Introduction to Plant Gas
Exchange Measurements
LI-COR Inc., Lincoln, NE, USA
Who Measures Photosynthesis?
• Mainly scientists measure photosynthesis
• Crop producers (farmers, horticulturalists)
do not usually measure photosynthesis
• In the paste, improvements in crop
production were achieved by lengthening
the growing period, by selecting higher
grain:foliage ratio, by the application of
fertilizer and irrigation - without
understanding photosynthesis.
Gas exchange versus agronomic
measurements
• Gas exchange – short term, high
sensitivity – e.g. reducing PAR reduces A
• Agronomic – longer term, integrative
(final yield, biomass production, LAI, etc.)
Analogous to monitoring heart, blood
pressure, sugar etc. versus monitoring
weight, height of a child
Practical applications of gas exchange
measurements examples (I):
• Cold tolerance of Maize genotypes
• Screening for fungicides, insecticides, with
least harmful effect on crop
• Screening for selective herbicides
Practical applications of gas exchange
measurements examples (II):
• Correct drought stress for growing sweet
grapes by monitoring stomatal conductance
• Finding optimum light levels for growing
medicinal herbs – absence of active
compounds under high light conditions
• Screening for reduced photorespiration
Basic Research
Should we never study anything
unless it has an immediate practical
application?
Historical examples of basic research
• History of electricity - Michael Faraday’s
experiments in electromagnetic induction
• Rutherford’s comments on nuclear science in 1936
“of no practical value”
• Mendel’s experiments on the genetics of sweet
peas. He was told to “go plant more flowers in the
garden”
Basic research applications of gas
exchange measurements:
• Basic research on understanding photosynthesis a reaction on which all life depends
• Scientists want to study how plants grow, how
ecosystems work.
• Global Change research: how rising level of CO2
and temperature could affect agriculture, as well
as the ecology (C3:C4 species balance).
Applications of gas exchange
• When choosing a topic for
research, it is important to pick
something which interests you.
Checking the LI-6400
• How do you know if the LI-6400 is working
properly?
• Would you test it on a leaf to see if it reads
photosynthesis correctly?
LI-6400 system checklist
Checking the LI-6400 Calibration
• User calibration - setting zero and span
• Does the LI-6400 IRGA needs factory calibration?
• New internal chemicals?
• How do you know?
Examples of data with weaknesses
Data Quality – avoiding noisy
measurements
Measurement precision and IRGA noise
A
u (c R  c S )
S
Photo 
 mol S  1 . mol mol

2
m

1



  mol m
2
S
1
Flow *  CO 2
LeafArea
Typical IRGA noise of the LI-6400 is +/- 0.2 ppm. So
the ∆CO2 fluctuates by +/- 0.2 ppm
For a 5% measurement precision, DeltaCO2 should be
≥5 ppm (because 0.2/5 ≈ 5%).
Data Quality – avoiding noisy
measurements
• If DeltaCO2 is only 1 ppm, then noise in
photosynthesis will be 1 +/- 0.2 or 20%
• If in above case flow is reduced to half, then
DeltaCO2 will double to 2ppm, and noise in
photosynthesis will be reduced to 2 +/- 0.2 or 10%
• If in above case a 2 cm2 leaf area, is increased to 6
cm2 then deltaCO2 will increase to 6 ppm and
reduce noise in photosynthesis to 6 +/- 0.2 or 3%
Equation Summary
Transpiration
E 
u ( wi  wO )
S
 mol S  1 .mol mol

2
m

1



 mol m
2
S
1
Photosynthesis
A
u (ci  cO )
S
 mol S  1 . mol mol

2
m

1



  mol m
2
S
1
Intercellular Water Vapor
Water Vapor Mole Fraction
W 
e
P
and
Wi 
e sat (T L )
P
Water
Equation Summary -continued
Stomatal Conductance - obtained by restating transpiration in terms of Ohms law
E 
u (wR  wS )
S
 mol S  1 .mol mol

