (UV-2550 Shimadzu). Chl a, Chl b, and Car concentrations were calculated according to Wellburn (1994) (link).
Measurements of electron transport rate (ETR), photochemical quenching (qP) and non-photochemical quenching (NPQ) The youngest fully expanded leaves were harvested and electron transport rate (ETR), photochemical quenching (qP), and non-photochemical quenching (NPQ) were determined at 25 C in the laboratory. Prior to measurements, the leaves were pre-darkened for 15 min. By using the IMAGING PAM MAXI (Walz), images of fluorescence emission were digitised within the camera and transferred via ethernet interface (GigEVision®) to the PC for storage and analysis. Measurements and calculations of ETR, qP, and NPQ were determined as described previously (He et al. 2011) (link).
Measurements of electron transport rate (ETR), photochemical quenching (qP) and non-photochemical quenching (NPQ) The youngest fully expanded leaves were harvested and electron transport rate (ETR), photochemical quenching (qP), and non-photochemical quenching (NPQ) were determined at 25 C in the laboratory. Prior to measurements, the leaves were pre-darkened for 15 min. By using the IMAGING PAM MAXI (Walz), images of fluorescence emission were digitised within the camera and transferred via ethernet interface (GigEVision®) to the PC for storage and analysis. Measurements and calculations of ETR, qP, and NPQ were determined as described previously (He et al. 2011) (link).
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