1. Introduction to Plant Gas Exchange Measurements
LI-COR Inc., Lincoln, NE, USA

2. 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.

3. 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

4. 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

5. 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

6. Basic Research
Should we never study anything unless it has an immediate practical application?

7. 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”

8. 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).

9. Applications of gas exchange
When choosing a topic for research, it is important to pick something which interests you.

13. A recent Journal article gives an excellent description of protocols used to parameterize the model.
An Idealized A/Ci response. The rates of photosynthesis that would be achieved depending on whether Rubisco, (Wc), RuBP regeneration (Wj) or TPU triose phosphate utilization (Wp) are limiting

14. If there was no limitation to diffusion of CO2 by boundary layer and stomatal resistances then the assimilation rate would be A”…..however resistances do exist and when Ca =370 then Ci = about 240 in this figure and assimilation is lowered to A’

16. 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?

17. LI-6400 system checklist

18. 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?

19. Examples of data with weaknesses

20. Data Quality – avoiding noisy measurements
Measurement precision and IRGA noise
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%).

21. 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 +/ or 3%

22. Equation Summary
Transpiration
Photosynthesis

23. Intercellular Water Vapor
Water Vapor Mole Fraction
Water

24. Equation Summary -continued
Stomatal Conductance - obtained by restating transpiration in terms of Ohms law

25. 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):
Also it is known that gcs = gws/1.6

26. CO2 concentration in the mesophyll

27. 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
R: Sigma is the thermal emissivity of the leaf (0.95 for most leaves: Cotton (0.96), Tobacco (0.97), Cactus (0.98), Bean (0.96), and poplar (0.98)). Gamma is the Stefan-Boltzmann constant (5.67x10-8 W/m2 K).

28. 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

29. 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

30. 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

31. Photorespiration inhibition in a C3 leaf

32. Effect of O2 concentration of a C4 leaf

33. Diffusion Leaks

34. Custom Chambers

37. Stages of Photosynthesis

38. Leaf Structure

39. Chloroplast Structure
The Light Reactions occur in the grana and the Dark Reactions take place in the stroma of the chloroplasts.

40. Light Reaction Stages

41. 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

42. 6400-40 Leaf Chamber Fluorometer
Red (630nm)
Blue (470nm)
Far red (740nm)
Fluorescence Detection at >715nm<1000nm

43. Relative spectral outputs of the LCF

44. Pulse Amplitude Modulation (PAM)
Measuring on, Actinic on
Fm, Fm’
Light Intensity Fm
Fm’
After demodulation F
Measuring on, Actinic on Fs Fs Fo
Fo’
Time
Measuring On, Actinic off
Fo, Fo’

45. Fluorescence Parameters
Light-energized chlorophyll molecules release energy in one of three ways: Fluorescence, Heat or Photosynthetic photochemistry.
F + H + P = 1
As light intensity increases, P reaches a maximal value, and any additional light incident on the leaf is as Heat and Fluorescence. For any light above the saturation intensity the above equation thus becomes:
Fm + Hm + 0 = 1 So
Hm = 1- Fm
where Fm and Hm are the fluorescence and heat de-excitation energies from the modulated signal when the leaf is given enough light to saturate the photochemistry.

46. 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:
and
Also P = 1 - F - H

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

48. Other Fluorescence Parameters
Another relation similar to is:
The photochemical quenching of fluorescence, includes - photosnythesis and photorespiration
The non-photochemical quenching of fluorescence – heat, etc.
Another non-photochemical quenching parameter

49. A Fluorescence Induction Curve