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:

1. Use the Gilson respirometer to measure gas exchange reactions.

2. Describe the theoretical basis for the operation of the Gilson and gas exchange reactions

3. 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:

1. 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.

2. Weigh a group of 10 disks.  Record the weight and number of disk weighed in Table 2.

3. 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.

4. Place the pre-weighed leaf disks into your assigned flask. They should float in the buffer solution.  

5. With the operating valve open (UP, horizontal), attach the flasks to the respirometer.

6. Submerge the flasks in the bath in the dark and allow 10-20 minutes for the temperature to equilibrate.

7. 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.  

8. Turn on the lights, close the operating valves (DOWN, vertical).  Record the photosynthetic oxygen production (micrometer readings) at appropriate intervals in Table 2.

9. When finished with your measurements, OPEN the valves and remove your flask.  Dispose the contents in the proper receptacle.

10. Record ambient temp, water bath temp, and barometric pressure.  Record these data in Table 1.

Data:

Data Analysis and Conclusions:

1. 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. 

2. Calculate the rate of oxygen consumption (μL min^-1) for each dark sample (=respiration) from the slope of the lines.

3. Calculate the rate of oxygen production (μL min^-1) for each light sample (=photosynthesis) from the slopes of the lines.

4. Correct the rate of photosynthesis (μL min^-1) by adding the rate of respiration.

5. Calculate the mass specific rate of photosynthesis (μL O₂ � h^-1� g^-1 ) for each treatment.

6. 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)].

7. Calculate the μmol O₂  h^-1 � mg chl^-1 (obtain the chlorophyll data from the experiment below)
   

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:

1. Weight 1 (or more) leaf disks.  Record the weight and the number of disk in Table 5. 

2. 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..

3. Add a few milliliters of 80% acetone and grind more.  

4. 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).

5. Transfer the contents to a centrifuge tube, stopper and spin for 3 minutes on the highest setting.

6. Transfer the contents to a cuvette and measure the absorbance at 645 and 663 nm.

7. Complete the calculations in Table 5.

Data:

Data Analysis & Conclusions:

1. Summarizing your data by completing Table 6.

2. 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?

3. 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?

Last updated:  01/07/2009     � Copyright  by SG Saupe