Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu |
Photosynthesis
in Variegated Plant Tissues
Learning Objectives: Upon completion of this
lab you should be able to:
Use the Gilson respirometer to measure gas exchange reactions.
Describe the theoretical basis for the operation of the Gilson and gas exchange reactions
Measure the chlorophyll content of leaves using the Arnon
technique
Introduction: In
this lab we will measure the rate of photosynthesis in white and green sections
of a plant with variegated leaves. Photosynthesis
will be determined by measuring oxygen production with the Gilson respirometer.
The theory and procedures for the operation of this instrument are
provided in a separate technique notes.
Assignment:
At
the conclusion of the experiment, complete data tables 1- 6, the
statistical analyses, and write an abstract summarizing the results of this
experiment.
EXERCISE
1:
MEASURING PHOTOSYNTHESIS IN LEAF DISKS USING A GILSON RESPIROMETER
Background Information:
Gases participate in numerous metabolic processes such
as both respiration and photosynthesis. Thus,
assays that monitor the rate and magnitude of gas production and/or consumption
provide the physiologist with a valuable experimental tool.
Gas exchange reactions can be measured by a variety of techniques
including: (1) manometry such as a
Gilson respirometer; (2) gas-specific electrodes (i.e. oxygen electrode); and
(3) infra-red gas analyzer. The
latter two techniques have the advantage that specific gases can be measured
directly. Although manometric
methods only measure the total volume of gas exchanged, they can be adapted to
measure individual components. For
example, placing an alkali wick in the center well of a reaction flask will
adsorb any carbon dioxide produced in the vessel, trapping it as carbonate.
Thus, the manometric technique can measure just the rate of oxygen
consumption even though carbon dioxide production may also take place.
In this lab we will use the Gilson Respirometer to measure photosynthetic rates of green and white tissues of a variegated plant leaf. We expect that variegated regions will show less photosynthesis than green regions.
Question: Do the green regions of a variegated leaf exhibit more photosynthesis than white regions?
Hypothesis: Greater rates of oxygen production will be observed in green regions of the leaf.
Predictions: Leaf disks prepared from green regions of the leaf will have a higher rate of oxygen production than those from white regions.
Protocol:
Gently rinse and blot dry the leaves. Prepare 15 leaf discs with a hole punch. Float the discs in sodium bicarbonate/carbonate buffer (pH 9) as they are prepared.
Weigh a group of 10 disks. Record the weight and number of disk weighed in Table 2.
Prepare the Gilson respirometer: (a) set the operating temperature at 27 C; and (b) pipet 3.0 mL of sodium bicarbonate/carbonate buffer (pH 9) in the main compartment of each of two Warburg flasks.
Place the pre-weighed leaf disks into your assigned flask. They should float in the buffer solution.
With the operating valve open (UP, horizontal), attach the flasks to the respirometer.
Submerge the flasks in the bath in the dark and allow 10-20 minutes for the temperature to equilibrate.
Set the digital micrometer to 200. Close the operating valves (DOWN, vertical position) and record the rate of oxygen consumption (respiration) for about 10 minutes (Table 2, until a steady rate is observed. OPEN the valve and readjust the micrometer to 400.
Turn on the lights, close the operating valves (DOWN, vertical). Record the photosynthetic oxygen production (micrometer readings) at appropriate intervals in Table 2.
When finished with your measurements, OPEN the valves and remove your flask. Dispose the contents in the proper receptacle.
Record
ambient temp, water bath temp, and barometric pressure. Record these data in Table 1.
Data:
Table 1. Plant & Physical Data | |
Ambient temp (C) | |
Water bath temperature (C) | |
Vapor Pressure | |
Barometric pressure (mm Hg) | |
Plant species used | |
Source of Plant Material |
Table 2: Gilson Data for green and and white regions of a variegated leaf | ||||||||||||||
White | Green | |||||||||||||
1 | 2 | 3 | 4 | 5 | 6 | 7 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
Wt. Tissue (mg) | ||||||||||||||
# disks per treat. | ||||||||||||||
Dark (time / micrometer reading) | ||||||||||||||
Light (time / micrometer reading) | ||||||||||||||
Data Analysis and Conclusions:
Plot micrometer reading (μl) vs. time for each sample in both the light and in the dark. It may be easiest to plot two separate graphs, one for dark-treated samples and the other for light.
Calculate the rate of oxygen consumption (μL min-1) for each dark sample (=respiration) from the slope of the lines.
Calculate
the rate of oxygen production (μL min-1) for each light
sample (=photosynthesis) from the slopes of the lines.
Correct
the rate
of photosynthesis (μL min-1) by adding the
rate of respiration.
Calculate the
mass specific rate of photosynthesis (μL O2
� h-1� g-1
) for each treatment.
Calculate the μmol O2 h-1 � g-1 for each treatment. Use the equation: PV = nRT where P = pressure [atm; = (barometric - vapor pressure of water)/760); V = volume; n = moles; R = gas constant (0.0821 liter atm/mol degree); T = temperature (K)].
