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2 - Measuring through infrared gas analyzer (IRGA)
CHAPTER 4 Photosynthetic rates
wavebands caused by the presence of a gas between the radiation source and a detector. The reduction in transmission is a function of the concentration of the gas. The
primary role of IRGA is to measure the CO2 concentration. The IRGA is very sensitive to detect even a change of 1 ppm of CO2.
A leaf or a plant is enclosed in an airtight chamber and the CO2 fluxes are determined by measuring the CO2 concentration changes in the chamber atmosphere. The
major absorption peak of CO2 is at 4.25 mm with secondary peaks at 2.66, 2.77, and
14.99 mm. Both water vapor and CO2 molecules absorb IR radiation in the 2.7-mm
Procedure: The portable photosynthesis system is a portable IRGA and is designed to operate as an open system to measure the gas exchange parameters. It consists of separate IRGAs to measure CO2 and H2O vapor concentrations, an internal
air supply unit and the necessary software for the computation of gas exchange parameters. Li 6400 uses four independent infrared gas analyzers, two each for CO2 and
H2O. One pair of CO2 and H2O analyzers defined as reference measures the CO2 and
water vapor concentration in the ambient air that is sent into leaf chamber. Similarly
second pair, the analysis chambers measure the CO2 and water vapor concentrations
in the air that is coming from the leaf chamber. The difference between the reference
and the analysis IRGAs is computed. Deepa et al. (2012) measured physiological efficiency of greengram genotypes under moisture stress conditions in this laboratory.
A leaf is clamped to the leaf chamber. The leaf chamber is provided with suitable
pads to clamp an area of 2.5 cm2 under airtight conditions. Separate tubing is provided to send and withdraw air from the leaf chamber. These tubes are connected to
either of the reference or analysis IRGA for the determination of gas concentrations.
A quantum sensor is placed inside the leaf chambers transparent cover to measure
the actual light intensity in PAR range at the leaf surface. Blue and red light-emitting
diode (LED) is fixed on top of the leaf chamber. The LEDs emit light in the PAR
range and the intensity of which can be fixed and controlled at a required level. The
light source is capable of providing the photosynthetically active radiation in the
energy range of 0–2000 mmole m−2 s−1.
A CO2 cartridge normally carrying 8 g of pure CO2 in a liquid form is used to
get the requisite CO2 concentration in the leaf chamber. The system mixes ambient
air with the CO2 to obtain the requisite concentration in the leaf chamber. The path
of ambient air is provided with two scrubbers to remove moisture (drierite used as a
desiccant) and CO2 (soda lime to remove CO2).
IRGA Working Procedure (LI 6400): Usage of IRGA (Fig. 4.1) equipment by students and scientists often found complicated. Here is the operation protocol for easy
handling of the equipment both in greenhouse and field experiments.
1. First charge the batteries 1 day prior to record data using IRGA.
2. Load the charged batteries first.
3. Connect the CO2 tube to the inlet of the instrument.
4. All screws of this instrument must be in tight fitting.
4.2 Measuring through infrared gas analyzer (IRGA)
FIGURE 4.1 Infrared Gas Analyzer.
5. Connect the CO2 tube in a proper way. Connect this tube very tightly;
otherwise it shows leak (- ppm) in display.
6. The 2nd edge of this tube was kept in an empty thermocoal box and closed for
uniform entry of air into the tube.
7. Switch “ON” the instrument.
8. Display shows a. Welcome to loading open system.
b. Starting net working.
c. It shows fluorescence + WUE X m1 – press “enter.”
d. Is the chamber IRGA connected Y/S – Yes – press “Y.”
9. Open the IRGA leaf chamber one time and close it.
10. Select “New measurements” press (F4).
11. In display select “Open log file” press (F1).
a. Give file name and press “enter.”
b. Next—give sub file name and press “enter.”
c. Give date and press “enter.”
12. Next—CO2 matching.
a. Select Match (F5).
b. Wait up to we get equal values of reference CO2 and sample CO2.
CHAPTER 4 Photosynthetic rates
c. If we need close matching press “Match IRGA” (F5) after that press
In Display set the rows – m, n, c and 9.
a. If we want “m row”—press “m alphabet.”
b. If we want “n row”—press “n alphabet.”
c. If we want “c row”—press “c alphabet” it already exists.
d. If we want “9 row”—press “9 number.”
In this condition wait for 15–20 min for warming of instrument (before
inserting the leaf in IRGA chamber).
Leaf should not fold in IRGA chamber. If leaf get fold it shows negative
readings. Leaf should not have any moisture and dust before inserting leaf.
Insert the leaf in IRGA chamber.
a. Give the “Dark pulse” (F3).
b. Press “zero” getting “zero” row.
c. Before going to next step, see the “F” value must be stable and df/dt value
d. Select DOF0Fm – (F3).
Select row no: 9: press “Actinic On” (F4).
Select row no: 8: press “Define actinic” (F3).
a. It shows “Actinic Definition – press “enter.”
b. Type 1000 (PAR value 1000) press “enter.”
Select “zero” row.
a. Before going to next step, see them, “F” value must be stable and df/dt
value is <5.
b. Select DOFsF0Fm – (F4).
If we want fluorescence value select “O” alphabet and note down the Fv’/Fm’
Now note down the IRGA readings (photosynthetic rate, transpiration rate,
Before taking next reading “Actinic is in OFF” (F4). Do as above for taking
every next reading.
Time taken for each reading is 10–20 min.
After taking of readings IRGA chamber must be in open condition (loose the
Replace the fluorescence chamber foam (White foam) at the time of entire
1. In every shut down process “Actinic” must be in “Off” condition.
2. Press “Escape button.”
3. Select “Utility menu – F5.”
4. Coming down using down arrow.
5. Select “Sleep.”
6. Give “Enter.”
4.3 Rubisco enzyme activity
7. It shows – Ok to sleep Y/N.
a. Press Yes – “Y” alphabet.
