Some Simple Experiments

If you have not had much experience making gas exchange measurements, you may wish to work through some of the experiments in this section. To do them, first establish a leaf in the chamber:

Do this first

1. Select a plant and leaf to measure
2. The preferred plant material is an adequately watered plant that is growing in full or partial sun. By contrast, measurements will be more difficult if done on a dry, neglected house plant that has only seen dim (for the plant) fluorescent lights its whole life.
2. Do Steps 1 through 6 in the previous section (see Clamping Onto the First Leaf)
3. Set the controls (light, flow, CO₂, temperature), area, and stomatal ratio.
3. Observe the CO₂ concentrations
4. Note CO2S_μml. Is it below CO2R_μml? If so, that’s good, because it means there is more photosynthesis than respiration. (Net photosynthetic rate will be on display line c, under Photo.) CO2S_μml should stabilize (within 0.2 or 0.3 μmol mol^-1) after 30 seconds or so of clamping onto the leaf. If it’s not stable, check the stability of CO2R_μml. Perhaps the mixer hasn’t stabilized yet, or you need a buffer volume. Consult Unstable Photosynthetic Rates if you need help fixing this instability. If CO2S_μml is not below CO2R_μml, then perhaps you need to match (Matching the Analyzers).
4. Observe the humidity values
5. The RH_S_% value on display line b is the relative humidity in the sample IRGA. It’s calculated from the water IRGA signal, which is H2OS_mml. Other things being equal, you want to make gas exchange measurements in as high a humidity as possible, without letting the flow rate (which is determining that humidity) go too low. 200 or 300 μmol s^-1 is a reasonably low flow range; but you can drop to 100 if you need to. (Leaks are a bigger problem at low flow rates - see Leaks for more details.)

You are now ready to do some or all of the following elementary experiments.

Humidity Control Experiments

Step 9 in Revisit the flow control asks you to decide how you wish to operate while making measurements: either a fixed flow rate (with a potentially variable humidity), or constant humidity (with a potentially variable flow rate). The following experiment will acquaint you with the capabilities and trade-offs involved.

Experiment #1 Finding the Humidity Limits

If you are using the CO₂ mixer, set it to control the reference concentration at a value slightly above ambient, such as 400 μmol mol^-1 if you’re outdoors.

1. Operate in fixed flow mode
2. With the desiccant midway between scrub and bypass, operate in a fixed flow mode at 400 μmol s^-1.
2. Match the IRGAs
3. When CO2S_μml and H2OS_mml become stable, match the IRGAs.
3. Note the conditions
4. After matching, note the values that pertain to photosynthesis (CO2R_μml, CO2S_μml, ΔCO2, and Photo) and the values that pertain to conductance (H2OR_mml, H2OS_mml, RH_S_%, and Cond).
4. Find the upper humidity limit
5. Set the desiccant on full bypass, and the flow rate to 100 μmol s^-1. Wait about one minute, then observe the water numbers. The value of H2OS_mml will be about as high as you’ll be able to achieve with this leaf at this stomatal conductance.

Question #1: How can the RH_S_% value (as opposed to H2OS_mml) be further raised and lowered?

Lower or raise the temperature using the coolers.

Water the plant.

Adjust the RH knob.

Correct. Relative humidity is also a function of temperature, so using the coolers to bring down the chamber temperature will increase the RH_S_% value (or warming the chamber will decrease it) even though H2OS_mml remains unchanged. Conversely, raising the chamber temperature will lower the RH_S_%.

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6. Note that we just dropped the flow rate F by a factor of 4 for this step. Since A = (ΔCO₂)/ F and E = (ΔH₂O) / F (the complete equations for photosynthesis A and transpiration E are in the Equation Summary) it might lead you to believe that ΔCO₂ and ΔH₂O should each have also increased by a factor of 4.

Question #2: Did you observe a 4-fold increase in ΔCO₂? How about ΔH₂O? If you didn’t, why not?

There was an increase much larger than 4-fold.

Neither one changed by that much, but they did change.

There was no change.

