When infiltrating with buffer alone, carbonate must diffuse into the mesophyll before PS can get going. When the infiltration is done at normal room lighting intensities, very little, if any, PS takes place, so the assay will begin when the syringe is placed in a more intense light.
An effective way to pursue this exercise is to have different groups of students design and perform different experiments with an emphasis on the predictions and the design of the experiment to promote clear thinking.
The suggested variations all work, although I recommend trying these variables using your particular lab materials prior to class. I have not as yet gotten the prism to work, although filters can be substituted if of similar density. A light meter might be useful for actually measuring light intensity.
Using simply a ruler, syringes were placed at different distances from light sources, e.g., 10 cm, 14.1 cm, and 20 cm, to produce intensities based on the unity of light intensity at the first syringe, of 1, 1/2, and 1/4. The inverse square law is not obvious to most undergraduate students.
Varigated geraniums with an achlorophyllous margin worked very well. Not only can you cut "white" disks, but you can cut disks that approximate half&half. Several students thought of that & made the correct prediction about PS rate.
In one 3-hour laboratory period, students should be able do one cookbook assay in the first hour, then work out an experimental protocol for one of the suggested variables, and test their predictions with at least one run. Another approach is to introduce the assay at the end of one period, then have them make predictions, design experiments, and have their ideas worked out, and then use a second lab period to run their experiments one or more times. In a more open investigative approach, students could pursue several variables, designing several experiments based on the FLDA, presenting all of their data and conclusions in a report.
The best argument for using the FLDA is that it is so simple, students understand how it works almost immediately, and the system easily manipulated so they can learn about PS and RS.
Sorenson Phosphate buffer (pH 6.5-7.5)
Solution A: 0.2 M NaH2PO4.H2O (27.6 g/L)
Solution B: 0.2 M Na2HPO4 (28.4 g/L)
Mix X ml of A with Y ml of B, diluted to 200 ml with distilled water.
Add 1.5 ml of 1.1% CaCl2.
Check pH.
pH 6.5 (X=68.5, Y=31.5)
pH 6.6 (X=62.5, Y=37.5)
pH 6.7 (X=56.5, Y=43.5)
pH 6.8 (X=51.0, Y=49.0)
pH 6.9 (X=45.0, Y=55.0)
pH 7.0 (X=39.0, Y=61.0)
pH 7.1 (X=33.0, Y=67.0)
pH 7.2 (X=28.0, Y=72.0)
pH 7.3 (X=23.0, Y=77.0)
pH 7.4 (X=19.0, Y=81.0)
pH 7.5 (X=16.0, Y=84.0)
Photosynthesis is the metabolic process used by many autotrophic organisms to capture light energy and convert it to chemical energy in the form of carbohydrate molecules. The actual energy capturing molecule is chlorophyll, and generally organisms possessing this green pigment are called plants.
Although numerous intermediary reactions are involved, the overall photosynthetic reaction is simple. Carbon dioxide combines with the hydrogen from water yielding a carbohydrate, the 6-carbon sugar (hexose) glucose, and oxygen. Balence the equation below (Hint: How many carbons are needed to make the carbohydrate?).
_____CO2 + _____H2O ----> C6H12O6 + _____O2
The photosynthetic production of oxygen and our knowledge of leaf anatomy allow us to construct 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. In essence this is our experimental method, however, 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, wavelength, CO2 concentration, plant adaptations, respiration, and chlorophyll content change the rate of photosynthesis? FLDA has also been used by the Pennsylvania Department of Agriculture in field surveys for detection of herbicide resistant weeds (5).
One problem in measuring a rate of photosynthesis (PS) is that there is a competing process occurring at the same time, respiration (RS), 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 RS can be measured separately, a simple calculation can determine gross photosynthesis.
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.
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. An instructor will demonstrate the proper arrangement of a lamp, a heat filter, and a test tube rack. Turn off the light.
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. The time required for 50 percent of the leaf disks to float is called the photosynthetic effective time, shortened to PS ET-50, sort of an average rate. The larger the PS ET-50, the slower the rate of PS; the smaller the PS ET-50, the faster the rate of PS. Use graph paper to plot the percent disks floating as a function of time and determine the PS ET-50 for each experimental treatment and control you use. PS ET-50s can be easily compared.
5. You now should have at least one syringe with 10 floating leaf disks.
a. What do you predict will happen if a syringe with floating leaf disks is now placed in the dark?
6. 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 per cent of the leaf disks to sink. This is called the RS ET-50, or the respiratory effective time for 50 percent of the leaf disks to sink. The larger the RS ET-50, the slower the rate of respiration; the smaller the RS ET-50, the faster the rate of respiration. Use graph paper to plot the per cent disks floating as a function of time.
