Laboratories for Measuring Physiological
Processes in Plants and Animals

Plant Phisiology
Measurement of nitrogenase activity, electron
allocation efficiency and effects of O₂

Plants require a number of different mineral elements for healthy growth, elements which may already be available in the soil or may need to be added in the form of fertilizers. Some of these elements are needed in relatively large amounts (the macro-nutrients) and others in very small amounts (the micro-nutrients). One of the most important macro-nutrients, one that is frequently in short supply, is nitrogen. This may seem strange when the atmosphere contains 80% nitrogen. Unfortunately this nitrogen is in a gaseous form (N₂) that plants are unable to use. Plants must obtain nitrogen in the form of nitrate (NO₃^-) or ammonia (NH₃) and these forms are much less abundant.

There are organisms that can "fix" or convert gaseous nitrogen into ammonia and they are referred to as 'nitrogen fixers'. They are able to make large amounts of the nitrogen in the air available to plants through this activity. All of these nitrogen-fixing organisms are prokaryotes; some are free-living in soil or water and others live in a symbiotic relationship with higher organisms. The most well-studied symbiotic association exists between legumes (beans, peas) and the bacterium Rhizobium. The Rhizobium cells live in nodules on the roots of the plant. It is within the bacteria that live inside these nodules that the following reaction occurs:

N₂ + 8H⁺ + 8e^- + 16ATP   →  2NH₃ + H₂ + 16 ADP + 16 P_i

This reaction is catalyzed by the enzyme, nitrogenase. As you can see from this equation, the reduction of N₂ to ammonia is a very expensive process energetically. It requires a large number of electrons and ATP molecules to reduce a single molecule of N₂. The process is made more expensive by an unavoidable side reaction which simultaneously reduces protons (H⁺) to molecular H₂. While the latter reaction is a wasteful one for the plant, it does provide us with a way to indirectly measure the activity of the nitrogenase enzyme using a hydrogen sensor. By pumping air through the nodulated root system of a legume in a sealed pot, we can measure the amount of H₂ evolved by the activity of nitrogenase. This is not a direct measurement of the rate of N₂ fixation but it gives us the Apparent Nitrogenase Activity (ANA) expressed as µmol H₂/hr. This is because a minimum of 2 of the 8 electrons used by nitrogenase actually go to H₂ production; the remaining 6 electrons are used to reduce N₂ to NH₃. Thus by measuring H₂ production we can estimate the proportion of enzyme activity that is being used to reduce protons. While this proportion should represent at least 25% of the total nitrogenase activity (according to the equation above), the allocation of electrons between N₂ and H⁺ reduction can vary. To measure the Total Nitrogenase Activity (TNA), expressed as µmol H₂/hr, we must replace the N₂ in the air passing through our system with an inert gas such as argon (Ar). In the presence of Ar (and the absence of N₂) all of the electrons previously used to reduce N₂ will now be used to produce H₂ and the rate of H₂ production should increase. The maximum rate of H₂ production after the switch to argon will represent the TNA. The rate of nitrogen fixation that was occurring in air can be calculated as follows:

N₂ fixation rate = (TNA - ANA)/3

The portion of electrons going to N₂ reduction (rather than H₂ production) is determined by subtracting the ANA from the TNA. This number is then divided by 3 because N₂ reduction requires 3 pairs of electrons compared to H⁺ reduction which requires only one pair of electrons. The N₂ fixation rate would be expressed in µmoles N₂ reduced per hour. The other important parameter of nitrogen fixation is the Electron Allocation Coefficient (EAC) which describes the allocation of electrons between proton (H⁺) reduction and N₂ reduction.

EAC = 1 - (ANA/TNA)

Remember that at least 25% of total electron flow through the nitrogenase enzyme is used to reduce H⁺ to H₂ but this value is not constant. Often more than 25% of the electrons are used to reduce H⁺, which is energetically very wasteful for the plant. Thus it is important to determine the EAC as an indicator of the efficiency of nitrogenase activity. Higher values of the EAC indicate that a higher proportion of nitrogenase activity is being used to provide ammonia to the plant.

The purpose of this experiment is to demonstrate that nitrogenase activity and the rate of N₂ fixation in legumes can be determined by measuring the rate of H₂ evolution from nodulated roots. It will also investigate how N₂-fixing nodules regulate their activity with changing physiological and environmental conditions. The nitrogenase enzyme is extremely O₂ labile and a very low O₂ concentration is maintained in the infected cells of the nodule to prevent nitrogenase inhibition. This experiment will show that the nodule is capable of regulating its internal O₂ concentration to maintain active nitrogenase.

