In both experiments, seedling height and photosynthesis were measured weekly on each individual. Maximum photosynthesis (Amax, [photosynthetic photon flux density of 1400 µmol m−2 s−1], flow rate 400 µmol s−1, 400 ppm CO2) rates were measured using a Li‐6400XT photosynthesis system (LI‐COR Inc., Lincoln, Nebraska, USA) on a subset of 10 randomly selected individuals from each treatment. Plant height was measured weekly and used to calculate absolute growth rate (AGR cm=height at time of measurement) and relative growth rate (RGR %=height at time of measurement x height at start of experiment−1). We focused on height growth because occupancy of the canopy was considered the ultimate measure of success. At the conclusion of both experiments, all individuals were harvested and measured for leaf, stem, root biomass, and total leaf area. Biomass was dried at 60°C for 48 h. Leaf tissue of five individuals from each treatment was analyzed for nutrient content (percent nitrogen) and δ13C isotopic content using a Thermo Scientific Finnigan Delta‐C Elemental Analyzer‐Infrared Mass Spectrometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at the Florida International University Stable Isotope Lab. Carbon isotope ratios were used to test for indication of water and light stress.
Li 6400xt photosynthesis system
Manufactured by LI COR
Sourced in United States
The LI-6400XT is a portable photosynthesis system designed for measuring gas exchange in plants. It is capable of measuring net photosynthesis, transpiration, stomatal conductance, and other related parameters. The system includes a leaf chamber, control unit, and accessories for reliable and accurate measurements of plant physiological processes.
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Lab products found in correlation
Li 6400 40 leaf chamber fluorimeter, by LI COR (1 mentions)
Chlorophyll meter model spad 502, by Konica Minolta (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Fluorcam, by Photon Systems Instruments (1 mentions)
Li 250a light meter, by LI COR (1 mentions)
Spad 502 m, by Konica Minolta (1 mentions)
Spad 502, by Konica Minolta (1 mentions)
15 protocols using li 6400xt photosynthesis system
1
Measuring Growth and Photosynthesis in Plants
2
Leaf Gas Exchange Measurement Protocol
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Net photosynthesis rate (A), stomatal conductance (gs), and transpiration rate (E) were measured at the end of the experiment in the youngest fully-expanded leaf of each plant (six plants per treatment), using a LI-6400XT photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA). Leaf gas exchange was measured as described by Martinez et al. [6 (link)]
3
Evaluation of Photosynthetic Parameters
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Gas-exchange measurements were done on the newest, fully expanded, undamaged leaf from each plant, using a LI-6400 XT photosynthesis system (LI-COR Inc., Lincoln, LE, USA), which is a portable infra-red gas exchange system, with an incorporated leaf chamber. Three plants were randomly selected for the gaseous exchange investigation. To take the measurements, the leaf chamber was set with an air flow per unit leaf area of 500 μmol s-1, leaf temperature of 22–23°C, ambient thermal reading of 20–23°C with humidity at 30%. Net photosynthesis and the stomatal conductance was recorded after each leaf had reached a steady state, where assimilation and stomatal conductance had stabilized, which required 2 to 4 minutes.
The maximum photochemical yield of Photosystem II (Fm/Fv) was measured using a MINI PAM (MINI–PAM II Photosynthesis Yield Analyser., WALZ Mess-und Regeltechnik., Germany). The measurements were taken directly from each plant on three fresh and fully expanded, dark adapted leaves. The leaves were allowed 10 minutes to become dark adapted before the measurements were taken.
The Fm/Fv value was calculated using the formula:
Where Fm is the maximal fluorescence obtained with a 0.8 saturation flash and F0 is the dark fluorescence yield. The three Fm/Fv readings were then averaged to produce a single comparative estimation of Fm/Fv for each plant.
The maximum photochemical yield of Photosystem II (Fm/Fv) was measured using a MINI PAM (MINI–PAM II Photosynthesis Yield Analyser., WALZ Mess-und Regeltechnik., Germany). The measurements were taken directly from each plant on three fresh and fully expanded, dark adapted leaves. The leaves were allowed 10 minutes to become dark adapted before the measurements were taken.
The Fm/Fv value was calculated using the formula:
Where Fm is the maximal fluorescence obtained with a 0.8 saturation flash and F0 is the dark fluorescence yield. The three Fm/Fv readings were then averaged to produce a single comparative estimation of Fm/Fv for each plant.
4
Stomatal Conductance Measurement Using LI-6400XT
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Stomatal conductance was measured using a LI-6400XT photosynthesis system (Li-Cor) (Wang et al., 2021 (link)). In brief, stomatal conductance was measured in a 2 × 3-cm leaf chamber with a red-blue LED light set to 400 μmol mol−1 CO2 and 200 μmol m−2 s−1 photosynthetic photon flux density.
