Photorespiration: A classroom demonstration1 D. D. Wolf and E. W. Carson2

ABSTRACT This paper presents several modifications of a simple experiment to demonstrate differences be- An easily implemented test to show biochemical tween photorespiring (high CO compensation) differences between plant species in photorespiration 2 has proved to be an effective addition to the formal and non-photorespiring (low CO2 compensation) classroom lecture. The principle of the test is that plants. The method is less costly and less time con-

leaves in a closed system cause the CO2 concentration suming than that described by Moss (3). The meth- (^CO?) to reach characteristic compensation values. od has also been used in a survey to categorize The test uses a sodium bicarbonate indicator solution species and is modified from that described by which is blue when in equilibrium with low CO2 atm (created by non-photorespiring species) and green in Tregunna et al. (4). high CO2 atm (created by photorespiring species). METHODS AND MATERIALS Additional index words: Visual aid. Carbon dioxide. Compensation value. Teaching aid. Two 50-ml beakers (other containers can be used) are placed side-by-side on the bottom of a 1 liter (1 qt) wide-mouth glass jar. About 30 ml of "D LANTS within both the Monocotyledoneae water are placed in one beaker. The leaf material and Dicotyledoneae classes have distinct ana- (preferably with open stomata and a leaf area of at tomical and -biochemical differences, and are sub- least 10 cm2) is detached, recut under water, and classified into photorespiring and non-photorespir- placed in the beaker containing water. About 5 ml ing groups. Characteristic of the non-photorespiring of a sodium bicarbonate indicator solution (5 X s group are Kranz type, tropical, and C4-dicarboxyIic 10~ M) containing 1% (v/v) Universal Indicator acid plants; plants having low CO2 compensation; (Fisher Scientific Co.) is placed in the other beaker. and L syndrome. The identifying term of "non- The indicator solution will be greenish-yellow and photorespiring," however, seems to have gained can be stored. A closed system is created by secur- wide popular acceptance (2, 4, 5). Characteristic of ing the lid on the jar. A cover made of two layers the photorespiring group are C3 and Calvin cycle of polyethylene plastic, such as a plastic bag held in plants; temperate species; and plants having high place by a rubber band, also seals the jar. The plant CO2 compensation. Including the concept of material is continuously illuminated by a fluorescent photorespiration in introductory plant science desk lamp having two 40-W bulbs. A change in courses stimulates student interest in the bio- chemistry of plant -growth, and can help them to Contribution of the Agronomy Department, Virginia Polytechnic Institute and State University, Blacksburg, VA relate crop yields to efficient photosynthetic and 24061. respiratory mechanisms. 2 Associate Professor and Professor. 114 JOURNAL OF AGRONOMIC EDUCATION, VOL. 4, AUGUST 1975 color of the solution indicates photosynthetically 5. The Universal Indicator is a complex of pH- active tissue andPcO2 reduction. A green solution specific color indicators that show the pH of the indicates a PCO2 °f greater than 50 ppm (photo- equilibrium buffer solution and, thus, indicate the respiring plant), whereas a blue color indicates a concentration of CO2 in the atmosphere of the con- GSS tainer. low PCO2 °f I than 5 ppm (non-photorespiring plant). The system can be checked by placing a Plant material selected should be grown under sodium hydroxide solution in the beaker instead of soil, water, and environmental conditions that ) water. Since sodium hydroxide creates a low/ CO2> would encourage open stomata. With open stomata the indicator will be blue at equilibrium. Once this and in a closed system, the atmospheric CO2 con- experiment is assembled, the demonstration can be centration will be reduced to compensation levels set up in a few minutes. by photosynthesis within about 15 min if sufficient leaf area is present. Gentle swirling of the jar will RESULTS AND DISCUSSION help mix the indicator solution and speed the equilibrium. Time needed for color change will de-

Visual demonstrations of biochemical differences pend on rate of CO2 exchange with the solution between species help to stimulate interests in plant and usually will occur in less than 4 hours. The characteristics, e.g., the efficiency of yield capabili- leaves maintain equilibrium for several days. Al- ties. Any plant material that is photosynthetically ternatively, the lights can be turned off to demon- active can be used, but the most effective way to strate respiration. With lights turned on again, the standardize the system is to compare the color pro- original equilibrium returns. duced by two plants whose CO2 compensation The effect of oxygen on photosynthesis and CO2 points are known, for example, corn (low) and bean compensation value (1) can also be demonstrated in (high). the following way. Set up two of the jars contain- This procedure builds upon and applies a number ing similar leaves of a photorespiring species. Flush of basic concepts—diffusion, chemical equilibria, one jar well with nitrogen before closing the lid. pH, organic indicators, respiration, and photo- The leaves in the oxygen-free environment will synthesis—which usually have been presented previ- create a lowPCQ2 value (blue color) demonstrating ously in chemistry, physics, and biology courses. blockage of photorespiratory processes, while the The basic principles which should be stressed to leaves in normal air will photorespire and create a students are: high/*CO2 value (green color). m 1. Under equilibrium conditions, the PCQ2 the closed atmosphere will depend upon the physi- ology of the species being tested (photorespirer or non-photorespirer).

2. The concentration of CO2 in the atmosphere of the closed container and the concentration of

CO2 dissolved in the indicator solution will attain an equilibrium due to simple diffusion into or out of the solution. 3. The final (equilibrium) concentration of CO2 in the indicator solution will be directly propor- tional to the concentration of CO2 in the container atmosphere.

4. The dissolved CO2 will chemically react with the water to produce carbonic acid:

+ + 2 CO2 + H2O ^ H2CO3 - H + HCO3" - H + CO3 ~