In this experiment you will investigate the paradox faced by nodules with respect to O₂. Oxygen is required for aerobic respiration in support of nitrogenase activity, but it is also a potent inhibitor of the nitrogenase enzyme. Therefore, there must be stringent control of the O₂ status of the infected cells so that a sufficient flux of O₂ is maintained to support nitrogenase-linked respiration, while a low O₂ concentration is maintained to avoid nitrogenase inhibition. This control of O₂ concentration is achieved, in part, by the presence of a "diffusion barrier" in nodules that is able to vary its resistance to O₂ entry depending on changes in both O₂ supply and demand. The barrier restricts entry of O₂ into the nodule, and the high rate of infected cell respiration (O₂ consumption), helps to maintain a low O₂ concentration in the nodule central zone. In this experiment, you will observe how nodules under different conditions respond to changes in external O₂ concentration.

You must read the section "Measurement of Nitrogen Fixation using H₂ Sensor" on the web. before proceeding with the experiment.

Materials

A nodulated soybean in a growth pot cuvette
Qubit Systems Nitrogen Fixation Package

Procedure - Part 1: Electron allocation efficiency

1. Attach the outlet of the desiccator column directly to the H₂ sensor. Turn on the computer and select the N₂ Fixation package and the "N₂ FIXO₂" icon.
2. A command box will appear asking "Do you want to load the calibration saved with this experiment?" Click on the Yes button.
3. Two graphs will appear on the screen. The upper graph will display data from the H₂ sensor, and the lower graph will display data collected from the O₂ sensor. At the bottom of the screen the outputs of the two sensors will be displayed digitally.
4. The output of the O₂ sensor should read 20.7%. If it does not, use a small screw driver to adjust the gain control on the O₂ amplifier box until the numerical display reads 20.7%. The O₂ sensor is now calibrated. It is not necessary to calibrate the H₂ sensor.
5. The default setting for the H₂ axis is 0-3.0 volts. Set the time axis for 60 minutes. DO NOT stop the computer until you have completed your measurements.
6. Attach a gas bag containing Ar:O₂ (80:20) to the inlet of the pump and the black flow restrictor to the outlet and flush this gas through the system. Click on the Start button to begin data collection.
7. A trace will appear on the graph that represents the ZERO value for your experiment in Ar:O₂. Note that this value is not 0.0, but a positive value that must be subtracted from your measured data during data manipulation. Allow the zero reading to proceed for approximately 5 minutes, or until the trace is stable.
8. Detach the bag of Ar:O₂ from the pump and flush room air through the system. Allow the H₂ trace to reach a new steady state value. This is your ZERO value for measurements made in air, and it may be different from that for measurements made in Ar:O₂.
9. Seal the plant in its growth pot and attach the outlet of the flow restrictor to the gas inlet port at the base of the pot. Attach the end of the tubing from the gas outlet port to the inlet of the desiccant column.
10. When the pot is attached to the system, observe the increase in the voltage output of the H₂ sensor. This is shown numerically at the far right of the bottom of the screen and is labeled "Cor. H₂", for corrected hydrogen. Allow this to reach steady-state.
11. Reduce the flow rate through the system by replacing the black restrictor with the red flow restrictor. Observe the change in output of the H₂ sensor and when this reaches a steady state record the Cor. H₂.
12. Increase the flow rate through the system again by replacing the red flow restrictor with the black flow restrictor and wait for the Cor. H₂ to reach a steady state.
13. Attach the bag of Ar:O₂ to the inlet of the pump and observe the increase in the Cor. H₂ value. When this reaches a steady-state, record the value.
14. Remove the Ar:O₂ and let room air flow through the plant roots for about 2 minutes. In the meantime stop data collection and save your data by selecting "Save as . ." from the File menu. Save your data in your designated folder.

Procedure — Part 2: Effects of oxygen

1. Repeat the above experiment with the same plant. Without the plant attached, pump room air through the system using the black flow restrictor. The trace on the graph represents the ZERO value for your experiment in room air. Note that this value is not 0.0, but a positive value that must be subtracted from your measured data during data manipulation. Allow the zero reading to proceed for approximately 5 minutes or until the trace is stable.
2. Attach a gas bag containing N₂:O₂ (70:30 or 30% O₂) to the inlet of the pump and measure zero again. Note that the zero reading will decline. Wait until this reading is stable.
3. Detach the 30% O₂ bag from the pump and allow room air to flush through the system again.
4. Attach the plant in its growth pot, which you have previously sealed, to the system to observe the change in the Cor. H₂ value until a steady state is reached.
5. Attach the gas bag containing 30% O₂ to the inlet of the pump. You should see the Cor. H₂ value decline swiftly, and then recover slowly over the following 30 min. Note the minimum value attained, and the steady state value attained while the root system is exposed to 30% O₂. If the gas supply runs low during your experiment, unplug the pump, attach the reserve gas bag to the inlet, and restart the pump. Fill the empty gas bag as your experiment proceeds.
6. When the output from the H₂ sensor has reached a steady value at 30% O₂, detach the gas bag from the system and flush with room air. Stop data collection.