8. Switch off the system.
9. Disconnect the CO2 tube.
10. Keep batteries for charging.
Parameters recorded from IRGA:
1. Photosynthetic rate (Photo): mmole CO2 m2 s−1
2. Stomatal conductance (Cond): mole H2O m2 s−1
3. Transpiration rate (Trmmol): m.mole H2O m2 s−1
4. Intercellular CO2 concentration (Ci): mmole CO2 mole−1
5. Chlorophyll Fluorescence (Fv′/Fm′ values)
where Fv′ = variable fluorescence; Fm′ = maximum fluorescence.
4.3 RUBISCO ENZYME ACTIVITY
The enzyme Rubisco (ribulose bisphosphate carboxylase/oxygenase) has some special features of its own. It is the only enzyme which can catalyze carboxylation or
oxygenation reaction depending upon the molecular concentration of CO2 or O2. As
a carboxylase enzyme, it catalyzes combination of RuBP and CO2 resulting in the
formation of two molecules of 3-PGA. As an oxygenase, it plays the key role in the
production of p-glycolate, the first intermediate in the photo-respiratory pathway.
Rubisco constitutes more than 50% of soluble leaf protein. This indicates how important this enzyme into the plant.
In C3 plants, Rubisco is located in the stroma of all chloroplasts. However, in C4
plants it may be restricted to the chloroplasts of the bundle sheath cells.
There are eight active sites per molecule of Rubisco. The Michaelis constant (Km)
value of Rubisco for CO2 is around 10–20 mM and for O2 is around 200 mM. High
Km values reflect low affinity.
Rubisco is one of the most difficult enzymes to assay. This is because it is converted from an inactive to an active form by reaction with CO, and Mg++ and inactivation readily occurs in their absence. Another complication is that the extracted
enzyme appears to be cold inactivated in certain plants such as wheat (Coombs et al.,
1987). The amount of active Rubisco in a leaf is an important factor regulating the
rate of photosynthetic carbon fixation (Servaites et al., 1984).
4.3.1 MEASUREMENT OF RUBISCO ACTIVITY
Principle: Assay of Rubisco is generally carried out by radiometry technique where
radioactive CO2 is used as a substrate and traces of radioactivity in the products are
counted as a measure of enzyme activity.
1. Tris-hydroxymethyl amino methane
2. Hydrochloric acid (HCl)
CHAPTER 4 Photosynthetic rates
3. Magnesium chloride (MgCl2)
4. Dithiothreitol (DTT)
5. Acetic acid
7. 2,5-Diphenyl oxazole
8. 1,4-bis-2-methyl, 5-phenyl oxazolyl-benzene
1. Collect the leaf sample on sunny days and bring it in a butter paper bag, kept in
an ice box.
2. Weigh 0.2 g of freshly cut leaf pieces after blotting them dried.
3. Grind the sample in a prechilled pestle with mortar using 5 mL of ice cold
grinding medium [the grinding medium consists of Tris-HCl buffer, 50 mM (pH
8.0), MgCl2, 5 mM, and dithiothreitol (DTT), 10 mM].
4. Centrifuge the homogenate at 25,000 × g at 0–4°C for 5 min.
5. Take the supernatant, measure its volume, put it in a sample vial fitted with lid/
cap, and keep it under ice.
1. Measure the initial activity at 25°C by injecting 50 mL of soluble leaf extract
into an assay mixture containing 50 mM Tris-HCl (pH 8.0), 20 mM MgCl2,
0.1% (w/v) BSA, 0.5 mM RuBP, 10 mM NaH14CO3 (1 m Ci/assay) in a total
volume of 0.5 mL (the initial enzyme activity is measured immediately after
2. Measure the total activity in a similar manner with an exception that 50 mL of
soluble leaf extract and 350 mL of the assay mixture are incubated together at
25°C for 10 min in a shaking water bath before 100 mL of 2.5 mM RuBP is
3. Stop the reaction by adding 0.1 mL of 6 M acetic acid.
4. Dry tightly covered sample vials at 65°C.
5. To this, add 10 mL of cocktail [cocktail contains 2 g of PPO
(2,5-diphenyloxazole) and 50 mg POPOP (1,4-bis-2-methyl, 5-phenyl oxazolylbenzene) dissolved in 500 mL of toluene].
6. Determine acid stable (3-PGA) radio-activity in a liquid scintillation counter.
7. Run a blank similarly without RuBP (instead RuBP, add 0.05 mL of buffer).
8. Measure total soluble protein content in the crude enzyme extract by Bradford
9. Rubisco activity is expressed as [mmol CO2 (g protein)−1 s−1].
As mentioned earlier, Rubisco must be activated fully to achieve maximal activity (Lorimer et al., 1976). According to Kobza and Seemann (1988) percent activation of Rubisco can be calculated using the following formula
Activation (%) = (Initial activity/Total activity) × 100
4.4 Chlorophyll fluorescence ratio (Fv/Fm values)
4.4 CHLOROPHYLL FLUORESCENCE RATIO (FV/FM VALUES)
Chlorophyll fluorescence is the most widely used technique in photosynthesis and
plant stress research. Photochemical efficency of Photosystem (PS)-II is measured
as chlorophyll fluorescence in terms of Fv/Fm ratio (variable fluorescence/maximum
fluorescence). The extent of photoinhibition induced by any environmental stress
can be rapidly assesed by measuring the maximum photochemical effiency of PSII.
Chlorophyll fluorescence is measured directly by IRGA as explained earlier in this
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