JoVE Lab Manual
Lab Bio
Lab Bio
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Have you ever wondered why every autumn leaves on trees change from green to shades of yellow, orange, and red? What gives leaves their bright colors in the first place? The answer lies in the plant organelles called chloroplasts which contain pigments that absorb certain wavelengths in sunlight and reflect others. One particular pigment, chlorophyll, is the most abundant in summer. It absorbs high-energy, purple, blue, and red wavelengths from sunlight, and reflects green wavelengths giving leaves their green appearance. There are other pigments in leaves, such as carotenoids, which reflect red and yellow light. In autumn the leaves stop replenishing their pigments. Since chlorophyll degrades faster than the other pigments, the colors of these carotenoids are unmasked.
The presence of different pigments in a green leaf can be demonstrated with chromatography paper, a hydrophilic polymer that separates molecules based on their solubility in a particular solvent. First, leaf extract is loaded onto the paper. When the paper is dipped into an organic hydrophobic solvent, the solvent travels along the paper due to capillary action and along the way it separates different pigments in the leaf extract. The pigments that are the most hydrophobic are carried further up the paper. Whereas hydrophilic pigments bind to cellulose which hinders their movement. After all of the pigments are sorted by their hydrophobicity, we can calculate the retention factor or Rf values. The Rf value is the ratio of the distance traveled by a pigment to the distance traveled by the solvent. Each pigment has a unique Rf value and we can easily identify pigments by comparing calculated values to standards. Photosynthesis, the process by which plants convert carbon dioxide, water and light energy into chemical energy and oxygen, is carried out primarily in the leaves of a plant, and chlorophyll plays a critical role in this process. Chloroplasts contain dozens of chlorophyll molecules, each performing a specific task and interacting in complex ways. Ultimately, light energy causes chlorophyll molecules to give up electrons which are utilized in other metabolic processes. Therefore, chlorophyll needs a continuous supply of electrons to replace the ones it loses. These replacement electrons come from splitting water molecules into protons, electrons and oxygen molecules. At high rates of photosynthesis water is split faster to replenish electrons and oxygen is generated rapidly.
This phenomenon helps us to assess the rate of photosynthesis in the lab by simply suspending leaf discs in a bicarbonate solution where bicarbonate acts as a rich source of carbon. At the beginning of the leaf disk experiment the gases are forced out of the leaf discs by applying negative pressure with a vacuum in a syringe. The leaf discs with their gases expelled become heavier and sink to the bottom of the bicarbonate solution when transferred to a beaker. When photosynthesis takes place water in the environment is split in order to replenish chlorophyll electrons. The resulting oxygen makes the discs lighter causing them to float to the surface over time. Environments that allow for higher rates of photosynthesis have discs that float faster.
In this lab you will first separate and identify pigments in spinach leaves using chromatography paper. Then you will assess the rate of photosynthesis in water and in bicarbonate solution with a leaf disk experiment.
光合作用
Autotrophs
Almost all living organisms on Earth depend on photosynthesis, which is the process that converts sunlight energy into a simple sugar called glucose. This molecule can be used as a short-term energy source or to build more complex carbohydrates like starches for long-term energy storage. Autotrophs are organisms that capture light energy using photosynthesis. Also known as primary producers, they provide the energy necessary to the organisms that eat them, which are known as consumers.
Organisms that can photosynthesize are rather diverse, including cyanobacteria, some protists such as algae, as well as plants. In eukaryotic cells, photosynthesis takes place in an organelle called the chloroplast, which appear green due to its high content of the pigment chlorophyll. Pigments are molecules that absorb light at certain wavelengths. The light that is not absorbed by a pigment is reflected as visible light that can be observed as the color of the pigment. Plants produce multiple pigments with various functions that absorb light from the sun at different wavelengths. For example, chlorophyll absorbs light at red and blue wavelengths while it reflects wavelengths that correspond to green. Some other pigments such as carotenoids, anthocyanins, and betalains commonly reflect light at low-energy wavelengths between 600 and 800 nm, and thus appear yellow to red. In temperate regions, the decrease in chlorophyll in autumn reveals these pigments when leaves change color to reds, yellows, and oranges.