Data Analysis

Determine the appropriate values from your experiment and record them in the Results and Discussion section by following the procedure described below:

1. Open your experimental file. A command box will appear asking you whether or not you wish to load the calibration stored with your data file. Answer "Yes". Your data will appear on the screen exactly as it appeared when you saved it at the end of the experiment.
   
   
2. Select Examine from the Analyze menu. A vertical line will appear on your graph which can be moved along the data by moving the mouse. Note that as you move the vertical line, the digital display on the bottom of the screen will change to show you the Cor. H₂ value from the H₂ sensor and the time value at the point on the graph where the line is situated.
   
   
3. The Cor. H₂ measurements are shown in volts and should be converted to rates of H₂ production using the following equation:

H₂ production (µmol/h) = voltage x K x flow rate

K is a factor for converting voltage readings to measurements of H₂ concentration in units of µmol/liter. Flow rate has units of liters per hour. Since the H₂ sensor has a different response in N₂ and Ar, the value of K differs in each gas.

K = 0.75 µmol/liter per volt in air

K =1.0 µmol/liter per volt in Ar: O₂

K = 0.95 µmol/liter per volt in N₂:O₂ (30% O₂)

You must use the correct value of K in your calculations in air, Ar:O₂ and 30% O₂. Also, remember that you must subtract the appropriate zero reading from your H₂ voltage measurements before using the equation above.

From Qubit Systems Inc

Results and Discussion

Part 1

	Low Range Flow Rate	=	________ L/hour
	High Range Flow Rate	=	________ L/hour
	Cor. H2 Zero Value in Air	=	________ Volts
	Cor. H2 Zero Value in Ar:O2	=	________ Volts

 

Flow Rate (L/hour)	Cor. H2 in Air	Cor H2 in Ar: O2	Cor H2 - Zero	K	H2 Evol’n (µmol/h)
		------

 

 

1. Calculate values of ANA and TNA from your experiment, and use these to calculate the EAC of your plant. Why does EAC have a maximum value of 0.75? Show your calculations.
   
   
2. What happened to the H₂ content of the gas flowing from the pot when flow rate was reduced? Can you suggest a reason for this response? How did this affect ANA?
   
   
3. Given that 2 electrons are required to reduce 2 protons to H₂, and 6 electrons are required to reduce one N₂ molecule to 2 NH₃, use your data to calculate the rate of N₂ fixation in your plant in terms of µmol N₂ fixed/h. Show your calculations.
   
   
4. Legumes have a maximum EAC value of 0.75. What would be the effect on plant yield if this value declined to a very low level.

 

 

Part 2

	Low Range Flow Rate	=	________ L/hour
	High Range Flow Rate	=	________ L/hour
	Cor. H2 Zero Value in Air	=	________ Volts
	Cor. H2 Zero Value in 30% O2	=	________ Volts

 

	Values of Cor. H2
	Stable at 20% O2	Minimum at 30%O2	Maximum at 30% O2
Plant # 1
Plant # 2

Using these values of Cor. H₂, calculate rates of H₂ production and record these in the table below. Make sure to subtract appropriate zeros from the Cor. H₂ values, to use the appropriate value of K, and to use the correct flow rate for each of your calculations.

 

	H2 Production Rate (µmoles H2/hour)
	Stable at 20% O2	Minimum at 30%O2	Maximum at 30% O2
Plant # 1
Plant # 2

 

1. When O₂ concentration is raised from 20% to 30%, the increased gradient of O₂ from the rhizosphere to the nodule interior causes an increase in internal O₂ concentration which, if maintained for a long period, would cause irreversible inhibition of nitrogenase activity. Did you observe such an inhibition when O₂ concentration was raised in the roots of the control plant? Comment on your results.
   
   
   
2. Under natural conditions, the O₂ concentration of the soil never increases to 30%. Therefore, the treatment used in this experiment to induce an increase in nodule O₂ concentration is non-physiological. Under what circumstances would you expect nodule O₂ concentration to increase under natural conditions?
   
   
   
3. Measurement of the rate at which H₂ is produced by the nodules provides a means for measuring nitrogenase activity. However, this provides only an estimate of Apparent Nitrogenase Activity (ANA) since under most circumstances the major part of the enzyme's activity is involved in reducing N₂ gas to ammonia. At least 25% of the activity of nitrogenase is involved in H₂ production, but this value may increase with environmental or physiological conditions. Given this information, do you think that your experiment provides a reliable indication of the effects of O₂ concentration on nitrogenase activity? How would you improve the experiment to validate your results?