5
Photosynthetic Performance Assay
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Net photosynthesis rate (CO2 assimilation rate), stomatal conductance, transpiration rate, maximum efficiency of photosystem II under light (Fv/Fm), and the actual photochemical efficiency of photosystem II (Φ PSII) were measured with a LI-6400XT photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA) equipped with a LI-6400-40 Leaf Chamber Fluorimeter and a LICOR 6400-01 CO2 injector [5 (link)]. Leaf gas exchange was measured in a 2 cm2 leaf cuvette. During these measurements, air CO2 concentration was controlled using the injection system and compressed CO2 cylinders with a CO2 concentration of 400 μmol mol−1 CO2. Measurements were taken at a saturating light of 1000 μmol·m−2·s−1 and at ambient air temperature and relative humidity every week, with recording points at 0 days (before the stress treatment started), 7 days (one week after the stress treatment started) and 14 days (2 weeks after the stress treatment started). Data reported are the mean ± SE 6–8 biological replicates per treatment.
6
Photosynthesis and Chlorophyll Fluorescence Measurement
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The different photosynthetic and chlorophyll fluorescence parameters were measured at the end of the experiment in the youngest fully-expanded leaf of each plant (six plants per treatment), using a LI-6400XT photosynthesis system (Li-Cor, Inc., Lincoln, NE, USA). The conditions established in the LI-COR were as described in Martinez et al. (2018) (link).
7
Gas Exchange Measurements and Photosynthesis
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Gas exchange parameters, including the stomatal conductance (gs) and intercellular carbon dioxide concentration (Ci), were measured by an LI-6400XT photosynthesis system (LI-COR Inc., Lincoln, NE, USA) from 9:00-11:00. The leaf chamber temperature was set to 25°C, the relative humidity was set to 65%, and the CO2 concentration was kept at 390 μmol mol–1. Photosynthetically active radiation (PAR) was set to 1800, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 150, 100, 50, and 0 μmol m-2 s-1, respectively. The stomatal limit (Ls) value was determined by equation (1).
Where Ca indicates the atmospheric CO2 concentration.
And
The PNmax was obtained by the photosynthesis-light response curves based on the photosynthetic electron transport of photosystem II in C3 and C4 species (Ye, 2012 (link); Ye et al., 2013 (link)).
SPAD value was measured by Chlorophyll Meter Model (SPAD-502, Konica Minolta, Japan).
Where Ca indicates the atmospheric CO2 concentration.
And
The PNmax was obtained by the photosynthesis-light response curves based on the photosynthetic electron transport of photosystem II in C3 and C4 species (Ye, 2012 (link); Ye et al., 2013 (link)).
SPAD value was measured by Chlorophyll Meter Model (SPAD-502, Konica Minolta, Japan).
8
Quantifying Photosynthesis and Chlorophyll
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For chlorophyll, ethanol extractions were done as in Yang et al. (2016) . Determinations were done by measuring optical density at 645 nm and 665 nm using an Epoch Microplate Spectrophotometer (www.biotek.com ). Chl a=5.21A665−2.07A645; Chl b=9.29A645−2.74A665. Values were combined for the ‘total chlorophyll’ value. Photosynthetic rate was measured using a portable LI-COR LI-6400XT Photosynthesis system (www.licor.com ). Photosynthetic rate was measured every 5 min for a total of 15 min to allow for acclimation and plateau (light, 1500 µmol m−2 s−1; temperature, 20 °C; CO2, 450 ppm). The first or second true leaf was measured for all plants tested. For the stomatal index, epidermal peels were done by applying clear nail polish to the first or second true leaf of 3-week-old plants for 30–60 s. A strip of clear packing tape was then applied over the nail polish and promptly removed and placed on a microscope slide. Differential interference contrast (DIC) images were taken using a Leica DMI300B inverted microscope, and stomatal and pavement cells were counted manually using Image J software. For fresh and dry weight, the aboveground tissue of at least six plants per genotype were cut and weighed, dried at 70 °C for 7 d, and weighed again. For seed yield, at least six plants of each genotype were harvested and all viable seeds were manually counted.
9
Leaf Gas Exchange Measurements
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Gas exchange, including leaf stomatal conductance and leaf transpiration, were measured in three randomly selected plants by a LI-6400XT photosynthesis system (LI-COR Inc., Lincoln, NE, USA) during 9:00-11:00. The photosynthetic active radiation was fixed at 1000 μmol m-2 s-1, CO2 was fixed to 400 μmol mol-1, and flow rate was 500 μmol s-1.
10
Chlorophyll Dynamics in Seedling Photomorphogenesis
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Germinated seeds (3 days after soaking) were incubated in the dark for 7 days at 22°C in a culture room and then exposed to light (16-h day, light intensity of 1000 μmol·m−2·s−1) (Yuan et al., 2010 (link)). Approximately 3-cm sections of the first leaf were sampled from five individuals per genotype at four time points (0 h, 24 h, 48 h, and 72 h post onset of illumination), and the content of total chlorophyll was determined for each sample using the acetone extraction method (Wei et al., 2021 (link)). Chlorophyll fluorescence and F0 and Fv values were captured with a FluorCam instrument (Photon Systems Instruments, Czech Republic). Plants at the six-leaf stage grown under normal conditions (22°C/16-h day, light intensity 600 μmol·m−2·s−1) were used to measure the net photosynthetic rate with an LI-6400XT photosynthesis system (LI-COR, USA) under a light intensity of 1000 μmol·m−2·s−1 and 500 μmol·s−1 CO2 flow conditions.
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