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

LeafArea

COFlowPhoto 2*

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%).

122

11.)(

Smmol

m

molmolSmol

S

ccuA SR

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

122

11.)(

Smmol

m

molmolSmol

S

wwuE Oi

122

11.)(

Smmol

m

molmolSmol

S

ccuA Oi

Transpiration

Photosynthesis

Intercellular Water Vapor

P

TeWand

P

eW Lsat

i

)(

Water Vapor Mole FractionWater

Equation Summary -continued

Stomatal Conductance - obtained by restating transpiration in terms of Ohms law

)( Sis wwgE

121

12

)(

Smmolmolmol

Smmol

ww

Eg

Sis

122

11.)(

Smmol

m

molmolSmol

S

wwuE SR

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):

)( iaCS CCgA

Also it is known that gcs = gw

s/1.6

wS

ai g

ACC

6.1

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 (Tleaf-Tair), 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%

-4.00

-2.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00

Ci (ppm)

Ph

oto

(u

mo

l/m

2/s

)

21% Oxygen

2% Oxygen

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)

-5

0

5

10

15

20

25

30

35

0 50 100 150 200

Ci (ppm)

Ph

oto

(u

mo

l/m2/

s)

21% Oxygen

2% Oxygen

Diffusion Leaks

Effect of CO2 diffusion - empty chamber measurements(Room [CO2] was approximately 500 ppm)

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 100 200 300 400 500 600 700 800 900

Sample chamber CO2 (ppm)

Ap

par

ent

CO

2 ex

chan

ge

(um

ol/

m2/

s)

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)

Fo, Fo’0

Fm, Fm’

Measuring On, Actinic off

Measuring on, Actinic on

Light Intensity

After demodulation

Fm

Fs

Fm’

Fo

Time

FMeasuring on, Actinic on Fs

Fo’

Fluorescence Parameters - continued

Fm

FmFH

)1(

Fm

FmFF

)1(1

Also if it is assumed that the ratio of heat:fluorescence de-excitation remains constant (for a given state of the leaf), then:

Fm

Hm

F

H

and

Also P = 1 - F - H

Fm

FFm

Fluorescence Parameters - continuedIf the F is measured on a dark-adapted leaf, then it is referred to as Fo and P becomes:

Fm

Fv

Fm

FoFmdarkP

)(

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:

PSIIFm

F

Fm

FsFmlightP

'

')(

Other Fluorescence Parameters

'

''

'

'

Fm

FsFm

Fm

Fv

'

'

FoFm

FmFmqN

Another relation similar to is:

''

'

FoFm

FsFmqP

PSII

'

'

Fm

FmFmNQP

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