Detached leaves of mature plants grown under standard conditions for five weeks were floated on water or 2.3 mM lincomycin for 16 h in darkness after which they were illuminated under with high light of 1000 μmol photons m−2 s−1 for 1.5 h. Thereafter, the water samples were moved to standard growth conditions for recovery. PSII efficiency (FV/FM) was measured after a 20 min dark-adaptation using FluorPen FP 110 (Photon Systems Instruments). The amount of D1 protein was measured by western blotting with protein samples collected at indicated timepoints. Total protein samples were isolated by homogenizing frozen leaf material in 100 mM Tris-HCl pH 8.0, 50 mM Na-EDTA, 0.25 mM NaCl, 0.75 (w/v) % SDS, 1 mM DTT followed by 10 min incubation at 68 °C. The extracts were clarified by centrifugation at 12,000 × g for 10 min. Protein concentration was determined using BioRad’s DC Protein assay according to the manufacturer’s protocol. Solubilized protein samples were separated by SDS-PAGE (12% acrylamide, 6 M urea) and immunoblotted with rabbit D1 DE-loop antibody91 (link) used as a 1:8000 dilution. LI-COR Goat anti-rabbit IRDye® 800CW 2nd antibody was used for detection according to manufacturer’s instructions.
Fluorpen fp 110
Manufactured by Photon Systems Instruments
Sourced in Czechia
The FluorPen FP 110 is a portable, handheld fluorescence measurement device. It is designed to measure the fluorescence properties of various samples, including plants, leaves, and other materials. The device provides accurate and reliable data on parameters such as chlorophyll fluorescence, which can be used for a range of applications in plant science, environmental monitoring, and related fields.
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6 protocols using fluorpen fp 110
1
Measuring PSII Efficiency and D1 Protein
2
Assessing Plant Growth and Physiology
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After 8 weeks, the disease severity of the “daughter” plants was estimated and the plant height, crown diameter, chlorophyll fluorescence, chlorophyll level (SPAD), and fresh and dry weights of shoots were examined.
A chlorophyll meter (SPAD-502, Konica Minolta Inc., Osaka, Japan) measured the SPAD. The chlorophyll fluorescence parameters were measured using a portable fluorometer (FluorPen FP110, Photon Systems Instruments, Drásov, Czech Republic). The fresh weight was measured with an electronic scale (EW 220-3NM, Kern and Sohn GmbH., Balingen, Germany). The samples were rinsed with distilled water and dried using a forced air-dry oven (Venticell-222, MMM Medcenter Einrichtungen GmbH., Munich, Germany) at 70 °C for 72 h before the dry weights were recorded. The dried leaves were further used to determine the nutrient contents.
A chlorophyll meter (SPAD-502, Konica Minolta Inc., Osaka, Japan) measured the SPAD. The chlorophyll fluorescence parameters were measured using a portable fluorometer (FluorPen FP110, Photon Systems Instruments, Drásov, Czech Republic). The fresh weight was measured with an electronic scale (EW 220-3NM, Kern and Sohn GmbH., Balingen, Germany). The samples were rinsed with distilled water and dried using a forced air-dry oven (Venticell-222, MMM Medcenter Einrichtungen GmbH., Munich, Germany) at 70 °C for 72 h before the dry weights were recorded. The dried leaves were further used to determine the nutrient contents.
3
Measuring Plant Flowering and Photosynthesis
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For chemical treatment, the NF (Sigma-Aldrich) stock solution was prepared in ethanol. For the measurement of flowering time, diluted NF solution was applied to plants by spraying them every two days until the sprayed plants bolted. For short-term treatments to test the effect of NF on gene expression, the NF solution was added to the Murashige and Skoog (MS) medium 24 h before sampling.
Plants were grown in soil or on standard MS medium at 23°C and 16°C under LD conditions (16:8-h light:dark) and at 23°C under SD conditions (8:16-h light:dark). Chlorophyll a and b levels were measured using ethanol as a solvent (Lichtenthaler, 1987 (link)). The photosynthetic parameters Fo, Fm, and Rdf were measured using the FluorPen FP 110 (Photon System Instruments).
For the measurement of flowering time, total leaf number was counted when the primary inflorescence reached approximately 5 cm. Boxplots were constructed to represent flowering time distribution (Postma and Goedhart, 2019 (link)). The data are shown as a boxplot, with the box indicating the interquartile range, the whiskers showing the range of values that are within 1.5 × interquartile range, and a horizontal line indicating the median. The P values were obtained using Student’s t-test or one-way ANOVA followed by Tukey’s range test.