Correct! Most likely, neither ΔCO₂ nor ΔH₂O changed by a factor of 4. The ΔCO₂ value won’t because we are holding the reference CO₂ constant, so decreasing the flow lowers the ambient CO₂ for the leaf, and the photosynthetic rate drops (unless you are on the flat part of a CO₂ response curve, such as C4 plants at high CO₂). If, however, we had been maintaining a constant sample cell CO₂, we would have seen a 4-fold increase in ΔCO₂, at least until some stomatal changes occurred. The ΔH₂O value, on the other hand, can’t increase by 4 because the transpiration rate has to fall as the humidity increases in the chamber.

That would be unusual. Try again!

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5. Find the lower humidity limit
6. Now set the desiccant on full scrub, and increase the flow to 800 μmol s^-1 (it probably won’t achieve that value). Give it a minute or so to stabilize, and observe the new set of values. This H2OS_mml value represents your lower humidity limit for this leaf.
7. How is stomatal conductance (Cond) behaving: steady, dropping, or increasing?

Question #3: We just lowered the humidity in the chamber. If there are water sorption effects on the chamber walls, will they make stomatal conductance too high or too low?

Too low.

Too high.

No change.

Correct! If the chamber walls were equilibrated at the higher humidity, a drop in humidity will cause water to come off the walls and be added to the air stream. This would make the ΔH₂O too big, and the conductance high.

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Question #4: How might you differentiate real stomatal changes from water sorption effects?

You cannot.

There is no relation.

The effects happen on different time scales.

Correct! Both leaf conductance and water sorption affect the water vapor in the chamber, but they have different time scales. Water sorption effects will be most pronounced in the first minute or two after a change in chamber humidity. Stomata usually take many minutes to change. Therefore, apparent conductance changes in the first minute after a large humidity change are mostly due to water sorption; after that, it’s probably stomatal change.

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6. Return to starting Conditions
7. Return the flow to 400 μmol s^-1, and the desiccant to mid-range between scrub and bypass.

Experiment #2 Maintaining a Constant Humidity

Often it is desired to make measurements or to conduct an experiment at a consistent chamber humidity. This experiment lets you see the automatic humidity control in action.

1. Pick a humidity target
2. Start out in fixed flow mode at 400 μmol s^-1, with the desiccant adjustment knob midway between scrub and bypass. When H2OS_mml is stable, change to constant humidity control (use the H option: constant H₂O mole fraction), and target that current value of H2OS_mml. Your flow rate should settle into the 300 or 400 μmol s^-1 range. (If flow jumps to either extreme, and messages appear about targets being too dry or too wet, make sure you selected the H option and entered a reasonable value, in mmol mol^-1.)
3. Note the CO2S_μml value.
2. Dry the incoming air
3. Once the chamber humidity is on target, and the flow rate is stable, turn the desiccant knob to full scrub. Observe what happens to reference humidity, flow rate, and sample humidity (H2OR_mml, Flow_μml, and H2OS_mml). Reference humidity should go to zero, flow will decrease, and H2OS_mml will remain unchanged. Also, keep an eye on how quickly or slowly CO2S_µml gets to its new value.

Question #5: What does it mean if the reference humidity (H2OR_mml) doesn’t get within 0.5 mmol mol^-1 of zero?

Nothing.

You should wait up to an hour longer.

There is an issue to address.

That is correct! It either means the desiccant isn’t very good, or the IRGA is not properly zeroed.

Try again!

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Question #6: Will the sample cell CO₂ (CO2S_μml) increase or decrease during this step? Why?

Increase.

Decrease.

No change.

That is correct! The incoming air is drier, so the flow rate drops (we’re maintaining a constant chamber humidity), so the leaf has more time to remove CO₂ from the air as it passes through the chamber.

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3. Moisten the incoming air
4. Now turn the desiccant knob to full bypass, and watch the flow increase, reference humidity increase, and the sample cell water mole fraction remain unchanged. The sample cell CO₂ will go the other direction from how it changed in the previous step (not to give the answer to Question #6 away…).
4. Change to constant RH
5. Return the desiccant knob to midway, and after the flow rate stabilizes, note the value of RH_S_%. Now change to constant RH control, targeting that value of RH_S_%.
5. Turn on the coolers
6. Turn on the temperature controllers (f4 level 2), by setting the block temperature for a target about 5°C below its present value (display line h). But, before you do this, try another question:

Question #7: What will cooling the chamber do to flow rate (owing to the constant RH control mode) and why?