Calculate
the μmol O2
h-1
� mg chl-1 (obtain the
chlorophyll data from the experiment below)
Table 3. Data Summary for White Regions of a Variegated Leaf | ||||||
Sample | O2 consump (dark; μL min-1; from slope) | O2 prod. (light; μL min-1; from slope) | Ps Rate (μL O2 prod min-1; corr. for respiration) | Ps Rate (μL O2 prod h-1g fw -1 | Ps Rate (μmol O2 prod h-1g fw -1 | Ps Rate (μmol O2 prod h-1g chl -1 |
1 | ||||||
2 | ||||||
3 | ||||||
4 | ||||||
5 | ||||||
6 | ||||||
7 |
Table 4. Data Summary for Green Regions of a Variegated Leaf | ||||||
Sample | O2 consump (dark; μL min-1; from slope) | O2 prod. (light; μL min-1; from slope) | Ps Rate (μL O2 prod min-1; corr. for respiration) | Ps Rate (μL O2 prod h-1g fw -1 | Ps Rate (μmol O2 prod h-1g fw -1 | Ps Rate (μmol O2 prod h-1g chl -1 |
1 | ||||||
2 | ||||||
3 | ||||||
4 | ||||||
5 | ||||||
6 | ||||||
7 |
EXERCISE 2: MEASURING
CHLOROPHYLL CONTENT IN LEAF DISKS
Background Information:
Chlorophyll can easily be quantified with a spectrophotometer.
Based on the Beer-Lambert Law and the extinction coefficient for
chlorophyll, Arnon (1949) devised the following equations
for quantification of the total chlorophyll, chlorophyll a and
chlorophyll b content in an 80% acetone extract:
Total chlorophyll (�g/ml) = 20.2 (A645) + 8.02 (A663)
Chlorophyll a (�g/ml) = 12.7 (A663) - 2.69 (A645)
Chlorophyll b (�g/ml) = 22.9 (A645) - 4.68 (A663)
If the absorption is greater than 0.8 then the solutions should be diluted with fresh 80% acetone and remeasured.
A quicker, although slightly less accurate method for determining the
total chlorophyll content in a pigment extract is to determine the absorption of
an 80% acetone extract at 652 nm. The
absorption spectra of purified chlorophyll a and chlorophyll b intersect at this
wavelength. By substitution the
extinction coefficient (ε)
for chlorophyll (36 ml cm�1
mg�1) into the Beer-Lambert equation (A=εcl)
and solving for concentration the following equation results:
c (mg/ml) = (A652)/36 x l
where l = path length (cm)
Question: Do green regions of a variegated leaf have more chlorophyll than the white regions? How much chlorophyll a, chlorophyll b and carotene do these regions have? What is the ratio of chl a / chl b?
Hypothesis: Green regions will have considerably more chlorophyll than white regions. The chlorophyll a/b ratio will be about 2 (according to literature reports).
Predictions: The
chlorophyll content of green sections will be significantly greater than white
areas.
Protocol:
Weight 1 (or more) leaf disks. Record the weight and the number of disk in Table 5.
Place the disk in mortar and grind gently thoroughly with a pestle. You should work under the hood in dim light - and you must wear goggles and gloves..
Add a few milliliters of 80% acetone and grind more.
Transfer
the contents to a small graduate cylinder.
Rinse the mortar with a little fresh 80% acetone and transfer the
washings to the same graduate cylinder.
Measure the final volume of the 80% acetone solution and record
(Table 5).
Transfer
the contents to a centrifuge tube, stopper and spin for 3 minutes on the
highest setting.
Complete
the calculations in Table 5.
Data:
Table 5. Chlorophyll Data | ||||||||||||
Treatment | Rep | # disks | fw disks (mg) | acetone vol (ml) | A645 | A663 | total chl (�g/ml) | ttl chl in leaf disks (�g) | ttl chl (�g/g fw) | Chl a (�g / g fw) | Chl b (�g / g fw) | Chl a / Chl b ratio |
White region | 1 | |||||||||||
2 | ||||||||||||
3 | ||||||||||||
4 | ||||||||||||
5 | ||||||||||||
6 | ||||||||||||
7 | ||||||||||||
Green Region | 1 | |||||||||||
2 | ||||||||||||
3 | ||||||||||||
4 | ||||||||||||
5 | ||||||||||||
6 | ||||||||||||
7 |
Data Analysis & Conclusions:
Summarizing your data by completing Table 6.
Perform an unpaired t-test to determine if there is a statistically meaningful difference between photosynthetic rates in green and white regions of a variegated leaf. Record the null hypothesis for this test? What is the t value? What probability is associated with this t value? What does this probability value tell you about the null hypothesis?
Perform
an unpaired t-test to determine if there is a statistically meaningful
difference between the chlorophyll content of the green and and white
regions of a variegated leaf. Record
the null hypothesis for this test? What
is the t value? What
probability is associated with this t value?
What does this probability value tell you about the null hypothesis?
Table 6: Data Summary | ||
Green Regions | White Regions | |
Ps Rate (μmol O2 prod h-1g fw -1 ) | ||
Ps Rate (μmol O2 prod h-1g chl -1 ) | ||
Total Chlorophyll (�g/g fw) | ||
Chlorophyll a (�g/g fw) | ||
Chlorophyll b (�g/g fw) | ||
Chl a / chl b ratio |
| Top | SGS Home | CSB/SJU Home | Biology Dept | Biol 327 Home | Disclaimer | |
Last updated:
01/07/2009 � Copyright by SG
Saupe