Chl a fluorescence parameters were measured with a portable Chl fluorescence device (Mini-PAM, Heinz Walz, Effeltrich, Germany) as per the method of Kumari et al. (2005 (link)) after 1, 3, 5,7, 9 and 11 days of salt treatment. The light was provided by the internal halogen lamp of Mini-PAM and leaf temperatures were recorded simultaneously with a Ni/NiCr- thermocouple fitted with the Walz leaf clip holder. Leaves were darkened for 30 min and then subjected to actinic light intensity over 4 min in eight steps with increasing levels of light, each 30s apart (Rascher et al. 2000 (link)). Light-response curves of ΔF/Fm′ and ETR were obtained were calculated according to Genty et al. (1989 (link)).
Mini pam
Manufactured by Walz
Sourced in Germany
The Mini-PAM is a laboratory equipment device that measures the photosynthetic activity of plants and other photosynthetic organisms. It provides a reliable and quantitative assessment of photosynthetic performance.
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112 protocols using mini pam
1
Chlorophyll Fluorescence Analysis of Salt Stress
2
Photosynthetic Pigment Analysis and Efficiency
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Leaf chlorophylls (a, b, and total) and carotenoids were extracted and determined as in Spanò and Bottega [23 (link)]. The concentrations of pigments were expressed as mg g−1FW. Nineteen days after imbibition, before the collection of seedlings, photosynthetic efficiency was determined by analysing chlorophyll a fluorescence by a portable fluorometer (MINI-PAM Walz, Effeltrich, Germany), according to Sorce et al. [59 (link)]. Eight records per pot, each one on a distinct plant, were taken on leaves dark adapted for 30 min by dark leaf clips (Walz) before the measurement of Fo, Fm, and Fv/Fm; the latter is an expression of the maximum PSII quantum yield [60 (link)]. Consequently, each time, the value of each thesis was the average of 8 measurements ± SE.
3
Photosynthetic Pigments and Efficiency
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Chlorophylls (a, b and total) and carotenoids were extracted and determined as in Spanò and Bottega (2016) , according to Hassanzadeh et al. (2009) and to Lichtenthaler (1987) , respectively.
Pigment contents were expressed as mg g -1 FW. Photosynthetic efficiency was determined as in Pippucci et al. (2015) by analysing chlorophyll fluorescence by a portable fluorometer (MINI-PAM Walz, Effeltrich, Germany), at 7 and 14 days. Two records per individual were taken on sunexposed leaves, thus acquiring the operating Photosystem II (PSII) quantum yield (ΦPSII). Further two leaves were shaded with dedicated clips for 30 min, then measured to evaluate the maximum PSII quantum yield (Fv/Fm) (Genty et al., 1989) . All measurements were performed between 12 and 1 PM. The value of each thesis was the average of 24 measurements ± SE.
Pigment contents were expressed as mg g -1 FW. Photosynthetic efficiency was determined as in Pippucci et al. (2015) by analysing chlorophyll fluorescence by a portable fluorometer (MINI-PAM Walz, Effeltrich, Germany), at 7 and 14 days. Two records per individual were taken on sunexposed leaves, thus acquiring the operating Photosystem II (PSII) quantum yield (ΦPSII). Further two leaves were shaded with dedicated clips for 30 min, then measured to evaluate the maximum PSII quantum yield (Fv/Fm) (Genty et al., 1989) . All measurements were performed between 12 and 1 PM. The value of each thesis was the average of 24 measurements ± SE.
4
Chlorophyll Fluorescence Measurement Protocol
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The study of
Genty et al.59 (link) was followed for the measurement
of chlorophyll fluorescence attributes through a photosynthetic yield
analyzer (Mini-PAM, Walz, Effeltrich, Germany), while fluorescence
nomenclature was according to Maxwell and Johnson.60 (link) A 0.8 s saturating pulse of 8000 μmol m–2 s–1 in dark-adapted leaves was used to measure
the maximum fluorescence (Fm) with all PSII reaction centers closed,
while in light-adapted leaves, a second saturating pulse of 8000 μmol
m–2 s–1 was applied again.
Genty et al.59 (link) was followed for the measurement
of chlorophyll fluorescence attributes through a photosynthetic yield
analyzer (Mini-PAM, Walz, Effeltrich, Germany), while fluorescence
nomenclature was according to Maxwell and Johnson.60 (link) A 0.8 s saturating pulse of 8000 μmol m–2 s–1 in dark-adapted leaves was used to measure
the maximum fluorescence (Fm) with all PSII reaction centers closed,
while in light-adapted leaves, a second saturating pulse of 8000 μmol
m–2 s–1 was applied again.