Increase the flow rate.

Decrease the flow rate.

No change.

That is correct! Cooling will cause relative humidity to increase, so flow rate will have to increase to compensate.

Try again!

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6. Watch RH and flow
7. As the air temperature drops in the chamber, watch the flow compensate for the changing RH. Notice that the control is not quite as tight as when we were controlling on a constant mole fraction; RH will drift off target a little bit as the temperature changes. (Why? Read about RH control on page 7-10 in the instruction manual.)
7. Change to constant VPD
8. Note the current value of VpdA (display line d), the vapor pressure deficit based on air temperature. Then change to controlling to a constant VPD, based on Tair, and target that value. (See the VPD discussion on page 7-11 in the instruction manual.)
8. Change the temperature control to ambient + 5 °C
9. Before you do, test your understanding with this question:

Question #8: How will flow rate respond as the temperature increases, since we’re holding a constant vapor pressure deficit?

Flow rate increases.

Flow rate drops.

No change.

Correct! Warming the air will not directly change the vapor pressure of the air, but it has a profound effect on the saturation vapor pressure of the air. Therefore, warming will increase the vapor pressure deficit and lower the relative humidity. The flow rate will have to drop for the system to maintain the vapor pressure deficit and relative humidity. But there’s more: there will be increased transpiration (absent stomatal closure) into this drier air, which will dampen the need for decreased flow.

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10. Now try it, and see who is correct. Wait 2 or 3 minutes for things to stabilize.
9. Watch it warm
10. You might notice that warming is more efficient than cooling. You may also notice that the VPD gets away from the target a little bit as the temperature changes.

When the block temperature achieves its target, see that the VPD settles back to its target. Then set the target temperature to ambient, and bring the chamber back to normal.

You can turn the temperature control off if you like.

Controlling CO₂

The next two experiments are best done with a 6400-01 CO₂ mixer. If you don’t have one installed, you can still do approximate CO₂ control between ambient and zero by adjusting the soda lime tube flow adjust knob.

Experiment #3 CO₂ and Humidity Control Interactions

Start with the conditions described by Do this first. Make sure the desiccant knob is mid-range.

1. Set flow control for constant water mole fraction
2. Target the current value of H2OS_mml.
2. Change to constant sample cell CO₂
3. If you have a CO₂ mixer, switch over to controlling a constant sample cell concentration. Target the present value of CO2S_μml. Wait for CO2S_μml to stabilize.
3. Turn the desiccant knob to full scrub
4. Watch CO2R_μml and CO2S_μml. The latter is supposed to be held constant. CO2S_μml will drift well off target, then come back to where it was as CO2R_μml adjusts.

Question #9: Will CO2R_μml increase or decrease?

CO2R_μml will increase.

CO2R_μml will decrease.

No change.

Correct! CO2R_μml will increase. Here’s the sequence: desiccant knob to full scrub, incoming air dries, the humidity controller drops the flow rate, the decreased flow rate causes CO2S_μml to drop (if the leaf is photosynthesizing), so the CO₂ controller has to increase CO2R_μml to bring CO2S_μml back up.

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5. Note: Keep in mind that this test of abruptly changing the incoming humidity while trying to control both chamber humidity and CO₂ is an artificial worst case. Typically, the flow control system balances stomatal changes, which happen less rapidly, so the sample cell CO₂ control option isn’t faced with large swings in flow rate. The next two steps will illustrate a more typical sequence of events.
4. Return the desiccant knob to mid-range
5. Watch the sequence reverse itself. Also note the order in which things happen: Once Flow_μml stabilizes, then CO2S_μml can stabilize.
5. Shade the leaf
6. If you are using a light source, cut the light value in half. If you are not, just shade the leaf with your hand. Before you do, however, here’s another learning opportunity:

Question #10: What do you expect to happen to photosynthesis (Photo), stomatal conductance (Cond), and intercellular CO₂ (Ci) when you cut the light in half? How do you expect the control systems to compensate: specifically, how will flow rate change, and how will reference CO₂ change?

