Measurement of the Light Dependence of Photosynthesis:
Light response curves, photochemical efficiency and light compensation points of C3 and C4 Plants
The purpose of this experiment is to demonstrate that leaves produce
O2 during photosynthesis and to measure photosynthetic rate
by measuring the rate at which O2 is evolved from a leaf at
various light intensities. In addition the results will show that the
rate of photosynthesis increases with light intensity until a light saturation
point is reached. At that point, photosynthetic rate is limited either
by the ability of the leaf to transduce the light energy it absorbs to
chemical energy, or by the supply of some other factor required for photosynthesis,
such as CO2.
Materials
Leaves of a C3 and C4 species of higher plant
Qubit Systems photosynthesis laboratory package with oxygen sensor
Procedure
- Arrange the components of the photosynthesis package as described
in the introductory material, ensuring that the bottom edge of the light
housing is placed 11 cm from the surface of the leaf chamber. Ensure
that the light is off by sliding the dimmer control to its minimum setting
before proceeding further.
- Turn on the computer and calibrate the O2 and light sensors
as described in the introductory material. The computer screen will
show two graphs, the upper graph displaying percentage O2
plotted against time, and the lower graph showing photon flux (µmol
quanta/m2/s) plotted against time.
- Your experiment should take approximately 60 minutes to complete.
Adjust the time axis on both graphs to a maximum of 60 minutes by using
the mouse to highlight the maximum value present and then typing 60.
Press Enter. Adjust the range of values displayed on the y axis
of the upper O2 graph from a maximum of 21% to a minimum
of 17% O2.
- Ensure that the leaf chamber gaskets have a very thin coating
of vacuum grease. If any grease is visible it will be unnecessary to
apply more. With the light off, seal the leaf inside the leaf chamber
so that no part of the leaf is shaded by the O2 sensor or
the gas inlet and outlet ports. It does not matter if the leaf is too
large to be fully sealed within the chamber, and the "excess" may protrude
out of the chamber without influencing your results. When closing the
chamber turn the thumb-screws finger tight only.
- Place a 200-ml beaker of water on top of the chamber so that it covers
the major part of the leaf area.
- Using a drinking straw, inflate a plastic gas bag with your breath
being careful not to put pressure on the seams by over-inflating the
bag. Seal the bag with the clip provided. Depending on your metabolic
condition, your exhaled breath should contain between 16 and 18% O2,
and 3 to 5% CO2. Under normal conditions in humans, one molecule
of CO2 is produced in respiration for every molecule of O2
consumed, so if your breath contains 18% O2 it should also
contain 2.75% CO2, i.e. atmospheric O2 concentration
(20.7%) minus breath O2 concentration (18%) plus the CO2
concentration in the laboratory (typically 0.05%).
- Click on the Start button on the bottom left hand side of the
computer screen. The button will change to a Stop button and
data will begin to appear on the two graphs on the screen and as numerals
on the bottom of the screen. The initial O2 concentration
should be close to 20.7% O2, and the initial photon flux
should be close to zero. DO NOT stop the program at any time during
the experiment.
- Unseal the gas bag and attach its tubing to one of the gas ports
on the upper surface of the leaf chamber. Press the bag gently
so that your breath is flushed through the chamber. After approximately
30-45 seconds of flushing, remove the bag from the inlet port, seal
the bag and seal both ports of the leaf chamber with the plugs provided.
Observe the decline in the O2 reading on the computer screen
until this reaches a stable value.
- When the O2 reading on the screen has reached a steady
value, switch on the light by sliding the dimmer control to its maximum
setting. Make a note of the irradiance reading at the bottom of the
screen and then turn off the light immediately by sliding the
dimmer control to its minimum setting. This allows you to determine
the maximum output of the light source.
- Observe the changes in the O2 concentration within the
chamber. If the leaf has been maintained in near darkness (e.g. room
light) prior to the experiment, there will be little change in the O2
reading during the first 5-10 minutes of illumination. This corresponds
to the "induction period" of photosynthesis during which photosynthetic
metabolites are synthesized until they reach the critical pool sizes
required for photosynthesis to occur. Once this has been achieved, the
partial pressure of O2 in the chamber will increase as O2
is released in photosynthesis. After the photosynthetic induction period,
the pO2 in the chamber will rise slowly at first and then
will increase linearly.
- After observing the linear part of the rise for 3-5 min. switch off
the light and flush the chamber with gas from your gas bag for 10 seconds.
Reseal the chamber and the gas bag.
