FS561 – Fall 2009

Soil and Leaf Gas Exchange

Claire Phillips and Barbara Bond

Overview.

This activity will introduce you to gas exchange measurements using a LiCor 6400 Portable Photosynthesis Machine. You will use the 6400 to conduct soil respiration measurements and you will measure a light response curve for leaf photosynthesis. From your measurements (and supplemental data) you will analyze the results of the light response curve and you will investigate potential linkages between aboveground and belowground respiration.

Background. 

Relevance of Gas Exchange

The expression “gas exchange” is often used to refer to the uptake and loss of CO₂ and water vapor. In this lab we will focus primarily on above- and below-ground fluxes of CO₂ by measuring leaf photosynthesis of potted cottonwood seedlings and soil respiration from the pots they’re planted in. We will discuss water vapor flux insofar as it effects the measurement of CO₂ flux, but it will not be measured explicitly in this lab.

Why do we want to measure soil respiration in a plant physiology class?? Plants do not end at the soil surface, and root respiration accounts for 20-70% of soil respiration in forests.

Using the LiCor for Foliage and Soil Measurements

We will use the same instrument, the LiCor 6400, for foliage and soil measurements by making a few changes in plumbing. The LiCor is an IRGA, an Infrared Gas Analyzer. It measures CO₂ and water vapor concentrations by measuring the amount of infrared radiation that can be transmitted through a gas sample. Both CO₂ and water vapor are strong absorbers of infrared radiation (that is why they are greenhouse gases), and the level of infrared absorption is proportional to their concentrations. Separate detectors for water vapor and CO₂ contain filters that permit only wavelengths unique to each gas to reach the detectors. However, because there is overlap between the absorption spectra of each gas, the software corrects measurements of CO₂ concentration based on measurements of water vapor concentration.

To measure photosynthesis, leaves or needles are placed in a gasket-sealed cuvette while they are still attached to a stem. Instruments integrated into the cuvette control and measure light, temperature, and moisture levels. The concentration of CO₂ entering the cuvette can also be controlled.

Air is pumped through the LiCor and split into 2 pathways: one to the leaf cuvette and an attached sample IRGA, and the other to a reference IRGA. The reference IRGA measures the CO₂ concentration of air that enters the leaf cuvette.  The difference in concentration between the sample and reference IRGAs gives the amount of CO₂ taken up the leaf. This amount, multiplied by the air flow rate, gives the rate of photosynthesis in units of mmol CO₂ m^-2 s^-1(you might try to check the units to verify that this multiplication ends up with correct units). This so-called “open system” produces steady-state conditions– i.e new air is continuously pumped through the cuvette so over the course of a measurement the leaf encounters constant levels of incoming CO₂ and H₂O vapor

To measure soil respiration, the LiCor is re-plumbed as a “closed system,” and only the sample IRGA is used. The leaf cuvette is replaced with a 2 liter soil chamber that attaches to the sample IRGA. Soil respiration rate is determined by placing the chamber on the soil surface and measuring the rate of CO₂ accumulation in the chamber over time. The units of soil respiration are also mmol CO₂ m^-2 s^-1. During soil measurements the pump is only used for a few moments to move CO₂ free air into the soil chamber and draw down the CO₂ concentration below ambient levels. Then the pump is turned off and the rate of CO₂ accumulation is measured as the CO₂ concentration in the chamber approaches ambient levels. In contrast, photosynthesis rates are measured in a flow-through system, with the pump constantly moving new air through the sample and reference IRGAs.

This synopsis and the reading in the LiCor manual should provide you with a good understanding of how the LiCor works in both foliage and soil applications.

Specific learning objectives.  After you complete this laboratory activity and the write-up after the lab, you should be able to:

Photosynthesis

1. Explain the fundamental principles of how open system gas exchange measurements work.

2. Conduct photosynthesis and foliar respiration measurements with the LiCor 6400 (excluding set-up and calibration)

3. Construct a light-response curve, and estimate A_max (maximum net photosynthesis), R_d (dark respiration), quantum efficiency, and light compensation point from the curve. 

