The photosynthetic production of oxygen and our knowledge of leaf anatomy allow to construction of a system that can be used to experimentally investigate many of the photosynthetic variables. Many extracellular spaces exist within plant leaves which are normally filled with air for purposes of gas exchange; consequently, a leaf will float on water. If air is forced out and the intercellular spaces are filled with water, the leaf will sink. If we supply the necessary requirements for photosynthesis, the oxygen produced will form gas bubbles and the leaf would re-float. You will use small disks cut from leaves rather than a whole leaf to perform the floating leaf disk assay (FLDA).
This assay of photosynthesis may be used to answer many questions: What factors affect the rate of photosynthesis? How do changes in light intensity, CO2 concentration, plant adaptations, and chlorophyll content change the rate of photosynthesis?
One problem in measuring a rate of photosynthesis is that there is a competing process occurring at the same time, respiration, a process that uses oxygen. FLDA actually measures the rate of photosynthetic oxygen production minus the rate of respiratory oxygen use during the same time period. So FLDA measures the net rate of photosynthesis, that is, the energetic "profit" made by the plant. Actual photosynthetic activity is of course greater than this and is called the gross rate of photosynthesis. If respiration can be measured separately, a simple calculation can determine gross photosynthesis.
Your Task
You and your partners(s) will design and conduct experiments to determine which conditions (light, air and chlorophyll), or a combination of conditions best produces oxygen from plants. You will use a standard FLDA protocol (described below) to study the rate of oxygen production in plants. You will study the gross rate of photosynthesis to determine conditions which are most favorable to maximizing oxygen production. You may use any of the materials and equipment provided to complete your experiment.
Steps to follow
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1. In your own words, state the problem you are going to investigate, and write your statement of the problem on the page provided.
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2. Design one or more experiments to solve the problem. Describe your experimental designs on the page provided. Show your designs to your teacher before you begin your experiment.
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3. Cut the leaf disks and set up the environmental chamber first. Determine which variable(s) you will change to measure the time it takes to float the leaf disks.
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4. After receiving approval from your teacher, work with your partner(s) to carry out your experiments. While conducting your experiments, take notes on the pages provided. Include tables, charts and graphs. You must keep your own notes because you will not work with your partner when you write your lab report. All lab reports will be completed and evaluated individually.
GENERAL FLDA PROTOCOL
Materials:
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Geranium and Ivy leaves
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Plastic bag
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graduated cylinder
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60 ml of 0.2 % sodium bicarbonate solution.
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Paper towels
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single whole punch
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100 ml graduated beaker
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20 ml of phosphate buffer (pH 6.8)
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20 cc syringe
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Test tubes (4)
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light source
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metric ruler
A. Cutting Leaf Disks:
1. Use english ivy and geranium leaf disks in this experiment. Fresh leaves should be used because wilted leaves respond poorly. Collect leaves just prior to the assay and to prevent dehydration, keep them wrapped in moist paper towels in a plastic bag.
2. Use a common one hole paper punch to obtain leaf disks with a diameter of 6-7 mm. Major veins should be avoided as the presence of a vein may bias the photosynthetic rate of the disk. Place cut leaf disks between layers of wet paper toweling to keep them fresh.
B. Setting Up the Experimental Chambers
1. Take a 100 ml graduated beaker and add 20 ml of phosphate buffer (pH 6.8) and 60 ml of 0.2 % sodium bicarbonate solution. This mixture will reach equilibrium while you prepare the syringes used as assay chambers.
2. Prepare 2 (two) syringes as follows.
a. Remove the plunger from a 20 cc syringe and drop 10 leaf disks down the barrel of the syringe. Tap the syringe barrel so that the disks fall to the bottom (i.e. the tip end of the syringe).
b. An infiltration process can remove the air from the leaf disks and replace it with water. Carefully replace the syringe plunger. Do not crush the leaf disks. Pull 6 cc (1 cc = 1 ml) of buffered sodium bicarbonate solution into the syringe. Invert the syringe, tap a few times and push the plunger to the 4 cc mark to remove all air from the syringe. Air fills the intercellular space of leaf tissue (see Figure 1). In order to replace this air with water so the leaf disks will sink, a vacuum will be applied. Under vacuum, the extracellular air is drawn for the leaf disks and infiltration solution enters this space when the vacuum is released; the leaf disks will sink.
c. Hold the needle barrel of the syringe down firmly upon a rubber stopper. Pull the plunger up to the 10 cc mark and hold in this position. Shake the syringe and then release the plunger. Repeat this procedure several times until all the leaf disks sink.
d. After infiltration, invert the syringe and push out any bubbles that formed, then pull in additional solution to bring the volume to 16 ml. Plants can use the bicarbonate solution in place of the normal atmospheric CO2.
e. Determine the optimal arrangement of a lamp and a test tube rack.
f. One syringe with submerged leaf disks should be placed in the rack adjacent to the center of the lamp. The other identical syringe should be placed in an unlighted rack nearby.
3. To start a FLDA, simply turn on the light and note the time. Every minute thereafter count the number of leaf disks that are floating, then swirl the syringe so that all disks are suspended in a vortex. Record your data on a data sheet as number of leaf disks floating by minutes. The assay is complete once all or nearly all of the leaf disks are floating.
a. What do you predict will happen to the leaf disks in each syringe?
b. Which syringe setup should be called a TREATMENT and which a CONTROL? Why? Record your answers to these questions before continuing.
4. The time required for a leaf disk to float is an index of the net rate of photosynthesis in that leaf disk. However, since some leaf disks will be "early floaters" and others will be "late floaters", this variable can be reduced in significance by plotting the percentage of leaf disks floating as a function of time.
5. You now should have at least one syringe with 10 floating leaf disks. Turn off the light and record the number of disks still floating each minute. The time the disks take to sink in the dark is an index of the rate of respiration (RS). Since some of the leaf disks will be "early sinkers" and others will be "late sinkers", once again this variable will be dealt with by plotting the percentage of leaf disks floating as a function of time, and finding the time required for 50 percent of the leaf disks to sink.