The plant enters torpor and the instrument enters sleep mode.

No changes to the plant or instrument settings.

A cascade of responses by the plant and instrument.

Correct! When the light is reduced by half, photosynthesis will drop immediately, causing CO2S_μml to increase, so CO2R_μml will decrease to keep it on target. Stomatal conductance will initially remain the same, so Ci will increase, but then decrease as the stomata eventually start to close. When this happens, the flow rate will decrease, since we are doing constant water mole fraction control. It can take 10 or more minutes for all this to happen, however.

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6. Watch the response
7. Photosynthesis will immediately start to drop, and (if you wait 10 or 15 minutes), conductance will eventually decrease as well. Some species can react faster than this, however.
7. Restore the light
8. Return the leaf to its original light value and watch the control system respond to the changes.

Experiment #4 A Manual CO₂ Response Curve

CO₂ response curves are described in detail later (CO2 Response Curves), including how to generate them automatically. This experiment will give you a step-by-step guide to manually generating one. If you don’t have a CO₂ mixer, don’t despair1 Don't despair. Just buy one. It's worth it., you can still do the experiment.

As always, we start with the conditions described in Do this first.

1. Set the controls
2. Flow: Constant mole fraction, target the current value (Step 1 in Experiment #2 Maintaining a Constant Humidity).
3. CO₂: If you have a CO₂ mixer, set it to control reference CO₂ to a bit above ambient, such as 400 μmol mol^-1. If you don’t, set the soda lime knob on full bypass.
4. Temperature Control: Constant leaf temperature, and target the current value.
5. Light: Use 1000 μmol m^-2 s^-1. (If you don’t have a light source, do the experiments in a growth chamber, or outdoors. But beware: this experiment is meaningless without steady light).
2. Open Log File
3. Name it “Sample CO₂ curve”, or whatever you like. (f1 level 1)
3. Wait for stability, and log the first point.
4. When the CO₂ and humidity controls are stable and on target, log the starting datum (f1 level 1)
4. Next CO₂ value
5. If you are using the CO₂ mixer, lower the reference target by 100 μmol mol^-1. If you aren’t, turn the soda lime knob a bit toward scrub, so that the reference CO₂ drops to more or less what you want. Use these targets for reference concentration: 400, 300, 200, 100, and 30 (C3) or 0 (C4). The last point is designed to be below the compensation point.

Question #11: Notice when you change the CO₂ concentration (whether you did it with the mixer, or the soda lime tube) the indicated photosynthetic rate (Photo) becomes quite erratic. Why?

Entropy enters the system at random.

The plant has become proudly chaotic.

Sample and reference measurements are out of phase, leading to artifacts.

Correct! The sample cell and reference cell have different volumes, and different flow rates through those volumes. Thus, any change in incoming concentration will wash through the two cells at different rates, creating oscillations in the differential. The wildly fluctuating photosynthetic rate isn’t real - it’s just reflecting this phase difference. After a minute or two, it should stabilize, however.

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Question #12: If you are controlling CO₂ by varying the soda lime scrub knob, under what circumstances might you expect changes in this knob setting to affect the flow rate through the chamber? (Hint: it’s a humidity control question.)

Never.

When desiccant is bypassed.

Always.

Correct! The soda lime will release water vapor and change the humidity of the air stream. If the desiccant is largely bypassed, then these changes get through to the sample cell, and the humidity control system will respond.

