Measurement of Carbon Fixation Rates in Leaf Samples

Generation of a Light Curve

To address the hypothesis concerning photosynthetic efficiency it is necessary to expose sun and shade leaves to a range of light intensities long enough for them to fix significant amounts of carbon. It is necessary to expose identical surface areas under favorable conditions which are identical for all leaves except for light intensity (the experimental variable). A means of measuring the rate of carbon fixation is also necessary, of course.

Read over the recommended procedure for carbon-14 labeling of leaf disks, in which leaves are exposed to different light intensities in an atmosphere rich in the radioisotope carbon-14. Under favorable conditions, all newly-fixed carbon will include a proportion of labeled (radioactive) carbon. By measuring the amount of radioactivity in each disk, one can determine the amount of carbon fixed by each leaf.

After labeling the leaf disks, they must be extracted in order to make the new carbon compounds soluble. That way they can be assayed by liquid scintillation counting. Read over the extraction procedure, and peruse the articles on detection of radioactivity.

Carbon-14 Labeling of Leaf Disks

Objectives

This experiment will involve potentially hazardous substances and procedures. It is essential to be familiar with principles of laboratory safety before attempting this sort of procedure. Before working with radioisotopes, make sure you understand the basics of radioisotopes and their detection. Specific hazards will be pointed out at various points in the procedural outline.

In order to generate a light curve, you will expose leaf disks to various levels of light in the presence of radioactively-labeled carbon dioxide (carbon-14). Disks will be illuminated for 30 min., with some disks kept in the dark as controls. The rate at which a disk accumulates reduced carbon-14 in its tissues is proportional to its rate of photosynthesis, provided the rate of metabolism is relatively low. Therefore, total radioactivity will be counted for each disk. You know the time period of light exposure, the amount of radioactivity added to each incubation chamber, and you can calculate the total amount of carbon dioxide in the atmosphere in each chamber. Therefore you can determine the rate of carbon fixation in molecules of carbon fixed per unit time.

In the classroom you should work in pairs, however you will need data from the entire class to obtain significant results (replicate samples). Therefore you will have to organize and coordinate your work with other groups. You and your lab partner will be given a group number and a set of light levels. One or two other pairs will have the same group number. You and the other members of your group will share the same chambers for incubation of samples, and should start your incubations at the same time.

Chamber preparation

A 12 well cell culture dish (12 well cluster) makes an appropriate light exposure chamber. The lid is clear, and the interior is designed to maximum gas exchange among the wells. The inside edge of the lid can be lined with wax to make a gas tight seal, and prevent loss of label. Petri dishes (100 mm diameter) may be used as well.

Using cell culture dishes, plan to place no more than one leaf disk in any well, and reserve a central well for the radioactive solution. The lid will have a hole in it over the well that will receive label. Label as many pieces of Whatman #1 filter paper as you will have specimens of leaves. Wet the pieces with distilled water and place them in appropriate positions in the chamber(s) as instructed. The paper should be completely damp, but there should be no excess water in the dish. The purpose of the water is to provide a humid atmosphere to prevent dessication of the leaf disks, which can no longer replace water lost to transpiration. Prepare the chamber lid by lining the inside edge with wax, but don't seal it yet. Don't smear up the clear lid - that would interfere with the entry of light into the chamber.

Use a paper punch and forceps to remove leaf disks from a single sun leaf, avoiding large veins (why?). Place one or more disks in the chamber(s) on the moist paper in appropriate positions. Your instructor will advise you as to how many duplicate disks to use per chamber. Repeat for a shade leaf. The disks should be placed with the cuticle (top surface) up. Try to keep the disk from flipping over prior to and during exposure to light. If a disk should flip over after sealing the lid, make a note of it. That may or may not make a difference. Seal the chamber(s).

Exposure to light and carbon-14

Place your chamber(s) underneath the light source (turned off for now). Clear the immediate area of any books, pencils, or anything other than your chamber materials. Always use bench paper and keep unnecessary objects away from any radioisotope experiment in case of a spill. Once your dishes are prepared and in position you are ready for the radioacitve material. EXCEPTION: If natural sunlight is used, the chambers must be charged (label added) in a dim area, and then transported in the dark to the outside. Timing of exposure would then begin the moment they were exposed to light.

One person should be designated to handle the dishes for your group, and must put on gloves and a lab coat. Steps that involve handling of radioactive chambers are to be carried out by that person only (under your supervision, of course). Obtain a syringe with 1 N citric acid. An instructor will add carbon-14 labeled sodium bicarbonate to each chamber. Record the amount added and the specific activity, as you may need to quantify the amount of labeled carbon later. The solution is at a high pH which reduces, but does not completely inhibit, exchange of bicarbonate anion with atmospheric carbon dioxide and water. Some radioactive carbon will be released into the atmosphere even before adding acid, so work efficiently.

In an aqueous solution, NaHCO3 reaches equilibrium with atmospheric CO2. (1) Under normal conditions NaHCO3 dissociates to [Na+] + [HCO3-], and H2O dissociates to [H+] + [OH-]. (2) [Na+] + [OH-] is, simply, ionized NaOH. (3) [H+] + [HCO3-] combine to form H2O + dissolved CO2. (4) Dissolved CO2 is exchanged with atmospheric CO2 by diffusion. All of the dissociations are reversible, and are influenced by pH.

