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Plant Detectives Manual: a research-led approach for teaching plant science

 

Activity 7: Gas exchange measurements on intact leaves — photosynthetic responses to light and CO2

7.1) Introduction and objectives

It is possible to determine rates of CO2 assimilation and water loss (transpiration) by measuring the flux of CO2 and water vapour from a leaf in a sealed chamber. This process, termed gas exchange (because CO2 is going in and water vapour is coming out) is more complicated than might be initially imagined. During photosynthesis, plants take up CO2 (which is converted to sugar) and produce oxygen. All the while they are respiring and releasing CO2 back into the cells. To make matters more complex, the enzyme that fixes CO2 (Rubisco) also ‘fixes’ oxygen, a reaction called photorespiration that releases CO2, but does not produce energy. Gas exchange is also influenced by light levels, because when more light is available, generally, more CO2 can be fixed.

 

In this activity you will measure gas exchange on your wild type and mutant plants and determine whether the plants differ in rates of carbon fixation or water loss at a set of standardised conditions. Your photosynthesis measurements will be done using an infrared gas analyser (IRGA) (Fig. 8). The IRGA uses infrared radiation to detect the concentration of H2O and CO2 in the air being pumped over a leaf in a chamber. In effect, the gas concentrations are compared before and after being passed by the leaf. Using information about the change in H2O and CO2,the IRGA calculates photosynthesis and stomatal conductance of water. Consider for a moment a well-lit leaf in the chamber — how would you expect the gas concentrations to change before and after exposure to the leaf? What if the chamber was darkened?

Examining the shape of the photosynthetic response to light reveals several important things about the biology of a leaf (Fig. 8C). In the dark, no photosynthesis takes place in C3 leaves and respiration (production of energy using O2 and producing CO2, just like animals do) is greater than photosynthesis. Therefore, plants show a net production of CO2. When light levels rise, photosynthesis starts and CO2 is taken up and fixed. The higher the light, the more CO2 can be fixed, up to a certain point when CO2 uptake is saturated. By determining the response of photosynthetic rate to light, we can identify the following parameters:

  1. Rd: respiration rate
  2. LCP: the light compensation point, or the light level at which respiration and photosynthesis balance each other
  3. LSP: the light saturation point, or the light level at which photosynthetic rate ceases to increase
  4. Φ: quantum yield
  5. photosynthetic capacity, or maximum rate of assimilation
  6. transpiration rate (T): although not a photosynthetic parameter, T is an estimation of the amount of water leaving the leaf per unit area per time
  7. stomatal conductance (gs): an estimation of how open stomatal pores are to allow flux of both water vapour and CO2 out and in the leaf

 

If time permits you may also be able to measure a CO2 response, or ‘A vs. Ci(ACi), where A is the photosynthetic rate and Ci the CO2 concentration inside the leaf. An ACi curve measures the rate of CO2 assimilation, generally at high light levels, and across varying internal CO2 concentrations. We use these curves to indicate the maximal photosynthetic capacity of the leaf in the absence of limitation by CO2 concentration. These curves can also be used to indicate electron transport capacity and characteristics of Rubisco kinetics. Your demonstrator will help you to measure and interpret an ACi curve.

 

If you are running out of time, you may want to measure assimilation only at one standard condition of ambient CO2 and high light and replicate these measurements to compare your mutant and wild type.

 

A porometer will be used to measure stomatal conductance (gs). The porometer has a time advantage over the IRGA, though both can tell you about plant water use. The porometer is a more simple piece of equipment that measures temperature and water content in the air around the leaf using a small, lightweight chamber. Because the porometer is quicker to use, more measurementsof gs in the abaxial side of the leaf (bottom side) can be made resulting in a large sample of data being available for statistical analyses.

 

Gas exchange is very sensitive to measurement conditions, including light level, temperature, CO2 concentration, water vapour/relative humidity in the chamber and, perhaps most surprisingly, time. When taking your measurements, record information about the measurement conditions. Also, remember that the porometer, unlike the IRGA does not have a light source. Consider the effect of light on stomata and make sure that your plants are in an appropriate light environment before performing the porometer measurements. Finally, be aware that plants often show a reduction in photosynthetic rates as the day progresses, sometimes ‘shutting-down’ entirely by mid- to late afternoon. We recommend that all gas exchange measurements are done early in the day. Alternatively, your instructor may grow your plants in a growth chamber with the timing of daybreak shifted relative to real time, so that your plants behave as if it is morning even when assayed in the afternoon.

