Measurement of the Light Dependence of Photosynthesis
Photosynthesis is the most important energy-capturing process
in both aquatic and terrestrial ecosystems. As the name suggests, photosynthesis
is a process by which light is converted to chemical energy and is used
to synthesize compounds within the organism. The green pigment chlorophyll
is the central light-absorbing pigment that makes this energy capture
possible. Chlorophyll, along with various other accessory pigments, is
located in chloroplasts within the cells of the green portions of a plant.
The leaves are the main organs of the plant housing chloroplasts and they
are very well designed to capture the maximum amount of light, as you
will see when you examine their structure in detail. The light energy
captured by the chloroplast pigments is converted into chemical energy
in the form of ATP and NADPH2. These compounds can then be
used to convert CO2 molecules into sugar molecules, which are
used to feed the plant and us. In the overall process of photosynthesis
CO2 molecules are absorbed by the leaf and O2 molecules
are released as a waste product of the process, as seen in the equation
below:
6 CO2 + 12 H2O → C6H12O6 + 6 O2 + 6 H2O
Thus the rate of photosynthesis can be determined by measuring
the rate of either O2 production or CO2 uptake.
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 or wavelengths. 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 convert the light
energy it absorbs to chemical energy, or by the supply of some other
factor required for photosynthesis, such as CO2. In addition,
you will determine the effect of altering the wavelength of light available
to the leaves.
You must read the file "Measurement of Photosynthetic Rate" in the
Photosynthesis Pack Folder or on the Web at www.grinnell.edu/courses/bio/qubitmanual/
before proceeding with the experiment.
Materials
Leaves of a higher plant
Qubit Systems photosynthesis laboratory package with oxygen sensor
Procedure
- 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.
Adjust the time axis on both graphs to 60 min by using the mouse to
highlight the maximum value present and typing in 60, then Press Enter.
- 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 beaker with 200 ml of water on top of the chamber so that
it covers the major part of the leaf area. This serves as a heat filter;
thus the water should be changed every 10-15 min to prevent heating
of the leaf. Position the light above the beaker so that its bottom
edge is 11 cm from the top of the leaf chamber. Turn on the light to
full intensity and record the irradiance reading at the bottom of the
screen.
- 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.
- 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 reflect the maximum output of the lamp.
- Unseal the gas bag and attach its tubing to one of the gas ports on
the upper surface of the leaf chamber. Unplug the other gas port. Press
the bag gently so that your breath is flushed through the chamber.
Observe the decline in the O2 reading on the computer screen
until this reaches a stable value. 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.
- Adjust the illumination of the leaf by sliding the dimmer control
to an intensity equal to 5% of the maximum output and observer 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 had 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 approx. 5 minutes
switch off the light and flush the chamber with gas from your gas bag
for at least 30 seconds. Reseal the chamber and the gas bag. DO NOT
stop the program at any time during this experiment.
- Switch on the light again, and increase its output to approximately
10% 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 photsynthetically induced. Measure the increase
in pO2 of the chamber for 5-10 min. and then repeat step
8.
- Increase the output of the lamp to 20% of initial, and repeat steps
8 and 9 until you have measured photosynthetic rate at a number of light
intensities equal to 5, 10, 20, 40, 60 and 80% of the initial light
output.
- If you are altering the wavelength (color) of light the leaf is receiving,
start with full intensity white light for your initial measurement.
Then alter the wavelength at maximum intensity by placing different
colored filters over the leaf chamber (under the beaker of water). Allow
each reading to proceed until a uniform rate is established, usually
about 5 minutes. Then turn off the light, replenish the water in the
beaker if necessary, and repeat steps 8 and 9 with each colored filter.
- 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.
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 in the box on 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 capturing the
portion to be analyzed inside the box that appears.
- 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 the values for m and b. The value for m
is the slope of the line, which is the rate of O2 production. Record
this in your data table. Close the box on the screen by clicking in the upper
right hand corner.
- Measure photosynthetic rate at the next 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. Again select Linear
Fit from the Analyze menu. The equation screen will reflect the new m
value for the range of data that you have selected. Record the new value for
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/sec. 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 mmole of any gas occupies 22.413 ml, so
at the temperature T of the laboratory, 10,000 x µl
of O2 contains:
µl O2/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 µmoles 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
Photon Flux
(µmol quanta/m2/s)
|
m
%O2/min
|
Photosynthetic Rate
(µmol O2 /m2/s)
|
|
5%
|
|
|
|
10%
|
|
|
|
20%
|
|
|
|
40%
|
|
|
|
60%
|
|
|
|
80%
|
|
|
When you have calculated rates of photosynthesis at each light intensity
used in your experiment, present your data 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. 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.
1) What might these factors be?
2) 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.
|
Color of Filter
|
m
% O2/min
|
Photosynthetic Rate
(µmol O2 /m2/s)
|
|
White
|
|
|
|
Red
|
|
|
|
Blue
|
|
|
|
Green
|
|
|
- How did photosynthetic rate
differ in each light color? Relate your results to the absorption spectra
of photosynthetic pigments.
- 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?
