How temperature affects the rate of photosynthesis.

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Biology A2 Coursework

How temperature affects the rate of photosynthesis

Contents:

Title:                                                                        Page number:

Abstract                                                                

Introduction                                                                

Preliminary experiment

Aim                                                                        

Prediction                                                                

Apparatus                                                                

Diagram                                                                

Method                                                                        

Key variables                                                                

Safety                                                                        

Results                                                                        

                        

Graph (individual)                                                                        

Graph (class)                                                        

Statistical analysis (t-test)                                                                

Conclusion                                                                

Discussion                                                                

Evaluation                                                                

Bibliography                                                                

Abstract: The aim of the experiment was to observe and analyse the affect of temperature on the rate of photosynthesis. This rate was measured by the rate of oxygen produced (mm3/min), by a photosynthesising plant (elodea), under different temperatures. A specialised apparatus called a potometer was used to observe this affect. A certain length of elodea (50mm) was cut at a slant at one end, and was placed in a test tube and was immersed in the pondweed water it was accustomed to. The cut end of the elodea was attached to a delivery tube which was connected to a measuring tube which was further attached to a syringe by rubber tubing. This whole apparatus (the potometer) was then placed in varying temperatures to measure the affect of the temperatures on the rate of oxygen production. For each temperature the length of the oxygen bubble released by the plant was measured by pulling it into the measuring tube of the potometer by using the syringe. This length was recorded and converted to the rate of oxygen produced by the plant per minute under that temperature (mm3/min). The same elodea was used to perform the experiment under the same temperature three times all together i.e. there were three repeats for each temperature. The temperatures used were: 0 oC, 15 oC, 25oC, 35 oC, 45 oC, 55oC, and 65 oC.

Many factors affect the rate of photosynthesis mainly carbon dioxide concentration (controlling the rate of the Calvin cycle), light intensity (Affecting the light stage) and temperature (affecting the kinetic energy of all the molecules, including enzymes involved in the photosynthetic reactions and reducing the rate of photosynthesis by denaturing the enzymes after a certain temperature). Temperature was the only factor to be varied, which meant that the other two limiting actors had to be available constantly and in plenty. Excess carbon dioxide was provided by adding equal amounts of sodium hydrogen bicarbonate to each test tube which dissolved in water to provide carbon dioxide and the light intensity was controlled by providing only one artificial source of light i.e. a lamp which was kept at the same distance of 50mm from each test tube (with the elodea in it).

The shape of the graphs and the results obtained made me realise that the rate of photosynthesis mainly depends on the functioning of enzymes. The graphs showed an increase in the average rate of oxygen production between 0 oC -45 oC (with peaks forming at approximately 42 oC i.e. at the optimum temperature). As the temperature increased beyond 45 oC, the rate of oxygen production decreased steeply and the lowest rate was recorded at 65 oC. The t test value was calculated for 0 oC and 35 oC, 25 oC and 55 oC and 35 oC and 65 oC.

Valid conclusions were made and most of the results were in accordance with the prediction although there were some anomalies present. The errors and limitation of the experiment were evaluated and certain improvements were suggested.

Introduction: Photosynthesis (photo=light, synthesis=putting together) is defined as the trapping or fixation of carbon dioxide and its subsequent reduction to carbohydrate (sugars), using hydrogen from water. These sugars can then be converted into other essential substances like fats, proteins etc- which plants need to live and grow.  [Text adapted from OCR A2 Biology Textbook]

Photosynthesis occurs mainly in the leaves of green plants which contain a light trapping pigment called chlorophyll. The raw materials needed for photosynthesis to occur are carbon dioxide (CO2) which is obtained from the air via stomata by diffusion and water (H2O) from the soil which is transported up to the leaf via the root hair and the xylem tissue. These act under the action of light energy trapped in the chlorophyll of the leaf to form the sugar glucose (C6H12O6) which is a carbohydrate and oxygen (O2), which is released as a by product through the stomata into the atmosphere.

