MEASURING PHOTOSYNTHESIS BY GAS EXCHANGE SYSTEMS

(LI-6400)
/Petra Majer/

Goals of the practice:

 Learn principles of gas exchange measurements and infra red gas
analysis
 Get familiar with infrared gas exchange systems, in particular with the
LI- 6400
 Make photosynthesis measurements on different tobacco leaves

PRINCIPLES

Gas exchange measurements

Measuring gas exchange is the most commonly utilized technique at present for
commercial and research purposes in order to measure photosynthesis of individual
leaves, whole plants or plant canopy. Gas exchange measurements provide direct
measure of the net rate of photosynthetic carbon assimilation.
Main advantages of gas exchange measurements: instantaneous, non-destructive,
direct.
CO2 exchange systems use enclosure methods, where the leaf in closed in a
transparent chamber. The rate of CO2 fixed by the leaf enclosed is determined by
measuring the change in the CO2 concentration of the air flowing across the chamber.
Because ambient atmospheric CO2 concentration is only 0.04 % (400 ppm), it is
difficult to measure photosynthetic CO2 uptake and sensitive sensors are needed.

Infrared gas analysis

Heteroatomic gas molecules absorb radiation at specific infrared (IR) wavebands,
each gas having a characteristic absorption spectrum. Infrared gas analyzers (IRGAs)
measure the reduction in transmission of IR wavebands caused by the presence of
CO2 between the radiation source and a detector. The reduction in transmission is a
function of the concentration of CO2. The only gas normally present in the air with an
absorption spectrum overlapping that of CO2 is water vapour. Since water vapour is
usually present in the air at much higher concentrations than CO2, this interference is
significant, but may be overcome simply by drying the air or measuring H2O
concentration by another IRGA.
PHOTOSYNTHESIS GAS EXCHANGE SYSTEMS

General parts of a gas exchange system:

- leaf chamber
- flow meter
- means of generating and controlling air flow over the leaf
Open versus closed systems
In closed systems the signal from the sample cell is compared to the zero gas
reference signal to provide an absolute measurement of CO2 concentration. A leaf is
enclosed in a chamber, sealed to avoid gas exchange with the atmosphere, and the rate
at which the CO2 and H2O concentration changes in the chamber are monitored.
Major disadvantages of closed IRGA system:
 Photosynthesis measurements must be made within a few seconds after
closing the leaf chamber (once the leaf is sealed in the chamber CO2
concentration in the leaf chamber is continually decreases and water vapour
increases).
 The operator has limited control over environmental conditions within the
chamber.
Open systems are configured to allow air from a single source to enter both the
analysis and reference lines. Air is continuously passed through the leaf chamber (to
maintain CO2 in at fixed concentration) and measurements of photosynthesis and
transpiration are based on the differences in CO2 and H2O in an air stream that is
flowing into the leaf cuvette (reference cell) compared to the air stream flowing out of
it (sample cell). The rate of CO2 uptake is used to assess the rate of photosynthetic
carbon assimilation, while the rate of water loss is used to assess the rate of
transpiration (counted on a leaf area basis).

Schematic illustration of open and closed IRGA systems

(Illustration by Alan Rhodes, Mulkey & Smith 1998)
LI-6400 Portable Photosynthesis System (LI-COR, Lincoln, Nebraska, USA)

LI-6400 is an open system design photosynthetic gas exchange system. IRGAs (H2O
and CO2) are in the sensor head, that gives a possibility for a tight control of
conditions in the leaf chamber response to leaf changes. The incoming air stream can
be conditioned for CO2 concentration, humidity and temperature. There are chemical
tubes for scrubbing CO2 and H2O, and air can be diverted through these tubes in any
proportion desired. CO2, however, is best controlled by scrubbing all of it from the
incoming air, and using CO2 source to provide a stable concentration at the desired
value (optional CO2 injection system). The system is equipped with Peltier
thermoelectric coolers for temperature control and light sensors. LI-COR offers a
variety of interchangeable leaf chambers (coniferous, broadleaf, soil). The newest
version, LI-6400XT is also capable of measuring fluorescence and gas exchange
simultaneously over the same leaf area.

Schematic of LI-6400 (Source: Using the LI-6400, Manual, version 5.3)

The reference analyzer measures incoming gas concentrations and is located directly
below the sample analyzer. The sample and reference analyzers can be matched at any
time, either manually or automatically, without altering conditions in the leaf
chamber.

A B

The LI-6400 device (A) and the sensor head (B) (Source: Using the LI-6400, Manual, version 5.3)
Main parts of the LI-6400 device:
Console: a data logging computer with keypad and LCD screen
Sensor Head/IRGA: leaf chamber (for different leaf areas), spring-loaded latching
handle, 2 Peltier thermoelectric coolers, sample and reference IRGAs, light sensor
(external quantum sensor is optional)
Chemical Tubes: a tube containing soda lime (to remove CO2 from incoming air
stream) and a tube containing Drierite, a desiccant (to remove water vapour)
CO2 source: cartridge + injector or tubing from a buffer volume

LI-6400 console (Source: Using the LI-6400, Manual, version 5.3)

What can we measure with LI-6400?