Soil Respiration

1. Explain the fundamental principles of how closed-chamber gas exchange measurements work.

2. Measure soil respiration with the LiCor-6400.

Supplementary reading:

1. Li6400 Instruction Manual pp 4-24 through 4-28, “Light Response Curves”

2.  Using the LI 6400 v.5

    pp 7-2 through 7-20, “Environmental Control”

    pp 28-2 through 28-6, “Using the 6400-09 Soil Chamber”

3. LiCor Application Note #124 “Considerations for Measuring Ground CO2 Effluxes with Chambers”

Lab Activities. 

The lab will have three parts.  First, Claire will give a brief introduction to the lab and to general principles of gas exchange.  Then we’ll divide the class into two groups.  Both groups will measure both photosynthesis and soil respiration on a hybrid poplar and a native black cottonwood.  One group will work on photosynthesis and the other on soil respiration.  The 2 groups will switch places about half way through the class. You will compare the measurements of photosynthesis and respiration for both the control and the transgenic plants. 

PHOTOSYNTHESIS

A. Preparation

You will be using potted Populus plants for this lab; half are hybrids and the other half are black cottonwood (P. trichocarpa).  You will measure a light response curve for at least one leaf of each type.  Choose the first fully mature leaf near the top of the plant for this measurement. 

Trace the outline of the leaf on the grid attached to the end of this lab (in order to estimate its area), and count all of the leaves on the plant, roughly estimating their area relative to the leaf you outlined.  Assume that the grid is 0.5 cm (it is close to that).  From this and the number of leaves you counted for each plant, estimate the total leaf area of the entire plant.

B. LiCor 6400 Setup

·         Commands in the “MEASUREMENTS” menu are made from 6 sub-menus (or “levels”) numbered at the bottom left of screen. Levels may be selected by scrolling with “LABEL” keys or by hitting 1-6. Within the different levels, commands are made by hitting command keys (f1, f2, etc)

• Open a log file and name file such as “fs561 foliage-group#-treatment (transgenic or control)” (f1 Level 1)
• Set “FLOW METER” at 500ums (f2 Level 2).
• Set “LEAF AREA” to 6.
• Set “STOMATAL RATIO” to “0” (f2 Level 3; 0 = one-sided, 1 = two-sided).  This ratio refers to the area of stomata on the upper, or adaxial surface divided by the lower, or abaxial, surface.  Douglas-fir have no adaxial stomata, so the ratio is 0 for this species). 
• “GRAPH QUICK PICK” select: the default graph of “standard gas exchange” (f2 Level 4) which plots photosynthesis and stomatal conductance. This will help to determine stability prior to logging measurements.

C. Photosynthesis Measurements for light response curve

• Set “MIXER” CO₂S (CO₂ sample) at 365ppm (f3 Level 2)
• Set “LAMP” at quantum flux of 1500 umol m^-2 sec^-1 (f5 Level 2)
• Open cuvette and clamp onto leaf.
• View graph and wait for values to stabilize (f3 Level 4)

·         Select “MATCH” and follow on screen instructions for matching IRGAs (f5 Level 1). Be sure to allow water vapor values to match before leaving match mode.  It is critical to match irgas periodically (especially when CO₂ levels or temperature have changed much) and a good idea to match just before each measurement.

• Take a measurement (black button by handle or “LOG” (f1 Level 1).
• Repeat the above steps, changing the light level between each measurement.  Try the following light levels:  1500, 900, 500, 250, 100, 50, 25, and 0.  Wait until the gas exchange stabilizes after each change.  

RESPIRATION

Demonstration of soil respiration measurements

You will make a set of 3 soil respiration measurements on each of the potted seedlings that you use for photosynthesis measurements.

1. Open a new log file

Open a new log file (f1 level 1) and type in an appropriate name.

2. Turn prompts on

Turn prompts on to enter the pot ID with the measurement (f4 level 3).

3. Position the ring stop

Move the metal ring on the outside of the soil chamber up or down so that when the chamber is placed inside a soil collar the chamber will be 0.5-1.5cm above the soil surface. Tighten the thumb screws well so it doesn’t slip!

4. Insert the soil temperature probe

Insert the probe near the collar but out of the way of where the chamber needs to be. Be gentle as it bends easily.