The general equation for photosynthesis in a green plant is:

Light energy

Carbon dioxide    +     water                          glucose      +      oxygen

Chlorophyll

Light energy

6CO2    +    6H2O                C6H12O6    +    6O2

Chlorophyll

Photosynthesis takes place in two steps. These are light dependant reactions which require light energy and the light independent reactions, for which light energy is not needed.

Light energy drives the light dependant reactions and the products formed at the end of it account for the functioning of the light independent reactions. Light energy is trapped from the source like the sun, by two types of photosynthetic pigments:

  1. The light-dependant reactions: The light independent reactions take place in the grana of the chloroplast. These reactions include:
  • Synthesis of ATP from ADP in photophosphorylation.
  • Splitting of water by photolysis to give hydrogen ions
  • Production of reduced NADP

Photophosphorylation of ATP can be cyclic and non cyclic, which depends on the pattern of electron flow in the photosystems.

Cyclic Photophosphorylation: Light energy is absorbed by P700-photosystem I, by the accessory pigments and is transferred to the primary pigment i.e. chlorophyll a. Electrons in the chlorophyll a molecule are excited and reach a higher energy level and are emitted. This excited electron is captured by an electron carrier and channelled back to chlorophyll a molecule through a chain of electron carriers. The electrons lose energy as they pass through electron carriers. This energy is used to synthesise ATP from ADP and an inorganic phosphate group (Pi). This ATP is required in the light independent stage i.e. the Calvin cycle. The electron is sent back to photosystem 1 via electron carriers after ATP is formed, thereby completing the cycle.

Non cyclic Photophosphorylation: Non cyclic photophosphorylation involves both photosystem 1 and photosystem 2. Light energy is absorbed by both photosystems and excited electrons are emitted from the primary pigments of both reaction centres (P680 and P700). These electrons are absorbed by electron acceptors and passed along electron carriers leaving the photosystems positively charged. The electrons emitted from photosystem II travel to photosystem 1 and stabilise it. [Text adapted from Page 20 of OCR A2 Biology Textbook]. To stabilise photosystem II, photolysis of water occurs to form hydrogen ions, oxygen and electrons are released. This reaction is catalysed by a water splitting enzyme present in the photosystem itself.

                            H2O                       2H+ + 2e- + ½O2

These electrons produced during the photolysis of water are absorbed by P680 and stabilise it. The hydrogen ions combine with the electrons released from photosystem I (P700) and a carrier molecule called NADP to from reduced NADP. This reduced NADP is of importance in the Calvin cycle or the dark stage. As in cyclic photophosphorylation ATP is synthesised as electrons lose energy while passing along the electron carriers throughout. The formation of reduced NADP is shown by the following equation:          

                  2H+ + 2e- + NADP Reduced NADP (NADPH)

[Diagram taken from  http://content.answers.com/main/content/wp/en/6/6d/Z-Scheme.PNG]

As photolysis is carried under the action of a water splitting enzyme, temperature would have an effect on the rate of splitting up of water and therefore the rate at which electrons are released i.e. rate at which photolysis takes place.  This is because the action of enzymes depends on the temperature as a low temperature would reduce there action (low kinetic energy) whereas a high temperature of about 40 degrees would increase the rate at which they carry out their processes (high kinetic energy). But a temperature higher than 40 degrees can denature them, and the enzymes can stop functioning.

Oxygen is the only measurable product of photolysis, and the release of oxygen depends on the enzyme controlled photolysis of water. So temperature affects the functioning of enzymes and therefore the rate at which oxygen is produced during the light stage of photosynthesis. The rate at which oxygen is evolved in a fixed amount of time can be used to measure the rate of photosynthesis in the experiment under varying temperatures.

