Effects of Temperature on Cell Respiration. (B4-1)
Cell respiration can be viewed as a series of enzyme catalyzed reactions in which carbohydrates, proteins, and fats are broken down to carbon dioxide and water with the release of energy. During the process, hydrogen is removed from the fuel molecules and oxygen is consumed. With this background information, students measure oxygen consumption and hydrogen liberation in germinating barley at different temperatures. The program provides eight calibrated respirometers for measurement of oxygen consumption and the chemicals required to perform a graphic dye reduction assay. The exercise introduces students to a fundamental biological process and provides insight into seed structure and germination.
Here is a sample from the student guide.
Experiment B4-1. Effects of Temperature on Cell Respiration
A. CELL RESPIRATION
Carbon dioxide and water are two of the most prevalent and energetically stable compounds found in nature. Thus, energy is liberated when complex carbon and hydrogen containing molecules are converted to carbon dioxide and water. The burning of fuel provides a dramatic example of this type of energy yielding reaction. When methane is burned in air, oxygen is consumed and carbon dioxide, water, and energy are released as shown below.
This is an oxidation reaction because oxygen combines with another substance with the liberation of energy. More generally, oxidation refers to a reaction in which an atom loses one or more electrons. In the above example, electrons are shifted away from the carbon atom in methane as it is oxidized to CO2. Although the energy of this reaction is normally liberated as heat, it can also be used to perform work with an appropriate mechanical device. For example, steam produced by the burning of methane can be used to run a steam engine.
Cell respiration is the process by which organisms oxidize food materials to CO2 and H2O. Energy is liberated during respiration since the C and H atoms in the fuel molecules are electron rich as compared to the C and H atoms in CO2 and H2O. Respiration differs from combustion in a bonfire because it is a controlled oxidation that takes place in a large number of small steps. This sequential breakdown permits some of the energy trapped in fuel molecules to be transferred to "high energy" compounds like adensoine triphosphate (ATP) instead of being dissipated as heat. The high-energy compounds, in turn, are used to provide the energy that is needed to drive synthetic reactions in the cell.
The most common fuel for respiration in plants and animals is the six-carbon sugar glucose, which is often made available by the breakdown of complex carbohydrates. The complex carbohydrate starch is the major storage form of glucose in plants. In respiration, glucose is oxidized to carbon dioxide and with the release of energy according to the overall equation shown below.
C6H12O6 + 6 O2 →→→ 6 CO2 + 6 H20 + 674,000 calories
When one molecule of glucose passes through the entire sequence of reactions that comprise respiration, the net result is (1) six atoms of carbon are released from glucose and are evolved as CO2; (2) twelve hydrogen ions (H+) and twelve electrons (e-) are removed from the fuel and combine with six atoms of oxygen to form six molecules of water; and (3) energy is released which is transferred to form ATP or is lost as heat.
The oxidation of glucose proceeds through many reactions, each catalyzed by a specific enzyme. As respiration is an enzyme-catalyzed process, its rate is highly dependent on environmental temperature and increases sharply with increases in temperature in the biological range of 0°C to 40°C. Although the process appears complex, the basic patterns can be described in simple terms as outlined in Figure 2-1. Glucose is split into two molecules of pyruvic acid, each with three carbon atoms. These reactions occur in the cytoplasm by a process called glycolysis. Pyruvic acid then enters the mitochondria, where it is convened to the acetyl group (2-carbon) of acetyl coenzyme A. The acetyl group is then completely degraded to CO2 and water in the citric acid cycle and in the electron transport chain. During the citric acid cycle, the carbon atoms of the acetyl groups are liberated as CO2, while, the hydrogen atoms (protons + electrons) are transferred primarily to the carrier molecule NAL)* which is thereby reduced to NADH+11+. The electrons are then transferred from the NADH+1-1+ to a series of electron carrier molecules that comprise the electron-transport chain. During the course of this transfer, the electrons lose energy which is transferred to ATP. The terminal step in the chain is when the electrons and protons combine with molecular oxygen to form water.
B. MEASUREMENT OF THE RATE OF RESPIRATION.
In the laboratory, the rate of respiration can be determined by different procedures which are described briefly below.