- Switch on the light again, and reduce its output to approximately
80% of the initial intensity. This can be achieved by observing the
response of the light sensor display as the dimmer control is adjusted.
- After switching on the light, photosynthesis should begin almost immediately,
since the leaf is already photosynthetically induced. Measure the increase
in pO2 of the chamber for 5-10 min. and then repeat step
11.
- Reduce the output of the lamp to 60% of initial, and repeat steps
12 and 13 until you have measured photosynthetic rate at a number of
light intensities equal to 100, 80, 60, 40, 20, 10%, 5% and 2.5% of
the initial light output.
- After you have made all your measurements, stop the experiment by
clicking on the Stop button.
- Save your data by selecting Save as . . in the File menu. Give
your data an appropriate file name and save it to your data folder.
- Remove the beaker of water, detach the leaf from the plant, and detach
the leaf chamber from its mounting bracket with the leaf enclosed. Be
careful not to touch any hot surface of the lamp or its fitting while
doing this. Place the acetate grid on the surface of the chamber so
that it covers the leaf. Count the number of interstices completely
enclosed by the area of the leaf. Any interstices falling exactly on
the leaf margin should be given a value of 0.5. Sum the results and
divide the total by 4. The value you obtain is equal to the area of
the leaf in cm2.
Repeat the above procedure with the same leaf only this time alter the
light quality rather than the intensity by putting the colored filters
provided on top of the leaf chamber (under the beaker) and turning the
light to maximum intensity.
Data Analysis
The O2 sensor measures only the partial pressure of O2
present in the leaf chamber; it does not measure the rate at which this
O2 is produced. To measure the rate of photosynthesis in your
experiment, you will need to measure the increase in pO2 within
the leaf chamber as a function of time. This is achieved by measuring
the rate of the O2 response which, when the x axis of
your graphs is presented in minutes, will give a rate in %O2
per min. The procedure for analyzing your data is as follows:
- Open the file containing data from one of your experiments. 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.
- Select Examine from the Analyze menu at the top of the screen.
A vertical line will appear on your graphs which can be moved along
the data points on the graphs by moving the mouse. Note that as you
move the vertical line, the numerical display on the bottom of the screen
will change to show you the exact O2 concentration and time
value at the point on the graph where the line is situated.
- Measure photosynthetic rate during the linear part of the increase
in chamber O2 concentration. To do this, move the vertical
line to the point on your O2 data where you wish to start
the measurement, click on the mouse button and hold it down. Move the
mouse over the part of the data you wish to analyze, and then release
the mouse button. The selected part of the data will be highlighted
during this procedure.
- Select Linear Fit from the Analyze menu. In the command box
that is on the screen you will see the equation for a straight line,
y= mx+ b, along with values for m and b. The value
for m is the slope of the line which is the rate of O2
produciton. Record this in your data table. Coulse th box on the screen
by clicking in the upper right hand corner.
- Measure photosynthetic rate at the next lowest light intensity by
moving the vertical line to the linear part of the next set of data.
Select the next area of data to be analyzed by clicking and dragging
with the mouse. The previously highlighted data will disappear. Move
the line across the part of the data to be analyzed, and release the
mouse button. The equation on the screen will reflect the new m
values for the range of data that you have selected. Select Linear
Fit from the Analyze menu . Record the new value of m in
the Results section.
- Repeat this procedure for all of the light intensities and wavelengths
that you tested.
Calculations
Each m value from each regression that you performed represents
the rate of increase of O2 concentration in the chamber with
time. As such, each of these m values are rates of photosynthesis
expressed as %O2 per min. However, photosynthesis is usually
expressed in terms of µl O2/ m2/min. To make this
conversion the following procedure is required:
Let us assume that the m value was x, i.e. the O2
concentration of the chamber increased by x %O2/min.
x %O2 is equivalent to 10,000x parts per million
(ppm) O2 which, in turn, is equivalent to 10,000x µl
of O2 per liter of gas in the chamber.
x %O2/min = 10,000x µl O2/min/liter
of gas in the chamber
At STP 1 µmole of any gas occupies 22.413 µl, so at the temperature
T of the laboratory, 10,000 x µl of O2contains:
µlO2/min/liter/ [(273+T/273) X 22.413 =
µmoles of O2 /min/liter
To obtain the photosynthetic rate we must now multiply by the volume
of the chamber expressed in liters. The chamber is designed so that when
closed it has a fixed internal volume of 0.047 liters.