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5. Wait for stability, then match and log
6. Wait for a minute or so for the photosynthetic rate to stabilize, match the IRGAs, then log another record (f1 level 1).
6. Repeat until done
7. Repeat Steps 4 and 5 until you are done. Try to get about 4 or 5 points between your starting value and ending points. Go down to 30 μmol mol^-1 or so for C3 plants, and 0 for C4’s. (Hint: If you are using the CO₂ mixer, you get 0 μmol mol^-1 by turning the mixer off.)
8. At any point along the way, you can view a graph of your logged data by doing Step 8.
7. Finish the curve back at the starting point
8. Repeat the starting point. See how long it takes for the photosynthetic rate to return to normal. (Hint: don’t spend too long at lowest CO₂ value.)
9. If you have a CO₂ controller, do some points above ambient, such as 600, 800, and 1000 μmol mol^-1. (If global climate change is keeping you funded, go right on up to 2000.)
8. View the Graph
9. You can view your curve with GraphIt (press View File (f2 level 1 in New Measurements mode). If the axes are not defined for an A-Ci, press QuikPik Config (f1) and select “A_Ci Curve”. Press REPLOT GRAPH (f2) and draw it. Hint: If your low CO₂ point had negative photosynthesis (respiration), you may want to change the default A-Ci plot to automatically scale the axis minimum for photosynthesis. Otherwise, it won’t show that point.
9. Analyze the data
10. Use GraphIt to generate plots to answer these questions: What’s the CO₂ compensation point? Did humidity stay constant over the experiment? How much did the stomata change over the measurement?

Light Experiment

Photosynthesis is first and foremost driven by light, so a natural experiment is to measure this relationship.

Experiment #5 Sun and Shade Dynamics

For this experiment, select a fully sunlit leaf. An LED source is not required for this experiment; we’ll be changing back and forth between sunlit and shaded conditions, so you can simply use your hand to block the sun (or other light source) from the leaf when you need low light. It’s low tech, but effective.

We start once again with the conditions of Do this first.

1. Set the controls
2. Flow: Constant mole fraction, target the current value. (Step 1 on page 4-11). CO₂: If you have a CO₂ mixer, set it to control reference CO₂ to a little above ambient, such as 400 μmol mol^-1. If you don’t, set the soda lime knob on full bypass.
3. Temperature Control: Constant leaf temperature, targeting the current value. Light: If you have an LED source, set it to match full sun, or whatever the ambient light on the leaf is.
2. Clamp onto the leaf
3. Use Real Time Graphics
4. Set up strip charts for viewing photosynthesis, conductance, and Ci. The default configuration has the first two already defined, so you can add Ci to that screen, or put it on another one.
4. Simulate brief shade (fast cloud)
5. Activate the strip charts. When there are reasonably flat lines displayed (indicating stability), try decreasing the light by 80% (from 1500 μmol m^-2 s^-1) down to 300, for example) for 20 or 30 seconds, then returning it to its original state. (If you aren’t using the light source, do this by shading the leaf with your hand. If you are using the light source, do this by escape (to stop viewing the graph), then 2 f5 <low value> enter, wait 15 seconds, then f5 <high value> enter, then view the graph again by 4 f3.)

Question #13: How would you expect Photo, Cond, and Ci to react to this brief drop in light?

No change in any parameters.

Dramatic increases in all parameters.

Decrease, no change, and increase, respectively.

Correct! During this brief shade event, photosynthesis will drop, and conductance will not change, so Ci will increase. Once the light returns to normal, the reverse will happen.

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6. View the strip chart to see what really happened.
5. Simulate longer shade (slow cloud)
6. Now try decreasing the light by 80% for 2 minutes, then returning it to its starting value. Was this long enough to get the stomata to start to respond? (If you are patient, you might find out how long it takes for the stomata to stop responding when the light drops. That is, how long before they stabilize in the new conditions. It might be 10 to 15 minutes, or longer.)

Question #14: Why does stomatal conductance decrease when light is reduced, and what determines the degree of stomatal closure?

The leaf has responded to the chamber environment.

The leaf is fatigued.

The leaf is confused.

Correct! The leaf is not consuming CO₂ as fast in reduced light, so the stomata do not have to be so open to take CO₂ in. Water can thus be conserved. How much will stomata close? One notion is that plants tend to operate at constant Ci. This would mean that the stomata would close until the intercellular CO₂ concentration gets back down to where it “belongs”.

