As you may already know, you can investigate how different pigments are present in a single plant leaf via chromatography. These pigments help a plant absorb light energy. But what sort of factors affect how efficiently light energy is turned into chemical energy in the form of ATP? And how can biologists measure how these factors affect the rate of photosynthesis?
Chromatography is a lab technique that separates a mixture into the parts that make it up.
As mentioned in the Photosynthetic Pigments explanation, chromatography separates the different pigments that make up the colouring of leaves.
The redox indicator and the rate of photosynthesis
To investigate the rate of photosynthesis, we use a substance called a redox indicator. If you’ve already studied respiration, you’ll recall that ‘redox’ is short for ‘oxidation-reduction’, which is a term usually attributed to a kind of reaction where electrons are both lost and gained. Redox indicators are a type of chemical that you can add to a solution.
When this solution is reduced or oxidised, the redox indicator will cause that solution to suddenly change colour. Redox indicators are used in a variety of chemistry and biology experiments (including when we investigate the rate of respiration). In this experiment, we can use a redox indicator such as DCPIP or methylene blue.
How to investigate the rate of photosynthesis using redox indicators
Generally, photosynthesis is affected by three main factors: light intensity, carbon dioxide concentration, and temperature.
Equipment
Leaves from a single plant (spinach leaves are fairly easy to obtain and can be purchased in large enough quantities for an experiment).
An isolation medium (usually made up of a phosphate buffer solution, sucrose, and potassium chloride). Make sure this is ice cold so that the chloroplasts are not damaged.
Water baths at the following temperatures: 20°C, 30°C, 40°C, 50°C.
LED lights set at different distances from the plant. This will alter the light intensity.
Small test tubes.
Pestle and mortar.
Muslin cloth.
Funnel.
Beaker.
Fridge set between 0-4°C.
Stopwatch.
Pipette.
Centrifuge and centrifuge tubes.
Method
Crush the leaves using the pestle and mortar with about 20cm³ of the isolation medium.
Place four layers of the muslin into the funnel, and gently wet them with the cold isolation medium. Place the funnel above the beaker.
Filter the mixture of the crushed leaves and isolation medium through the funnel.
Pour the solution in the funnel into the centrifuge tubes, making sure that each tub contains the same amount of solution.
Centrifuge the tubes for around 10 minutes at high speed. This should produce a small pellet of leaf extract.
Pour off the extra liquid that surrounds the pellet, and resuspend the pellet with around 2cm³ of the isolation medium. This is your leaf extract solution.
In each test tube, add 0.5cm³ of the new leaf extract solution. Then, add 5cm³ of either DCPIP or methylene blue indicator into each test tube.
If you are testing the effect of temperature, then place each test tube in a different water bath.
If you are testing the effect of light intensity, then place each test tube into a separate dark room with a single LED light. Vary the distance of the LED light from the test tube. Make sure to place one test tube in an entirely dark room as a control.
Stir each test tube and note down the amount of time it takes for the solution to decolourise.
Note that the colour of the solution may change from blue to green instead of colourless due to the presence of chlorophyll.
Reaction mechanism
The light-dependent reactions of photosynthesis take place in the plant cell’s chloroplasts, along the thylakoid membrane.
During photosynthesis, chlorophyll will absorb photons. These are a type of light energy, which causes the electrons contained in the chlorophyll to move to a higher energy level, which allows them to hold that energy and participate in other reactions. These will then be picked up by an electron acceptor, an oxidising agent which accepts electrons transferred from another compound, moving down an electron transport chain (More on this in the Photosynthesis explanation).
However, if a redox indicator is present, the indicator will replace the electron acceptor and take up the high-energy electrons instead. This will cause the colour change that you will observe in the experiment. You can use the rate at which this colour change occurs to measure the rate of photosynthesis.