 

The objectives of Activity 7 are to:

  1. become familiar with a portable gas exchange system to determine the photosynthetic rates (A). Our protocol is based on a Li-Cor 6400 model IRGA, but can be adapted to any other instrument
  2. compare total photosynthesis and stomatal conductance between wild type and mutant plants
  3. run a ‘light curve’ for the wild type plant and, if time permits, for the mutant plant.

 

Image

Figure 8. Basics of gas exchange measurements and the light curve

A) Schematic representation of the gas exchange system LiCor. B) Basic equation for photosynthesis and transpiration. C) Light curve resulting from plotting rate of CO2 assimilation versus increasing light intensity (irradiance). Note that assimilation is first limited by the amount of light and then by the rate of carboxylation and recycling of the required precursors. Schemes courtesy of Susanne von Caemmerer, The Australian National University.

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7.2) Materials

  1. Li-Cor gas exchange system
  2. CO2 cartridges
  3. desiccant
  4. charged batteries for Li-Cor or adaptor for main power

7.3) Procedure

7.3.1) Measuring gas exchange parameters using an IRGA

It is likely that your instructor will operate the IRGA equipment for you. This is fine because it is a complicated piece of equipment that takes practice and experience to use. In the Plant Detectives Project we are more interested in you learning the principles than how to run the machine6.

 

The measurements will demonstrate the dependence of photosynthetic processes on measurement conditions and growth conditions, as well as allowing you to assess any differences due to the genetic make-up of the study plant.

 

Your instructor will aim to complete the following with you over the course of the exercise:

  1. a light response curve for a single wild type and mutant plant
  2. if time permits, measure photosynthesis at saturating light for two more replicates of the wild type and mutant. By combining your data with that from the light response curve from the same light level you will have three replicates to use in statistical analysis. If time permits you can measure an ACi (photosynthesis vs. CO2 concentration) curve on a single wild type and mutant plant to get an idea of how CO2 concentration affects photosynthetic rate and water loss.

Record the settings used for the measurement conditions in your laboratory notebook. We have previously found that the conditions in parentheses below work well for measurements of Arabidopsis plants grown in growth chambers or controlled conditions in a glasshouse:

  1. [CO2] reference: the CO2 level at which measurements are being made (400 parts per million (ppm))
  2. flow: how much the air is circulating in the system (300 ml min–1)
  3. Temperature: the temperature inside the measuring chamber (22 oC)
  4. PAR (µmol m–2 s–1): photosynthetically active radiation. For the light curve use in this order: 800, 500, 350, 100, 50 and 0.
  5. RH: relative humidity (or VPDl or [H2O]) — how much water is in the air. Aim to maintain between 50–60%

 

Record the following information into a table in your notebook or an Excel spreadsheet for each measurement you take. Your instructor may also provide you with a spreadsheet of all the data produced by the IRGA, but you’ll find the key measures are:

  1. plant number and whether wild type or mutant
  2. CO2 R: CO2 concentration in the reference cell (i.e., before passing over the leaf) — this is set by the operator
  3. PAR: this is set by operator
  4. temperature of the block: this is set by operator
  5. RH: this varies, but the operator aims to keep it between 50 and 60%
  6. photo: photosynthetic rate in terms of uptake of CO2, µmol m–2 s–1 (note, in the dark this will be negative, respiration releases CO2)
  7. cond (gs): rate of stomatal conductance to water
  8. Ci: internal concentration of CO2, ppm

 

Sample data sheet — fill all cells in the spreadsheet, even if it means repeating some information on subsequent lines (e.g., genotype and plant number will repeat over every line of a given set of light response curve measurements)

Genotype

Plant Nº

[CO2]Ref

PAR

Photo

Conductance (gs)

Ci

Comments

 

 

(ppm)

(µmol m–2 s–1)

(µmol m–2 s–1)

(mol m–2 s–1)

(ppm)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7.4) Expected outcomes

  1. You will be able to plot A (photosynthetic rate) vs light intensity and gs (stomatal conductance) vs light intensity for the individual plants measured. Make these plots and draw a curve by hand or using software if you prefer. Based on your light-response curve, determine what light level is needed to achieve ~90% of the maximum photosynthetic rate (e.g., where does the rate start levelling off?).
  2. Using the curve, approximate the compensation point, saturation point, quantum yield, and light-saturated photosynthetic rate for your wild type and mutant plants. Do they look very different?
  3. At saturating light, is there any difference in A or gs between the wild type and mutant plants? (If you managed to take some extra measurements you will be able to assess this statistically.)
  4. If you were able to measure it, what is the maximum photosynthetic capacity for your wild type and mutant plants in the absence of stomatal (CO2) limitation? How does this compare to the measurements at ambient CO2 levels?

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