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6. Change to sample cell CO₂ control
7. Change from controlling on reference CO₂, to controlling on the sample cell CO₂. Target the current value of CO2S_μml. Repeat Steps 4 and 5. How does the photosynthesis response differ from the first time you tried it?

Question #15: Suppose you want to do some sun / shade dynamics measurements, and you a) want the sample cell CO₂ concentration to be as consistent as possible, and b) don’t want the slower time response of the sample cell CO₂ control algorithm to interfere with your measurements. How could you do it?

Operate in variable flow mode with a low flow rate.

Wait patiently for things to look right.

Operate in fixed flow mode with as high a flow rate as you can, and set the mixer to control on reference CO₂.

That is correct! Operate in fixed flow mode with as high a flow rate as you can, and set the mixer to control on reference CO₂. High flow rates will do three difference in the sample cell CO₂ concentration as the photosynthetic rate changes; 2) minimize the time necessary to flush out the leaf chamber, which gives you the best dynamic response; 3) make the humidity in the chamber low. This last feature can be overcome by moistening the incoming air stream. See Humidifying Incoming Air on page 4-51, for example.

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7. Change to a shade adapted leaf
8. Change to a leaf that has been in the shade for some time. If you are using an LED source, don’t forget to adjust the light to a low value, before putting the leaf in the chamber. Change to CO₂ control back to a constant reference concentration.
8. Provide a brief sunfleck
9. Give the leaf full sun for 30 or 40 seconds, and observe the response of Photo, Cond, and Ci.
9. Provide a long sunfleck
10. Now give it full sun, and see how long (if ever) it takes for Photo and Cond to reach the values that you found for the sunlit leaf.

Experiment #6 Sun and Shade Leaf Survey

This experiment uses the LI-6400 in a survey mode in which a succession of leaves is measured, and each measurement lasts a minute or less.

Should you use the LED light source for this experiment? If you have this choice, here are some things to consider. This experiment will measure sun and shade leaves that are adapted to their radiative environment. If you don’t use the light source, you won’t be affecting that environment very much when you clamp onto the leaf with the clear chamber top. If you are using the light source, you’ll have to be sure and set the light to match this ambient value before clamping onto each leaf. If you have an external quantum sensor and a light source, you can use the Tracking mode in the New Measurements mode light control screen (f5 level 2), and have the source track ambient (if it is reasonably stable, of course) as measured by the quantum sensor.

Prepare the system: Use fixed flow at about 400 μmol s^-1, and control reference CO₂ to 400 μmol mol^-1. Match the IRGAs after stability is reached.

1. Open a log file
2. If you want, you can record your work. Open a log file, and name it “Survey Experiment”, or whatever you’d like.
2. Measure 5 sunlit leaves
3. Clamp onto another leaf, and wait for the photosynthesis and conductance values to stabilize. One minute is usually sufficient. Then press LOG (f1 level 1), or else press the button on the chamber handle for about 1 second. After logging, move on to the next leaf.
4. If you aren’t using a light source, be careful about shading these sunlit leaves. If a leaf is tipped away from the sun prior to measurement, the chamber walls will cast a shadow on the leaf when you place it in the chamber. Changing the orientation to avoid this shading will cause other problems since you’ve suddenly increased the light. For this experiment, it’s best to choose sunlit leaves that are directly facing the sun.
5. If your leaves aren’t filling the chamber aperture, be sure that the entered value of leaf area (f1 level 3) matches the actual one for each leaf.
3. Measure 5 shaded leaves
4. Now measure 5 leaves that have been well shaded for some time. If you are using a light source, remember to lower its value to match the typical shade leaf’s environment.
4. Plot the results
5. Enter GraphIt (f2 level 1) to view your data file so far. Try plotting it using the “Light Curve” configuration. When you are done, exit GraphIt and close the log file.

Where to Go From Here

This section has introduced you to survey. light response, and CO₂ response measurements that you can do with the LI-6400. The next sections describe these measurements in more detail, providing physiological and operational considerations to help guide you as you determine measurement protocols for your experiments. The remaining sections in this chapter describe some operational hints and considerations with which you should be familiar.