Light intensity will also have an affect on how fast the products of light dependant stage are formed. A high light intensity will mean more energy available for the electrons to be excited, so both cyclic and non cyclic photophosphorylation will take place at a higher rate. An increase in the rate of cyclic photophosphorylation will mean more ATP will be transported to the dark stage to be used. So the dark stage will also occur at a higher rate increasing the overall rate of photosynthesis. Because of the high rate of reaction of the non cyclic photophosphorylation, photolysis of water will occur at a higher rate and more oxygen will be produced. A low light intensity can reduce the amount of oxygen released because the electrons will be less excited and both the cyclic and non cyclic photophosphorylation will take place at a lower rate. Again photolysis of water will be slower and less oxygen will be produced. Also less ATP and reduced NADP (produced in the non cyclic photophosphorylation) will be available for the dark stage to use and this in turn will reduce the rate at which the dark stage produces its products. This would reduce the overall rate of photosynthesis and so light intensity will have to be considered during the experiment.

  1. The light independent reactions of photosynthesis: This stage is known as the Calvin cycle or the Dark stage as light is not required here and it takes place in the stoma region of the chloroplast. Carbon dioxide from the atmosphere is the raw material required to carry out these reactions. The reactions that take place are:

[The following bullet points are adapted from page 21-22 of OCR A2 Biology]

  • The first stage is the fixation of carbon dioxide called carboxylation. The carbon dioxide combines with a 5 carbon sugar- Ribulose Bisphosphate (RuBP) under the action of an enzyme called Ribulose Bisphosphate Carboxylase (Rubisco). The result is an unstable intermediate of 6 carbons.
  • This intermediate breaks down to form two molecules of a three carbon compound named Glycerate 3- phosphate (GP or PGA). Then GP is reduced to two molecules of a three carbon sugar called triose phosphate (TP) by using the energy from ATP and the hydrogen atom from the reduced NADP, both received from the light dependant stages of photosynthesis. As ATP is used, ADP and Pi are released and as the hydrogen atom from reduced NADP is used, NADP is released. ADP, Pi and NADP are returned to the light dependant stage to further from ATP and NADP respectively to be used in the Calvin cycle again.
  • Some of the triose phosphates condense to form six carbon sugars like sucrose, starch and cellulose or are converted to acetylcoenzyme A which forms amino acids and lipids. Other molecules of triose phosphate are used to regenerate Rubilose Bisphosphate (RuBP) which again combines with carbon dioxide under the action of Rubisco and the process starts again. The regeneration of RuBP also requires ATP, at the end of which ADP is reformed to be used in the light stage again.

At the end of the light independent reactions, carbohydrates (products of photosynthesis) are formed. The most important raw material for these reactions is carbon dioxide. Therefore the availability of carbon dioxide has a direct relation to the rate at which photosynthesis takes place. So during the experiment, the concentration of carbon dioxide should be controlled to make the test fair. Therefore external supplies of carbon dioxide should be provided like Sodium hydrogen carbonate (NaHCO3) which reacts with water to form carbon dioxide. This would make it available in excess for the plant to use. Also carboxylation requires the action of an enzyme (Rubisco). Again temperature will have a direct affect on the functioning of the enzyme Rubisco and therefore the rate at which photosynthesis occurs.

Diagram showing the Calvin Cycle

 

[Diagram taken from Page 21 - OCR A2 Biology]

Light intensity has an indirect affect on the reactions taking place in the light independent stage. As mentioned above, if the light intensity is low, then the electrons in the light dependant stage will be less excited. Therefore the rates of cyclic and non cyclic photophosphorylation will be low. This will result in less ATP and reduced NADP being formed from both phases of photophosphorylation. As these products are the key to the functioning of the light independent stage, a shortage can result in a lower rate of reactions in the Calvin cycle, thereby producing low amount of products like triose phosphate and to regenerate Ribulose Bisphosphate. This can result in an overall reduction in the rate of photosynthesis. So light intensity is a very important factor in the deciding the rate of photosynthesis and will have to be considered during the experiment.