5. Calculate the “insertion depth”

·         This number will be used to recalculate the chamber volume, which is part of the CO₂ flux calculation.

·         You need to determine the distance between the chamber rim and the soil surface. First measure the height of the soil collar above the soil, then the distance between the rim of the chamber and the foam gasket, and find the difference between the two measures. Since the soil surface is uneven, measure the height of the soil collar in a few locations and take an approximate average.

·         With the bottom row of menu options on level 7, press F5 to enter the insertion depth. Enter your measurement as a negative value because with soil collars chamber sits above the soil surface.

E. Determine the ambient CO₂ concentration

·         Lay the chamber on its side next to the collar, aiming upslope or upwind if possible to take advantage of air circulation.

·         When the readings have stabilized, enter the ambient concentration in “Target” (f1 level 7). You will then be prompted for DCO₂ (“Delta”), which is the range around ambient where you want to measure respiration. Entering 10 means respiration measurements will begin at 10ppm below ambient CO₂ and will end 10ppm above ambient. Enter 10 to start, and if respiration rates are very slow you can later switch to 5.

F. Start the measurement

·         Press Start (f3 level 7). If you have Prompts ON, you will get those now. The measurement cycle will begin.

·         Record the location, the starting time, and the final soil temperature, and all 3 flux rates.

G. Carefully remove the soil chamber without pulling out the collar.

Lab Write-up.  You don't need to provide copies of your raw data. 

1. (2)  This lab involves two kinds of comparisons.  One comparison is between the two Populus types.  The other comparison is between rates of photosynthesis and rates of soil respiration.  What kinds of differences would you expect (if any) in each of these comparisons?  Provide a rationale for your answers.  [note – we will not have time in the lab for you to conduct enough measurements to actually test these hypotheses, but we will see if there are any trends]. 
2. (1) For each of the leaves you used for photosynthesis measurements, plot the light response curve (i.e., net photosynthesis vs. light).  (You should have at least two curves, one for each Populus type).
3. (2) Using information provided in lecture on Tuesday (and any other supplementary information that you find useful), determine or estimate the following parameters from each light response curve:  Amax (maximum, light-saturated net photosynthesis), Rd (dark respiration), quantum efficiency, light compensation point. 
4. (1) Calculate “scaled up” estimates of maximum photosynthesis by the whole plant canopy.  To do this, first assume that the gas exchange characteristics for the entire leaf were the same as the portion you measured, and estimate the light saturated rate of photosynthesis for the entire leaf you measured for each plant.  Then, assuming that the gas exchange characteristics for every leaf on the plant were the same as the leaf you measured, estimate the light-saturated rate of photosynthesis for the whole plants.  [This is a very “coarse” way to scale up to the canopy.  If you have time you might want to do a few extra measurements of Amax on leaves in different positions to test whether this assumption is true]
5. (1) Using the data collected for soil respiration by the entire class (we will put the data on the chalk board in class as the measurements are made) calculate the mean and mean standard error for soil respiration for each of the two Populus types.   
6. (1.5) In a table or in text, summarize the differences in gas exchange characteristics that you found between the two Populus types.  Were these findings consistent with your hypothesis? 
7. (1.5) In a table, figure or in text, summarize any relationships that you found between respiration and leaf and whole-plant, light saturated photosynthesis.  Were these findings consistent with your hypothesis?

References

Field, Ball and Berry, Plant Physiological Ecology, (1989).  “Photosynthesis:  principles and field techniques”, chapter 11 in Plant Physiological Ecology:  Field Methods and Instrumentation, by R.W. Pearcy, J. Ehleringer and H.A. Mooney.  Chapman and Hall Press.

Li6400 Instruction Manual

ftp://ftp.licor.com/perm/env/LI-6400/Manual/Using_the_LI-6400-v5.3.pdf

LiCor application note #124, Considerations for Measuring Ground CO₂ Effluxes with Chambers. Presented at American Geophysical Union conference, 1998.

Making Measurements:  Light Response Curves (from the Li-6400 manual)

Taiz L.  Zeiger E. (2006). Plant Physiology, Fourth edition. Sinauer Associates.