5
Seawater Incubation for Photosynthesis
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Specimens were placed in 100 ml glass bottles completely filled with 0.2 µm-filtered seawater, with magnetic stirring, and tightly sealed to avoid gas exchange with the air. Assays were performed at saturating PFR (previously determined for each species by chlorophyll a fluorescence rapid light curves by means of a pulse-amplitude-modulated fluorometer; Mini-PAM, Walz) at their respective growth temperatures for specimens from stock cultures, or at 3±1 °C for the species collected in Kongsfjorden. Specimen size was adjusted to prevent self-shading and to ensure effective agitation of the medium. The pH was recorded using a thin glass electrode (CRISON 52 09, pH meter CRISON GLP 22) until it reached a stable reading (after ~24 hours of incubation), which represents the pH compensation point.
6
Chlorophyll Fluorescence Assessment of CO2 Impact
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Chlorophyll fluorescence parameters were measured using a pulse amplitude modulation fluorometer (MINI-PAM, Heinz Walz, Germany). Chlorophyll fluorescence measurements were performed using intact flag leaves (three plants from each CO2 treatment) at 9:00–11:30 at half-way anthesis stage (DC 65) (Zadoks et al., 1974 (link)). Generally, SH8675 reaches anthesis (213 d) earlier than ZYM (216 d). However, in the present study, both varieties reached anthesis on the same day under CO2 and eCO2 conditions. The leaves were dark-adapted for 20 min with leaf clips to determine the ambient temperature fluorescence of dark-adapted leaf when all reaction centers are open and closed (Fo and Fm, respectively). Fo was measured under a weakly modulated measuring light (< 1μmol photons m–2s–1), and the leaves were immediately illuminated with an intense saturating pulse light (8,000 μmol photons m–2s–1, pulse time, 1s) to obtain Fm. The leaves were then light-adapted for 20 min, then turn on the actinic irradiation until the fluorescence reaches a steady state, the steady-state chlorophyll fluorescence (Fs) was measured, and Fm′ in the light-adapted state was estimated under saturated pulse light. According to previous studies, other parameters were calculated using the formulae given in Table 2 .
7
Chlorophyll Fluorescence Measurement for Photosynthetic Stress
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Chlorophyll (Chl) fluorescence was measured using a portable pulse-amplitude modulated photosynthesis yield analyzer (MINI-PAM) essentially according to the manufacturer’s protocol (Heinz Walz, Germany). Chl fluorescence is a key indicator of the overall photosynthetic performance of a plant and also indicates whether a plant is experiencing stress23 (link). It also gives an insight into the use of excitation energy by photosystem II (PSII).
When leaves, that have been adapted to a brief dark period are exposed to a beam of low intensity light (0.1 μmol m2 s1), chlorophyll gets excited to a minimal level (Fo). Application of a saturating pulse (8000 μmol m2 s1) results in the formation of a maximum possible yield of fluorescence (Fm). The difference between Fm and F0gives a variability in fluorescence (Fv) and the effective quantum yield of PSII was calculated as Fv/Fm23 (link). The quantum efficiency of unstressed plants grown under normal conditions was in the range of 0.83–0.84. Low Fv/Fm values indicate that plants are under stress; higher values represent high quantum yield24 . Two readings were taken in all the transgenic plants along with WT after exposing them to a dark period of 30 min at an interval of 15 d each after withdrawing water. The mean of all these readings was plotted as a histogram.
When leaves, that have been adapted to a brief dark period are exposed to a beam of low intensity light (0.1 μmol m2 s1), chlorophyll gets excited to a minimal level (Fo). Application of a saturating pulse (8000 μmol m2 s1) results in the formation of a maximum possible yield of fluorescence (Fm). The difference between Fm and F0gives a variability in fluorescence (Fv) and the effective quantum yield of PSII was calculated as Fv/Fm23 (link). The quantum efficiency of unstressed plants grown under normal conditions was in the range of 0.83–0.84. Low Fv/Fm values indicate that plants are under stress; higher values represent high quantum yield24 . Two readings were taken in all the transgenic plants along with WT after exposing them to a dark period of 30 min at an interval of 15 d each after withdrawing water. The mean of all these readings was plotted as a histogram.