Guard cells: As photosynthesis takes place, carbon dioxide enters the plant (i.e. into the leaf) and oxygen leaves the plant (as a by product of photolysis of water). This gaseous exchange occurs through tiny pores present in the lower epidermis of the leaf called the stomata. These stomata also provide a gateway for water to move in and out during transpiration. Each stoma (singular) is bounded by two guard cells which regulate the opening and closing of the stomata. The wall of the guard cells next to the stoma is very thick and the wall furthest from the pore is very thin. When water moves into the guard cells, the thin wall bends more readily than the thick wall and this makes the guard cells turgid. The guard cells become curved and the stomata open. The reverse happens when water moves out of the guard cells and they become flaccid, thereby closing the stomata not allowing any gaseous exchange and transpiration to take place.

A diagram showing guard cells surrounding a stomata pore:

[Diagram taken from http://www.lima.ohio-state.edu/academics/biology/images/stoma.jpg]

Gaseous exchange depends on the opening and closing of the stomata which is controlled by the turgidity of the guard cells. In the experiment the amount of oxygen released will be measured, which moves out of the leaves via the stomata. During very high temperatures, the guard cells close due to loss of water by evaporation and the stomata are blocked, preventing any gaseous exchange to take place. Therefore no oxygen is allowed out of the leaf. Also no carbon dioxide will be allowed to enter the leaf and the reactions taking place in the Calvin cycle will cease or their rate will decrease. This affect of temperature in reducing the rate of photosynthesis can be considered while drawing conclusions during the experiment.

Enzymes: Enzymes are globular proteins and act as biological catalysts. Being catalysts, enzymes increase the rate of a particular reaction without being chemically altered themselves. “In the body, they increase the rate of reactions by a factor of between 106 to 1012 times, allowing the chemical reactions that make life possible to take place at normal temperatures”.

[Data adapted from http://www.biologymad.com/master.html?http://www.biologymad.com/Enzymes/enzymes.htm] 

Some stages of photosynthesis occur under the action of enzymes, like ribulose bisphosphate carboxylase (Rubisco), the water splitting enzyme for photolysis and ATP synthase for the synthesis of ATP.

As enzymes are proteins, they also have a primary, secondary and tertiary structure. The primary structure consists of the sequence of the amino acids joined together by peptide bonds to form a polypeptide chain. One or more polypeptide chains formed by the primary structure, coil into an alpha helix or a beta pleated sheets. This occurs due to the hydrogen bonding between the different amino acids present in the chain. The tertiary structure is formed when the secondary structure of the protein (enzyme in this case) is coiled and folded up to from a precise 3 dimensional shape. This shape of the molecule is very precise and is held in place by various bonds between the amino acids at different parts of the coiled polypeptide chains. These bonds include hydrogen bonds, disulphide bonds, ionic bonds and the hydrophobic bonds (or van der walls forces). [This text is adapted from OCR AS biology text book].

Each different enzyme has its own highly specific shape with a depression. This depression is called the active site. The active site for all molecules of one enzyme will be made up of the same arrangement of amino acids; it has a highly specific shape.  The active site is where the substrate i.e. the reactant molecule binds with the enzyme molecule. The product is called an enzyme-substrate complex. This 3 dimensional structure of enzymes is determined by the sequence of amino acids present in the tertiary structure of each molecule.

The active site of each enzyme molecule has is complementary to an individual substrate molecule. So only one type of substrate molecule can bind to the active site of a particular type of enzyme. In other words, each active site of an enzyme is specific to a substrate molecule. When the substrate molecule binds to the active site, temporary bonds are formed between the substrate and some of the enzyme’s amino acids and a complex is formed. Structural changes occur so that the active site fits precisely around the substrate. This mechanism is referred to as the “induced fit” mechanism as the substrate induces the active site to change shape.