Use the syringe to add about 5 drops of citric acid to the radioactive liquid, and immediately note the time. Immediately after the addition of acid, gently cover the opening with a bead of wax, being careful not to crack the lid. The acid favors the dissociation of the bicarbonate anion into water and carbon dioxide, thus shifting equation (3) to the right. This way, the radioactive carbon becomes a part of the atmospheric pool of CO2.

Expose the chamber(s) to light, and begin timing immediately. In the laboratory you can simply turn on a switch and expose them all simultaneously. Light intensity should be recorded using an appropriate meter as microeinsteins per m2 per sec. The einstein is appropriate because it is defined as the energy of one mole of photons of light of 700 nanometers (nm) wavelength.

Terminating the exposure

After a specific illumination period interrupt the light exposure, putting all chambers under a dark cloth for 10 min. Incubation in the dark permits the mechanisms involved in the "dark" reactions to complete the process of light-dependent carbon fixation, while still in a labeled CO2 atmosphore.

Put on gloves and lab coat and add 10 drops of 5 N KOH to the radioactive liquid. NOTE: read over the safety tip in the leaf extraction protocol about strong bases. The strong base dramatically reduces the rate of exchange of dissolved CO2 with bicarbonate anion.
Working over bench paper, open each chamber and use forceps to put each disk into a scintillation vial, previously labeled with a very small numeral and initial near the top of the vial. Radioactivity in the vials will be estimated by scintillation counting, which requires the recording of flashes of light. Large marks on the vials would interfere with the detection of light. Each individual should now work with one or more vials, each containing a 'hot' leaf disk.

Clean up the chambers.

Extraction of C-14 Labeled Leaf Tissue

Plant tissues should be placed in a clear screw-capped tube, such as a scintillation counting vial. All extraction procedures should be carried out within the vial, both to prevent contamination of benches and equipment as well as to retain all of the carbon-14 activity. Both the cap and the vial should be clearly labeled. Vials must be marked near the shoulder so as not to interfere with scintillation counting later.

For comparison of samples, all samples must be of the same surface area, since surface area defines the amount of incident light during the exposure. Usually, disks must be punched out of larger size leaf fragments, so that only healthy tissue is assessed for carbon fixation rate.

Tissue preparation

Wearing gloves and working over a piece of bench paper, the leaf tissue should be minced into fine pieces with scissors. No piece should be larger than 0.5 mm in any dimension. Mincing is best accomplished by keeping the scissor tips inside the vial and mincing the material in place. The scissor tips must be cleaned off with forceps and all pieces recovered, and the tips wiped off with a lab wiper between samples. All of the pieces must remain in the bottom of the vial for the count to be representative of the sample. With caps on but loose, the vials should be placed in a plastic beaker to prevent their falling over, and dried at 70 to 100 degrees C or under a heat lamp for 15 min.

The reduced carbon, incorporated into sugar molecules, must be extracted from the leaf tissue tobe dissolved in counting fluid for the measurement of radioactivity. Drying the tissue facilitates the extraction.

Extraction with nitric acid

Open vials must be handled with extreme care, because when the pieces dry they accumulate static charge, and may 'jump' out of the vial when disturbed. After allowing the vials to cool, 30 microliters nitric acid should be added to each vial, and the vials capped securely. Nitric acid is nasty, and is very efficient at destroying clothing, so it should be pipetted in the fume hood with the window at the lowest comfortable working position, so there is always glass between the investigator and the acid.

After placing the vials in a 70 degree (C) water bath, they should be removed at one minute intervals and votexed at high speed to expedite the extraction. Total extraction time is arbitrary, but five minutes is usually sufficient.

Safety tip - hazards of strong bases

You are probably well aware that strong acids are dangerous. The fact that they cause extreme skin irritation and sting if they get in your eyes is incentive enough to wear protective clothing and glasses. However strong bases, such as KOH or drain-cleaning solutions which are NaOH-based, are much more dangerous. They anesthetize nerve endings so that one does not feel the damage, even when the stuff gets in one's eyes. If you even think you might have come into contact with such solutions, rinse the affected area thoroughly. In the case of eye contamination, rinse with cold running water for a full 20 minutes.

Preparation for liquid scintillation counting

After allowing the vials to cool., 5 ml of scintillation fluid should be added (for 7 ml vials), using a repipet. After tightly capping the vials they should be vortexed thoroughly once more taken to the scintillation counter or vial rack, logged in and loaded up. Most counters continue to count samples while loading. Each vial may require as much as 10 minutes for an accurate count, so the raw data will be available the following day or later.

Safety tip - hazards of scintillation fluid

Scintillation fluid is volatile and may be carcinogenic. Always use a fume hood when working with scintillation fluid or any organic solvent, and work behind the glass shield. The scintillation "cocktail", as it is called, is usually delivered by means of a repipet. The repipet should be calibrated to deliver 5-6 ml for a 7 ml vial, or about 15 ml for a 20 ml vial.

Biodegradable scintillation cocktail is available. If the type and amount of isotope allows sink disposal under state regulations, the material can be flushed down the sink with plenty of tap water, with no harm to the environment. Since carbon-14 is a naturally-occurring radioisotope and is not highly hazardous, this method of disposable should be acceptable.