Plants were grown in soil or on standard MS medium at 23°C and 16°C under LD conditions (16:8-h light:dark) and at 23°C under SD conditions (8:16-h light:dark). Chlorophyll a and b levels were measured using ethanol as a solvent (Lichtenthaler, 1987 (link)). The photosynthetic parameters Fo, Fm, and Rdf were measured using the FluorPen FP 110 (Photon System Instruments).
For the measurement of flowering time, total leaf number was counted when the primary inflorescence reached approximately 5 cm. Boxplots were constructed to represent flowering time distribution (Postma and Goedhart, 2019 (link)). The data are shown as a boxplot, with the box indicating the interquartile range, the whiskers showing the range of values that are within 1.5 × interquartile range, and a horizontal line indicating the median. The P values were obtained using Student’s t-test or one-way ANOVA followed by Tukey’s range test.
4
NaB Impacts on Photosynthesis and Oxidative Stress
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The effects of NaB treatment on photosynthesis were determined through the analysis of chlorophyll fluorescence using a pulsed modular fluorometer (FluorPen FP110, Photon Systems Instruments, Drásov, Czech Republic).
The malondialdehyde (MDA) content was determined using a colorimetric assay and a thiobarbituric acid reaction and was expressed in nmol/mg of the fresh weight (FW). The MDA content was determined according to the absorbance coefficient of extinction (155 mM−1 cm−1) [37 (link)].
The malondialdehyde (MDA) content was determined using a colorimetric assay and a thiobarbituric acid reaction and was expressed in nmol/mg of the fresh weight (FW). The MDA content was determined according to the absorbance coefficient of extinction (155 mM−1 cm−1) [37 (link)].
5
Drought Stress Impacts on Transgenic Plants
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The 3-week-old transgenic (OX, OX-AS) and the control plants maintained in greenhouse conditions (22/18 °C, 16/8 h light/dark photoperiod) were subjected to 10 days of progressive drought stress. On the day 8, the fully expanded, top leaf from three plants of each line was dark-adapted using clamps for about 30 min. The standard non-imaging OJIP kinetics was measured using the FluorPen FP-110 device (Photon Systems Instruments, Brno, Czech Republic) in the dark-adapted leaves at 455 nm and saturating light pulse of 3000 μmol m−2 s−1. All the induced fluorescence (OJIP) parameters were recorded and exported to Microsoft Excel (Table S1 ). The mean values and standard errors derived from three plants of each line were used separately to compare maximum quantum yield of primary chemistry in PS II (Fv/Fm), initial fluorescence (Fo), performance index (Pi-Abs), absorption per reaction center (ABS/RC), and dissipation per reaction center (DI0/RC), which are considered as stress tolerance indicators.
6
Measuring PSII Efficiency and D1 Protein
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Detached leaves of mature plants grown under standard conditions for five weeks were floated on water or 2.3 mM lincomycin for 16 h in darkness after which they were illuminated under with high light of 1000 μmol photons m -2 s -1 for 1.5 h. Thereafter, the water samples were moved to standard growth conditions for recovery. PSII efficiency (F V /F M ) was measured after a 20 min dark-adaptation using FluorPen FP 110 (Photon Systems Instruments). The amount of D1 protein was measured by western blotting with protein samples collected at indicated timepoints. Total protein samples were isolated by homogenizing frozen leaf material in 100 mM Tris-HCl pH 8.0, 50 mM Na-EDTA, 0.25 mM NaCl, 0.75 (w/v) % SDS, 1 mM DTT followed by 10 min incubation at 68 °C. The extracts were clarified by centrifugation at 12 000 x g for 10 min. Protein concentration was determined using BioRad's DC Protein assay according to the manufacturer's protocol.
Solubilized protein samples were separated by SDS-PAGE (12% acrylamide, 6 M urea) and immunoblotted with rabbit D1 DE-loop antibody (Kettunen et al., 1996) used as a 1:8000 dilution. LI-COR Goat anti-rabbit IRDye® 800CW 2nd antibody was used for detection according to manufacturer's instructions.
Solubilized protein samples were separated by SDS-PAGE (12% acrylamide, 6 M urea) and immunoblotted with rabbit D1 DE-loop antibody (Kettunen et al., 1996) used as a 1:8000 dilution. LI-COR Goat anti-rabbit IRDye® 800CW 2nd antibody was used for detection according to manufacturer's instructions.
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