A diagram showing the “induced fit” mechanism:

[Diagram adapted from http://en.wikipedia.org/wiki/Image:Induced_fit_diagram.svg]

The reaction will take place and the product, being a different shape to the substrate, moves away from the active site. The active site then returns to its original shape and is free to react with another substrate molecule. During this reaction, bonds can be broken or be made. So the reactant molecules i.e. substrate can be one single molecule which can be broken down into two or more products, or it can be two or more molecules which bind to the active site and are formed into one product.

An enzyme speeds up a reaction by lowering the minimum amount of energy required to form the unstable intermediate known as the transition state which is a “hybrid” structure between the reactants and the product (the enzyme substrate complex). This energy is called the activation energy. The larger the activation energy, the slower the rate of reaction only a few substrate molecules will, by chance, have sufficient energy to overcome the activation energy barrier. When a substrate molecule binds to the enzyme, the active site changes shape and fits itself around the molecule, distorting it into forming the transition state, and so speeding up the reaction. This is referred to as the induced fit mechanism. So enzymes lower the activation energy by stabilising the transition state, and they do this by changing the reaction conditions within the active site of the enzyme. [The above text is adapted from http://www.biologymad.com/master.html?http://www.biologymad.com/Enzymes/enzymes.htm]

The way a substrate molecule binds to an active site is completely by chance. The collisions between the enzymes and substrate create this chance of a reaction to take place and more the number of successful collisions, faster the rate of reaction. The probability of successful collisions can only be created by increasing the number of collisions. The probability of the enzymes and substrate colliding and therefore increasing  the rate of the reaction, can be controlled by increasing the quantity of enzyme molecules (therefore number of active sites) or increasing the number of substrate molecules, or increasing the speed at which the enzyme and substrate molecules move within the reaction site. “As the speed of the molecules increases, they gain more kinetic energy and collide more often. Moreover they do so with more energy, enough to break or make the bonds and to form products.” [Text adapted from AS Biology for OCR]. So the rate at which a substrate molecule binds to an active site determines the rate of a reaction and this can only be controlled by high number of successful collisions.

Temperature has an effect on the working of enzymes and therefore it also controls the rate of reaction under certain conditions. A moving substance always has kinetic energy and temperature can be responsible for an increase or decrease in kinetic energy. At a low temperature, an enzyme and a substrate molecule will have a low kinetic energy and will be moving slowly. But as temperature increases, both the enzyme and substrate molecules start to vibrate and gain more kinetic energy. This means that they move faster around the environment they are present in and are more likely to collide with each other. This increases the chance of successful collisions i.e. a substrate molecule binding to the active site and reaction occurring. However an increase in the rate of reaction only happens until a certain temperature. Enzymes have a temperature where they work the fastest; this is called the optimum temperature. An increase in temperature makes the enzyme molecules gain more kinetic energy as they vibrate more and this increases the number of collisions and results in an increase in the rate of reaction. But if the enzymes gain a very large amount of kinetic energy, they being to vibrate uncontrollably and the bonds holding the tertiary structure together are weakened or they break. This causes the specific active site of the enzyme molecule to be distorted and the enzyme is said to be denatured. So the active site of the enzyme is no longer complementary to the substrate molecule and it doesn’t fit the active site no more. The enzyme is said to be denatured. Therefore the enzyme does not bind to the substrate molecule and does not catalyse the reaction and the rate of reaction is decreased. So temperature increases the rate of reactions controlled by enzymes but only until the optimum temperature of the enzymes is reached. The optimum temperature of enzymes to work is about 37.5oC.

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The experiment will investigate the affect of temperature on the rate of photosynthesis and temperature affects some important stages of photosynthesis like the Calvin cycle controlled by Rubisco. A high temperature accounts for the high rate of reaction at those stages, but a very high temperature (>40 C), will denature the enzymes and the reactions occurring at that particular stage will decrease or the reactions will no longer take place. This would result in less or no product being formed and the rate of photosynthesis to decrease.

Other factors affect the functioning of enzymes apart from temperature. ...

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