LI-6400 provides direct measurements of net photosynthetic rate and transpiration and
indirect assessment of stomatal conductance and many other variabes.
 Instantaneous gas flux measurements (13 computed and 55 measured
variables)
 Photosynthesis light response curves
 A/Ci curves: response of CO2 uptake to intercellular mole fraction of CO2
 Daily courses of gas exchange

Main variables: Abbreviations:

common LI-6400
Photosynthetic rate A Phot µmol CO2 m-2 s-1
Transpiration rate E Trmmol mmol H2O m-2 s-1
Stomatal conductance to H2O gs gsw mol H2O m-2 s-1
Intercellular CO2 concentration Ci Ci µmol CO2 mol-1
Photosynthesis light response curves (rapid/slow)
With increasing photosynthetically active radiation (PAR) chloroplast stroma becomes
more alkaline that leads to the activation of Rubisco, and an increase in ATP and
NADPH production, therefore to an increase in photosynthetic CO2 assimilation (A).
If we observe the relationship of photosynthetic CO2 assimilation and light, we can
see that initially A increasis linearly. It reaches the light compensation point at a
certain PAR level, where CO2 assimilation and respiration balances each other. The
slope of the linear shows the maximal quantum efficiency of photosynthesis (0.04-
0.06 mol CO2 mol-1 photon). In this part of the response curve, photosynthesis is
limited by the rate of electron transport, which is in turn limited by the amount of
available light. With further increase in light photosynthesis becomes CO2 limited,
until where the curve reaches a light saturation point, where A is not responding to
further increases in PAR level, and is limited by the carboxylation capacity of Rubisco
or by triose phosphate metabolism.
Rapid light response curves: ca. 2 minutes at each PAR, not enough time for
stomatal adjustment
Slow light response curves: 15-20 minutes at each PAR (till the stomatal adjustment
happens)

Amax

(PAR)
Photosynthetic light response curve in a C3 plant (Source: Taiz & Zeiger 2010)
PRACTICAL

Samples:
Tobacco (Nicotiana tabacum L. var Xanthi), 6-week-old
1, greenhouse grown at ca. 200 µmol m2 s-1 PAR
2, greenhouse grown at ca. 200 µmol m2 s-1 PAR and placed outdoor under solar
radiation for a few days
3, greenhouse grown at ca. 200 µmol m2 s-1 PAR, water withdrawal for a few days
(drought stressed)

Measurements:
1, Rapid light response curves (ca. 2 min per irradiation level)
a) greenhouse tobacco
b) outdoor tobacco
c) drought stressed tobacco
2, Effect of clouds
a) „fast cloud” (brief shade simulation)
b) „slow cloud” (long shade simulation)

DISCUSSION & QUESTIONS

Sources and further reading

Long SP, Farage PK, Garcia RL, 1996. Measurement of leaf and canopy photosynthetic CO2 exchange in
the field. Journal of Experimental Botany 47: 1629-1642.
Mulkey SS, Smith M, 1988. Measurement of photosynthesis by Infra Red Gas Analysis. IN:
Bioinstrumentation, C.T. Lange, (ed). 79-84. Publication of the American Biology Teachers Association.
Taiz L, Zeiger E, 2010. Plant Physiology, Fifth Edition. Sinauer Associates, Inc. Sunderland, MA, USA.
LI-COR, 2004. Using the LI-6400 Portable Photosznthesis System. Version 5 (2004) LI-COR
Biosciences, Inc. Lincoln, NE, USA
http://www.licor.com/env/products/photosynthesis/measurements.html
DATA SHEET

Plant PAR A E gs Ci
µmol m-2 s-1 µmol CO2 m-2 s-1 mmol H2O m-2 s-1 mol H2O m-2 s-1 µmol CO2 mol-1
100
300
500
greenhouse 700
tobacco 900
1100
1300
1500
1500
(30 s) 300
1500
(2 min) 300
1300
outdoor
1100
tobacco
900
700
500
300
100
100
300
500
drought
700
stressed
900
tobacco 1100
1300
1500

Rapid Light Curve: Rapid Light Curve:

30 30
A (µmol CO2 m s )

A (µmol CO2 m s )
-1

-1

25 25
-2

-2

20 20

15 15
10 10

5 5

0 0
100 300 500 700 900 1100 1300 1500 100 300 500 700 900 1100 1300 1500
-2 -1 -2 -1
PAR (µmol m s ) PAR (µmol m s )