The light-independent reaction is known as the Calvin Cycle. The Calvin Cycle needs carbon dioxide in order to form glucose (which needs carbon to form). First, one molecule of carbon dioxide is combined with a molecule named RubP. After they combine, they split in half producing two molecules of 3-phosphoglycerate. ATP and NADPH, both produced at the start of the light-dependent reaction, donate each a Hydrogen atom to 3-phosphoglycerate which transforms them into G3P (a type of sugar). The two molecules of G3P produced are able to form the glucose needed to fuel the plant.
The Calvin Cycle usually only uses six carbon dioxide molecules at a time. It’s only able to produce one molecule of glucose at the end and the leftover G3Ps are recycled back into RubP so the cycle can continue.
Which factors affect the rate of photosynthesis?
Photosynthesis is affected by a number of factors. However, the following factors can limit the rate of photosynthesis when they are in short supply:
Light
As the intensity of light increases, so does the rate of light-dependent reactions of photosynthesis. Therefore, increasing the intensity of light increases the overall rate of photosynthesis.
This is because more photons will fall on the leaf, therefore more electrons will be photoactivated (activated to a higher energy level by light waves), allowing the water to be oxidised faster. Therefore, the production of ATP and NADPH, a type of molecule which is used as a reducing agent in the Calvin Cycle, increases, and more cycles of the light-independent reaction will occur.
However, after a certain point, the rate of photosynthesis remains constant even if the light intensity increases, because one or more of the other factors run out which makes them a limiting factor.
Carbon dioxide concentration
Increasing the concentration of carbon dioxide will increase the rate of photosynthesis up to a certain point. If more carbon dioxide molecules are available, more cycles of the light-independent reaction will occur at a higher rate.
This means that more glucose molecules are produced, more NADPH and ATP are used up, more RuBP (known as the primary acceptor of carbon dioxide during the Calvin cycle) is produced, and the overall rate of photosynthesis will increase.
However, at a certain level, the rate of photosynthesis will be limited by other factors. There may not be enough light energy to produce enough NADPH and ATP to fuel more cycles of the light-independent reaction, so the rate of photosynthesis will not increase even if the concentration of carbon dioxide increases. Alternatively, there may not be enough heat energy available to catalyze the enzyme-controlled reactions in the light-independent reaction at a higher rate, so the rate of photosynthesis will not be able to increase with the concentration of carbon dioxide.
Study tip: you will notice that we did not vary carbon dioxide concentrations for this practical. This is because whilst carbon dioxide concentration does affect the rate of photosynthesis, it is not involved in the light-dependent reaction of photosynthesis. Therefore, its effect on the rate of photosynthesis can’t be measured using a redox indicator.
Temperature
Since enzymes control photosynthesis, temperature is an important limiting factor for the rate of photosynthesis. The rate of photosynthesis increases with temperature. However, unlike with carbon dioxide concentration and light intensity, the rate of photosynthesis reaches an optimum point before drastically declining.
The enzymes that control photosynthesis work at their best at around 35°C-40°C. However, if the temperature increases past this optimum point, the enzymes start to denature. The enzyme’s active site shape changes and the substrate no longer fits. This explains the sharp decrease in photosynthesis rate at higher temperatures. If the temperature is lower than 35°C, photosynthesis occurs at a slower rate because the enzymes do not move as fast. Thus, less reactions can occur because it’s harder to find the substrate.
Note: water is not a limiting factor for photosynthesis. There is very little water needed in the entire process of photosynthesis. However, even if there was a shortage of water to the point where photosynthesis would be restricted, the plant’s stomata would begin to close and absorb carbon dioxide at a slower rate. Therefore, other processes would stop before water could have a limiting effect on them.
Rate of Photosynthesis - Key takeaways
Redox indicators are a type of chemical that you can add to a solution. When this solution is reduced or oxidised, the redox indicator will cause that solution to suddenly change colour.
Examples of redox indicators include DCPIP or methylene blue.
To investigate the rate of photosynthesis, you need to combine a leaf extract with a redox indicator.
The redox indicator is used as it takes up the high-energy electrons that are usually taken by an electron acceptor. This will cause the colour change that you will observe in the experiment.
There are three different factors that generally affect the rate of photosynthesis: light intensity, temperature, and carbon dioxide concentration.
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