7. The relative rates of photosynthesis (PS) and respiration (RS) can be calculated. Put these formulae in your notes, and explain why these two rates are added together to calculate the gross rate of PS.
NET RATE OF PS = 1 / PS ET-50
RATE OF RS = 1 / RS ET-50
GROSS RATE OF PS = 1 / PS ET-50 + 1 / RS ET-50
For example, you may wonder how the concentration of CO2 affects the rate of PS, a concern of many scientists as human activities continue to increase the CO2 concentration in Earth's atmosphere.
What do you predict would happen to the rate of PS if the plant cells had, say half as much CO2 available? This could be done experimentally by adding 12 ml of bicarbonate solution to one syringe, and adding 6 ml of bicarbonate solution and 6 more ml of buffer solution to another syringe. What would be your control? The actual PS ET-50s could be obtained and compared.
A. Effect of CO2 concentration on PS.
OK, why not? What do you predict would happen to the PS if half as much CO2 were available? One fourth as much? Make your predictions and design your experiment.
B. Effect of light intensity.
What do you predict would happen to PS if two identical setups were placed at greater distances from the light source, say distances X, 2X, and 3X? Decide how to experimentally determine the results. Hint: Use ratios to compare your experimental results.
C. Effect of Wavelength
As you know, visible light is composed a various wavelengths corresonding to the colors of a spectrum. What do you predict would would happen to PS if the light source was directed through a prism in such a manner that different syringes could be placed in different colors of light? Remember ROYGBIV? Could you think of a different experimental setup? What variables might be a problem in such experiments?
D. How does chlorophyll content affect PS?
Chlorophyll is a membrane-bound pigment localized with chloroplasts. Leaf mesophyll cells contain numerous chloroplasts, and generally, the amount of chlorophyll is a function of the number of chloroplasts and chloroplast containing cells present. On average, leaf disks will contain a similar number of mesophyll cells and chloroplasts, and there is no way to extract chlorophyll or enhance the number of chloroplasts without killing or severely damaging the leaf. However, a naturally occurring phenomenon, VARIGATED LEAVES, allows us to seek an answer to the above question. Certain plants produce leaves with patches or areas of cells naturally devoid of chloroplasts.
Predict how does the RS of chlorophyllous and achlorophyllous leaf tissue will compare? Design an experiment to determine the results.
E. Shade versus Sun Adapted Leaves & Plants.
Certain plants have leaves adapted for low light intensities and others have leaves adapted for high light intensities. Some plants grow in shade and others in full sun; some plants, for example sugar maple, may have both sun and shade leaves on the same plant in different portions of the crown. Under higher and lower light intensities, how do you think PS sun and shade leaves, or sun and shade plants will compare? Certain plants have different adaptations for high light intensities, for example maize and sugar cane, both tropical grasses. How do you think such tropical plants' PS will compare with temperate zone plants or shade plants under high light intensities?
Design some experiments using FLDA and whatever materials are provided by your instructor.
F. Impact of herbicides on plant PS.
Herbicides are chemicals that kill plants, used to control agricultural and horticultural weeds. Herbicides fall into two large categories, those that kill "broad-leafed plants" (dicots) and those that kill "grasses" (monocots). So farmers with a wheat or maize field would use broad-leafed herbicides, while soybean fields would be sprayed to control grasses.
How do you think herbicides affect PS? How could the FLDA be used to assay the impact of herbicides on plant PS? For example, does a grass herbicide have any negative impact on the PS of a broad-leafed plant? This would be important to determine before deciding to use the herbicide or not. Design a protocol, an experiment, to determine whether or not a particular hericide has an impact on PS.
Should an herbicide be available, your instructor will give special instructions for using, handling, and disposing of the chemicals (5).
2. Steucek, Guy L., Robert J. Hill and Melvin P. Norbeck. 1985. An Assay for Photosynthesis. Carolina Tips, 48(12):45-47.
3. Tatina, Robert E. 1986. Improvements to the Steucek and Hill Assay of Photosynthesis. The American Biology Teacher, 48(6):364-366.
4. Juliao, Fernando and Henry C. Butcher IV. 1989. Further improvements to the Steucek and Hill assay of photosynthesis. The American Biology Teacher, 51(3):174-176.
5. Hill, Robert J. and Guy L. Steucek. 1985. Photosynthesis II. An Assay for Herbicide Resistance in Weeds. The American Biology Teacher. 47(2):99-102.