8
Chlorophyll Fluorescence Measurements in Plants
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Chlorophyll-a fluorescence was measured before the plants were transferred to the growth chambers and during the treatment period when it was placed alternately in each batch for three days. The measurement was done in the morning (2 h after the light was switched on) and in the afternoon (3 h before the light was switched off) using a plant efficiency analyser (PEA) (Hansatech Instruments, Kings Lynn, UK) after dark-adapting the leaves for 30 min using a leaf clip (Hansatech, Instruments, Kings Lynn, UK), and then subsequently exposing the leaves to 3000 µmol m -2 s -1 measuring irradiance to generate maximal fluorescence (Fm) [30] [31] [32] [33] , to measure the maximum photochemical efficiency Fv/Fm = (Fm -Fo)/Fm of dark adapted leaves. In parallel, a MINI-PAM (Walz, Effeltrich, Germany) was used to measure the PSII operating efficiency F′q/F′m = (F′m -F′)/F′m [34] and linear electron transport rate (ETR), which were calculated as described by Genty et al. [35] , at the ambient irradiance in the treatments.
9
Chlorophyll Fluorescence and Leaf Physiology
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Chlorophyll fluorescence was collected using a Mini-PAM portable fluorometer (PAM-2000, Heinz Walz GmbH, Effeltrich, Germany). A dark leaf clip was installed 15 min before measuring the data, and the second to third fully extended leaf from the top of main stem was a leaf sample. SPAD chlorophyll meter reading (SCMR) was measured via the SPAD-502 chlorophyll meter (SPAD-502, Minolta, Tokyo, Japan), which measured the same leaf position with a chlorophyll fluorescence measurement. Green and brown leaf numbers were also counted and compared between PEG and non-PEG treatments. All three non-destructive samples were done at 0%, 0.17%, 0.33%, 0.50%, 0.67%, 0.83% and 1.00% PEG application 1 and 2 mo after the recovery period.
At 0.67% PEG, photosynthesis rate and stomatal conductance were measured at the second fully extended leaf from the top of main stem using a portable photosynthesis system (LI-6400XT, LI-COR, Lincoln, Nebraska, USA). The measurements were done between 10:00 and 12:00 h.
For destructive samples, leaf water potential (LWP) was conducted with a pressure bomb device (Model 3005F01, Soil moisture Equipment Corporation, Santa Barbara, California, USA), collecting at 0%, 0.50% and 1.0% PEG 1 and 2 mo after recovery. The second and third fully extended leaves from the top of main stem were cut and processed between 10:00 and 12:00 h.
At 0.67% PEG, photosynthesis rate and stomatal conductance were measured at the second fully extended leaf from the top of main stem using a portable photosynthesis system (LI-6400XT, LI-COR, Lincoln, Nebraska, USA). The measurements were done between 10:00 and 12:00 h.
For destructive samples, leaf water potential (LWP) was conducted with a pressure bomb device (Model 3005F01, Soil moisture Equipment Corporation, Santa Barbara, California, USA), collecting at 0%, 0.50% and 1.0% PEG 1 and 2 mo after recovery. The second and third fully extended leaves from the top of main stem were cut and processed between 10:00 and 12:00 h.
10
Genetic Analysis of Rice Mutant spl42
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The rice spl42 mutant was isolated from rice cultivar (cv.) NingJing6. An F2 population derived from a cross between spl42 and N22 was used for the genetic analysis. Wild-type and spl42 mutant plants were grown in Nanjing (Jiangsu province) and Lingshui (Hainan province) under natural conditions. For temperature-sensitive treatments, plants in the growth chamber (GXM-258B, Ningbo) were treated with a 16 h light/8 h darkness photoperiod at a constant temperature of 20 °C, 25 °C, or 30 °C, respectively [44 (link)]. The thousand-grain weight, seed length, and width were examined by a seed phenotyping system (SC-G automatic test seed analysis software, Hangzhou). At the maturity of the wild type and spl42, the plant height, number of tillers, spike length, and leaf length and width were investigated. At seed harvest, agronomic traits such as the number of branches, seed set, number of grains per spike, grain length, grain width, and thousand-grain weight were investigated. The photosynthetic rate and other photosynthetic indicators of the wild type and spl42 mutant were measured at the maximum tillering stage using a portable photosynthesizer (Li6400XT, LI-COR, USA) and an ultra-portable modulated chlorophyll fluorometer (MINI-PAM, WALZ, Germany). Twenty plants were surveyed for each trait and the mean was taken.
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