Heat Production - Only a fraction of the energy given off during respiration is trapped as ATP. The remainder is liberated as heat which can be measured over time to provide an index of the rate of respiration. This "metabolic heat" is used by birds and mammals to maintain body temperature but is commonly viewed as wasted energy in plants because it is quickly dispersed into the environment.
Liberation of Carbon Dioxide - The rate of respiration can be determined experi¬mentally by measuring the amount of carbon dioxide evolved in a given time.
Oxygen Consumption - The rate at which oxygen is consumed by living tissue is a generally accepted index of the rate of respiration and you will use this method in today's laboratory. Measurements by this method can be made by using the simple respirometer diagramed in Figure 2-2. The respirometer consists of a sealed chamber attached to a calibration tube. Respiring tissue is placed in the chamber along with a tube containing potassium hydroxide. A drop of water is then introduced into the top of the calibration tube. As carbon dioxide is produced by the tissue, it combines with the potassium hydroxide to form potassium carbonate
(CO2 + 2 KOH → K2CO3 + H2O). Thus, as oxygen is consumed by the tissue, the volume of air in the chamber decreases because the CO2 in the respired air is absorbed by the KOH. The movement of the drop of water down the graduated tube provides a measure of this volume reduction, and by timing its movement, the number of ml of oxygen consumed per unit time is obtained.
Dye Reduction - As noted above, an important feature of respiration is the transfer of electrons and protons to "acceptor" molecules such as NAD+ which thereby become reduced. Artificial electron acceptor molecules can be introduced into respiring systems and such acceptors can be used to measure respiration. Tetra¬zolium is a colorless compound when it is in an oxidized form. However, electrons produced by cellular respiration reduce the dye to a red product as shown in the equation below.
HP + 2e
Tetrazolium →→→→→→→→→→ Triphenyl Formazan
(Soluble and Colorless) (Insoluble and Red)
Tetratazolium reduction has been used to localize respiring tissues in plants and animals and in this laboratory you will use this method to study respiration in a fascinating developing system.
C. SEED GERMINATION
The seeds of flowering plants are typically resistant structures in which embryonic plants are enclosed. The outer layer of the seed is called the seed coat which protects the embryo from adverse conditions. The structures of the seeds of flowering plants are similar in that each seed contains a seed coat, an embryo, and a food storage tissue. The structures in a mature corn seed are shown in Figure 2-3. The mature corn embryo has a single cotyledon, or seed leaf, which is made up of a food-adsorbing portion called the scutellum and the coleoptile which forms a protective cap over the shoot. Below the cotyledon is the plumule, the apex of the embryonic shoot. The basal end of embryo, the radicle, develops into the primary root when the seed germinates. The food for the development of the corn seedling is largely located in the endosperm. In fact, the embryo is embedded in the cellular endosperm, the cells of which are rich in stored protein and especially starch. The endosperm is surrounded by several layers of cells that form the aleurone layer, in which a variety of enzymes are produced during seed germination.
Mature seeds have a low water content and the cells of the embryo are biochemically dormant. The cells can be activated in seeds by environmental factors, especially an increase in the moisture content of the atmosphere. This activation is called germination which begins by the uptake of water (imbibition phase) and culminates in the protrusion of the embryonic root from the seed. In corn, germination requires 24-48 hours under ideal conditions, and can be divided into the following steps:
1. Imbibition of water and hydration of subcellular organelles
2. Activation and new synthesis of a variety of enzymes in the aleurone layer and in the embryo
3. An increase in the levels of digestive enzymes amylase and proteinases in the aleurone layer and scutellum. These enzymes break down the stored starch and proteins into glucose and amino acids in the endosperm.
4. The transport of the breakdown products (glucose and amino acids) from the endosperm to the embryo where they are used as a nutrient source for the growing embryo
5. An increase in oxygen uptake and respiratory activity of the embryonic cells
6. Cell division and differentiation in the embryo and the emergence of the root from the seed.
Objective: To measure oxygen consumption and dye reduction in germinating barley and corn seeds at different temperatures.
The experiment was designed for 8 groups of students and should take approxi-mately 90 minutes to complete.
- Germinating Corn Seeds (Note: the seeds must be soaked as described below for 2 days before the laboratory session.
