Photosynthesis
Autotrophs
Almost all living organisms on Earth depend on photosynthesis, which is the process that converts sunlight energy into a simple sugar calle…
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.
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.
View the full transcript and gain access to JoVE Lab Manual videos
Q1: Why do leaves change color in autumn?
Leaves change color because chlorophyll, the dominant green pigment, degrades faster than other pigments like carotenoids in autumn. As chlorophyll breaks down, the yellow and red wavelengths reflected by carotenoids become visible. In summer, chlorophyll absorbs high-energy blue and red wavelengths while reflecting green light, masking these other pigments.
Q2: What role does chlorophyll play in photosynthesis?
Chlorophyll absorbs light energy and uses it to excite electrons, which are then passed through an electron transport chain to generate energy-rich molecules like ATP and NADPH. When chlorophyll loses electrons during this process, they must be replaced by electrons from water splitting. This continuous electron cycling allows chlorophyll to repeatedly capture light energy for photosynthesis.
Q3: How does chromatography separate leaf pigments?
Chromatography paper is a hydrophilic polymer that separates pigments based on their solubility in an organic solvent. When leaf extract is loaded onto the paper and dipped in solvent, capillary action carries the solvent upward. Hydrophobic pigments travel farther, while hydrophilic pigments bind to cellulose and move slower, allowing identification by calculating retention factor (Rf) values.
Q4: What happens to leaf discs during photosynthesis in a bicarbonate solution?
Initially, leaf discs sink because gases are removed by vacuum pressure, making them heavier. During photosynthesis, water is split to replenish chlorophyll electrons, releasing oxygen gas. This oxygen accumulates in the leaf discs, making them lighter and causing them to float. Faster floating indicates higher photosynthesis rates, allowing researchers to studying photosynthesis leaf disc experiment procedures.
Q5: What is the relationship between photosynthesis and the carbon cycle?
Photosynthesis removes carbon dioxide from the atmosphere through carbon fixation, converting it into glucose and other biomolecules. This process is a critical step in the carbon cycle. Increased atmospheric CO2 from burning fossil fuels disrupts this balance, making forests and ocean algae essential for reducing CO2 levels and mitigating climate change.
Q6: How do light-dependent and light-independent reactions differ in location and function?
Light-dependent reactions occur in the thylakoid membrane and use sunlight to excite chlorophyll electrons, generating ATP and NADPH while splitting water. Light-independent reactions occur in the stroma and use the ATP and NADPH from light reactions to fix carbon dioxide into glucose. Together, they convert light energy into chemical energy stored in sugars.
Q7: What are autotrophs and why are they important to ecosystems?
Autotrophs are organisms that capture light energy through photosynthesis to produce glucose, serving as primary producers. They convert sunlight into chemical energy that flows through food chains to consumers. Diverse autotrophs including plants, algae, and cyanobacteria provide the foundational energy source for nearly all life on Earth.