µmoles of O2/min/liter X 0.047 liter =µmoles
of O2/min
However we would like to express the photosynthetic rate in µm O2/m2/min.
To do this we must perform the following calculations:
µmoles of O2 /min ÷ area of leaf in
m2 = µmoles of O2 /m2/min
In order to convert these units to µmoles of O2 /m2/sec,
the preferred units of photosynthetic rate used in the literature, divide
by 60:
µmoles of O2 /m2/min ÷
60 sec/min = µmoles of O2 /m2/sec
Results and Discussion
|
C3 Plant |
C4 Plant |
|
Photon Flux (µmol quanta/m2/s)
|
m% O2/min
|
Photosynthetic Rate (µmol O2 /m2/s)
|
m% O2/min
|
Photosynthetic Rate (µmol O2 /m2/s)
|
|
100%
|
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80%
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60%
|
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|
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40%
|
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20%
|
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10%
|
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5%
|
|
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2.5%
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When you have calculated rates of photosynthesis at each light intensity
used in your experiment, present your data for both the C3 and C4 plant
as a graph with photosynthetic rate (µmol O2/m2/sec)
plotted on the y axis and light intensity (µmol quanta/m2/s)
on the x axis. This will produce a photosynthetic light response
curve. An example of such a curve for a generalized leaf is shown
in the graph below. Be aware that the light intensities you will be using
are not the same as "incident illumination" give in the following graph,
but they are proportional to it.
From QUBIT SYSTEMS Inc Laboratory Manual
The photosynthetic light response curve of a particular plant is influenced
by many factors, and a study of the components of the curve can tell us a great
deal about the physiology and ecophysiology of the plant. The following are
important aspects of such a curve:
The Light Compensation Point. Extrapolate the linear portion of your
light response curve to intercept the x axis at the point where photosynthetic
rate is zero. The light intensity at this point is called the light compensation
point.
1) What does this point represent in terms of the physiology of the plant?
2) Compare the light compensation points in the C3 and C4 plants and
suggest reasons for any of the differences that you see.
The Rate of Dark Respiration. If the linear part of the light response
curve is extrapolated to intercept the y axis at zero light intensity,
the negative rate of photosynthesis at this point gives an estimate of "dark"
respiration rate.
3) What metabolic processes are included in "dark" respiration?
4) Do the rates of dark respiration differ in C3 and C4 plants? If
so, suggest reasons for this difference.
5) Criticize this method for estimating dark respiration and suggest
a way in which it may be measured directly.
Photochemical Efficiency. Photochemical efficiency may be defined as
the increase in photosynthetic rate achieved per unit increase in light absorbed
by the leaf.
6) What part of the curve would you examine to determine photochemical
efficiency?
7) Compare the photochemical efficiencies of C3 and C4 plants and explain
any differences that you observe. C4 species are usually restricted to
high light environments. Do your data indicate any reason for this?
8) In your experiment, you did not measure light absorbance by the leaf,
but only the amount of light transmitted through the leaf. Describe how
you would change the design of the experiment to make more accurate measurements
of photochemical efficiency.
The Light Saturation Point of Photosynthesis. The light intensity beyond
which the light response curve plateaus is called the light saturation point
of photosynthesis. At this point increases in light intensity do not cause
increases in photosynthetic rate, so other factors apart from the supply of
light must be limiting the photosynthetic process.
9) What might these factors be?
10) Did you measure the light saturation point in your experiment?
If not, why do you think the light saturation point was not reached?
If the light saturation point was reached, do you think that CO2
supply was the major factor limiting photosynthesis at this point? How
would you test this? Remember that your breath contains approximately
100 times the concentration of CO2 in the atmosphere.
11) Your experiment involved a C3 dicotyledonous plant and a C4 monocotyledonous
plant. Do you think this experiment allows you to make generalized conclusions
about the photosynthetic characteristics of C3 and C4 species? How would
you improve the experiment to make comparisons more informative?
12) Remove a leaf from the C3 and C4 plants and hold them up to the light.
What do you notice about the veins of each leaf? How does this anatomy correlate
with the mechanisms of carbon fixation in each species?
Effect of Light Quality
|
Color of Filter
|
m % O2/min
|
Photosynthetic Rate
(mmol O2 /m2/s)
|
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White
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Red
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Blue
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Green
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1) How did photosynthetic rate differ in each light color? Relate
your results to the absorption spectra of photosynthetic pigments.
2) What characteristics regarding the amount of light transmitted by the
filters would be important in order to make valid comparisons of the effect
of various wavelengths of light?