- Germinating barley seeds (Note: the seeds must be soaked as described below for 2 days before the laboratory session.
- Nongerminating barley seeds
- 8 respirometers - Each respirometer consists of a 25 ml glass tube, stopper with attached 1 ml calibration tube, and a 1.5m1 microtube for KOH (see Figure 2-¬2).
- *15% Potassium Hydroxide (Note: Observe extreme caution when handling the KOH.) The solution is very caustic and will damage clothes, furniture, and living tissue. If by accident, some gets on your clothes or hands, wash at once with water. Notify the instructor in any cases of spillage.
- Large Transfer Pipets - calibrated up to lml
- Small Transfer Pipets
- *Prepared as described in the Instructor Guide.
Materials Not Provided
Razor blades or scalpels
Distilled or deionized water
Forceps or tooth picks
Tissue (kleenex or Kimwipes)
Four pans for water baths
Ice and hot water
Four one liter beakers
Clock or watches with second hand
Eight medium sized (l0-20m1) test tubes for corn seed incubation with tetrazolium.
I. Prefab Preparation of the Germinating Seeds
A. Corn Seeds - Note: This procedure must be performed 2-3 days before the laboratory session. The procedure can be performed by the instructor, in which case about 60 seeds should be prepared, or by 8 groups of students who should prepare about 6-8 seeds each.
1. Place a layer of filter paper or paper towels in a petri dish and add about 5 ml of water to moisten the paper.
2. Add about 6-8 seeds and cover the dish. The seeds will be ready for analysis after about 2 days in the dish.
3. On the day of the experiment, place about 30 of the seeds in a beaker of water and boil for about 5 minutes. Place these seeds between moist paper towel and label "Boiled Corn Seeds".
B. Barley Seeds - Note: This procedure must be performed 2-3 days before the laboratory session.
Layer moist paper towels in the bottom of shallow pans and sprinkle about 80 g of barley seeds onto the towels until they form a seed layer of about 1/2 cm. Cover the seeds with wet paper towels and then cover the pans. Make sure that the towels remain moist for the 2-3 days before the experiment. On the day of the experiment, place about 10% of the seeds in a beaker of water and boil for about 5 minutes. Place these seeds between moist paper towels and label "Boiled Barley Seeds".
II. Preparation of the Four Water Baths
A diagram of one of the four water baths that you will assemble is shown in Figure 2-4. During the experiment, each bath will contain 1 to 3 respirometers and two tubes of corn seeds in a beaker.
Assemble each bath before the laboratory session as follows:
1. Obtain one pan or plastic tub at least 5 inches high and long enough to hold a 1 liter beaker and a collection plate.
2. Place the one liter beaker in the pan.
3. Fill the bath and beaker with water to a depth of about 5-6 inches and place a thermometer in the bath.
4. Mark the following on each of the four baths and on the reaction plates that will go into each bath. Bath 1, 0-3°C, Bath 2, 10-13°C, Bath 3, 20-25°C, and Bath 4, 35-40°C.
5. Adjust the four baths to the following temperatures: 0-3°C (Bath 1), 10- 13°C (Bath 2), 20-25°C (Bath 3), and 35-40°C (Bath 4). Add ice to baths I and 2 and hot water to bath 4 to adjust the temperatures. Ice and hot water should be added during the experiment to maintain these temperatures.
III. Addition of Tetrazolium to the Collection Plates
At the beginning of the experiment, four students should place about 3 ml of the tetrazolium solution into two test tubes. Using a waterproof pen mark one tube, "boiled corn seeds" and one tube "not boiled corn seeds". The solution can be dispensed with a large transfer pipet.
1. Your measurements of respiration using the tetrazolium reduction assay and the oxygen consumption assay should lead to similar conclusions with regards to the effects of temperature on respiration. Do they? (Compare tables in Data Analysis I VS II).
2. Seed-testing laboratories frequently determine the percentage of viable (living) seeds in a seed lot by growing seeds under prescribed conditions and observing the number that germinate. The ability of seeds to reduce tetrazolium during early germination forms the basis of a simple alternative test for seed viability. Why?
3. The temperature of the interior of a crate of celery is at least 10°C higher than the outside air. Why?