2
m

1



 mol m
2
E  g s ( wi  w S )
gs 
E
( wi  w S )
 mol m  2 S  1 

1 
 mol mol

 mol m
2
S
1
S
1
Calculating Ci
If assimilation is expressed in terms of Ohms law
(i.e. in terms of internal leaf to chamber air CO2
concentration difference and stomatal conductance):
A  g S (C a  C i )
C
Also it is known that gcs = gws/1.6
Ci  Ca 
1 .6 A
w
gS
CO2 concentration in the mesophyll
Energy Balance Leaf
Temperature Measurement
0=Q+L+R
• R: Net radiation, made up of solar (total leaf
absorption) and thermal (black body
radiation balance from Tleaf, Tair, , and )
• L: Latent heat of vaporization: transpiration
• Q: Sensible heat flux, a function of (TleafTair), specific heat capacity of the air, and
one-sided boundary layer conductance of
the leaf
• Express R in terms of L & Q, solve for
(Tleaf-Tair) to determine Tleaf
Configuring the LI-6400 for surveys
• RefCO2 - Ambient + expected Delta
• Flow – fixed, high, but still adequate Deltas
• Light – consider leaf and sun relation
• Use prompts for data identification
Configuring the LI-6400 for Light
Curves
• Constant Sample CO2 - not Reference CO2
• Why?
• If choosing constant humidity, then start
with high flow, and slow RESPNS
• Fixed temperature
• Going from high to low light levels is faster
Configuring the LI-6400 for CO2
Response Curves
• Allow plenty of time for leaf to acclimate to
the light level
• Matching IRGAs is very important
• Measurements can be very fast as there is
no need to wait for acclimation to changes
in light
• Diffusive leaks can be significant
Photorespiration inhibition in a C3 leaf
A-Ci curves for a bean leaf at 2% and 21% oxygen
(PAR=1500 umol/m2/s)
At ambient CO2, difference is about {(14-10)/10} = 40%
16.00
14.00
Photo (umol/m2/s)
12.00
10.00
8.00
21% Oxygen
6.00
2% Oxygen
4.00
2.00
0.00
0.00
-2.00
100.00
200.00
300.00
400.00
-4.00
Ci (ppm)
500.00
600.00
700.00
Effect of O2 concentration of a C4 leaf
A-Ci curves for a Maize leaf at 2% and 21% Oxygen
(PAR=2000 umol/m2/s)
35
30
Photo (umol/m2/s)
25
20
21% Oxygen
15
2% Oxygen
10
5
0
0
50
100
-5
Ci (ppm)
150
200
Diffusion Leaks
Effect of CO2 diffusion - empty chamber measurements
(Room [CO2] was approximately 500 ppm)
Apparent CO2 exchange (umol/m2/s)
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
0
100
200
300
400
500
600
-0.2
-0.3
-0.4
Sample chamber CO2 (ppm)
700
800
900
Custom Chambers
Stages of Photosynthesis
Leaf Structure
Chloroplast Structure
The Light Reactions occur in the grana and the Dark Reactions
take place in the stroma of the chloroplasts.
Light Reaction Stages
Fate of Absorbed Light
• Typical for low light
conditions:
– 97% Photochemistry
– 2.5% Heat
– 0.5% Fluorescence
• Under high light
conditions:
– low% Photochemistry
– 95+% Heat
– 2.5-5% Fluorescence
6400-40 Leaf Chamber Fluorometer
•
•
•
•
Red (630nm)
Blue (470nm)
Far red (740nm)
Fluorescence Detection
at >715nm<1000nm
Relative spectral outputs of the LCF
Pulse Amplitude Modulation (PAM)
Measuring on, Actinic on
Light Intensity
0
Fm, Fm’
Fm
Fm’
Measuring on, Actinic on
After demodulation
F
Fs
Fs
Fo
Measuring On, Actinic off
Fo’
Time
Fo, Fo’
Fluorescence Parameters - continued
Also if it is assumed that the ratio of heat:fluorescence de-excitation remains
constant (for a given state of the leaf), then:
H

Hm
F
and
Fm
H 
F (1  Fm )
Fm
Also P = 1 - F - H
 1 F 
F (1  Fm )
Fm

Fm  F
Fm
Fluorescence Parameters - continued
If the F is measured on a dark-adapted leaf, then it is referred to as Fo and P
becomes:
P ( dark ) 
Fm  Fo

Fm
Fv
Fm
Fv/Fm is the fraction of absorbed photons used for photochemistry for a dark adapted
leaf. For most plants Fv/Fm is around 0.8
Under non-saturating steady-state photosynthesis the above equation takes the form:
P ( light ) 
Fm '  Fs
Fm

F
Fm '
  PSII
Other Fluorescence Parameters
Another relation similar to  PSII is:
Fv '
Fm '
qP 
qN 
Fm '  Fs
Fm '  Fo '
Fm  Fm '
Fm  Fo '
NQP 
Fm  Fm '
Fm '

Fm ' Fs '
Fm '
The photochemical quenching of fluorescence,
includes - photosnythesis and photorespiration
The non-photochemical quenching of fluorescence – heat,
etc.
Another non-photochemical quenching parameter
A Fluorescence Induction Curve