Chloroplasts
Chlorophyll is the primary pigment in chloroplasts used in photosynthesis, whereas other pigments help channeling light energy to chlorophylls or protect the cell against light damage. Photosynthesis consists of two pathways, which are known as the light-dependent and light-independent reactions and occur at distinct locations within chloroplasts. These organelles contain three membranes: an outer membrane, an inner membrane, and the innermost thylakoid membrane, which forms long, disc-shaped folds within the chloroplast. The fluid-filled space between the inner and thylakoid membranes is called the stroma. The light-dependent reactions start when the energy in sunlight excites the electrons of chlorophyll pigments that are embedded within the thylakoid membrane. These high-energy electrons are then passed from one electron carrier molecule to another within the thylakoid membrane, collectively known as the electron transport chain. Each transfer within the electron transport chain takes the electron to a lower energy state, therefore releasing energy. Some of this energy is harnessed to synthesize small, energy-rich molecules such as Adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). The lost electron of the chlorophyll is replaced through hydrolysis (which means water splitting), when hydrogen and oxygen atoms are separated. Hydrogen atoms generated by hydrolysis donate their electrons to the chlorophyll whereas the oxygen molecules are released into the atmosphere. Stream of electrons through hydrolysis enables chlorophyll pigments to continuously absorb the light energy by repeatedly exciting new electrons and pass them down the electron transport chain.
The light-independent reactions take place in the stroma. This process uses the light energy indirectly by utilizing the energy from ATP and NADPH molecules generated by the light-dependent reactions. During this pathway, carbon dioxide (CO2) is used to build a three-carbon sugar, which then can be turned into glucose or other biomolecules. This process is also known as carbon fixation or carbon sequestration, because the carbon is sequestered from the atmosphere and fixed into biomolecules.
Photosynthesis and the Environment
Carbon sequestration during photosynthesis is an important step of the carbon cycle, in which CO2 flows from one reservoir to another at a relatively constant pace. Changes in the CO2 flow rate can shift the balance of it among reservoirs. More importantly, since CO2 is a greenhouse gas, increases in its atmospheric concentration contributes to rising temperatures. Most of this CO2 is emitted by burning fossil fuels, thus returning the carbon dioxide that was sequestered by photosynthesis hundreds of millions of years ago to the atmosphere at an unprecedented rate. Therefore, forests and ocean algae are evermore imperative in cooling the earth by reducing the increased CO2 levels1. This is one of many reasons why deforestation is a serious concern in a changing climate.
Much like how plants use photosynthesis to capture light energy into biomolecules, researchers are investigating artificial photosynthesis to create carbon neutral biofuels as an alternative to fossil fuels. Similar to solar panels, artificial photosynthesis methods obtain energy from the sun, and store it as chemical energy, some of which can be stored for long periods of time, instead of converting it directly to electricity2. Scientists have also been able to produce simple sugars and lactic acid using photosynthetic bacteria3. This approach has wide applications in manufacturing biomolecules, biofuels, and even biodegradable plastics with little to no harmful emissions. In addition, this approach may be used to remove excess CO2 from the atmosphere. A similar concept is being applied in the medical field, where photosynthetic bacteria are being used to produce physiologically active substances and medications, some of which have been applied to cancer diagnosis and treatment4. Future research may increase the efficiency of producing sustainable and carbon neutral molecules while remediating the effects of increased atmospheric CO2 levels.
References
- Bonan, GB. Forests and Climate Change: Forcings, Feedbacks, and the Climate Benefits of Forests. Science. 2008, Vol. 320, 5882 (1444-9).
- Liu, C, et al. Water splitting–biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis. Science. 2016, Vol. 352, 6290 (1210-1213).
- Niederholtmeyer, H, et al. Engineering Cyanobacteria To Synthesize and Export Hydrophilic Products. Appl Env Microbio. 2010, Vol. 76, 11 (3462-66).
- Sasaki, K, et al. Applications of photosynthetic bacteria for medical fields. J Biosci Bioeng. 2005, Vol. 100, 5 (481-8).
