Chapter 12 & 13 Energy and Respiration (PDF)

## 12 Energy and Respiration ### Now Test Yourself 5 Potassium cyanide prevents the release of the electron from the final electron carrier in the electron transport chain. Explain why cyanide prevents ATP synthesis. ### Respiration in Anaerobic Conditions In anaerobic conditions there is no oxy...

## 12 Energy and Respiration ### Now Test Yourself 5 Potassium cyanide prevents the release of the electron from the final electron carrier in the electron transport chain. Explain why cyanide prevents ATP synthesis. ### Respiration in Anaerobic Conditions In anaerobic conditions there is no oxygen. - If oxygen is not available, oxidative phosphorylation cannot take place, because there is nothing to accept the electrons and protons at the end of the electron transport chain. - Reduced NAD is not reoxidised, so the mitochondrion quickly runs out of NAD or FAD that can accept hydrogens from the Krebs cycle reactions. - The Krebs cycle and the link reaction therefore come to a halt. - Glycolysis, however, can continue, so long as the pyruvate produced at the end of it can be removed and the reduced NAD can be converted back to NAD. ### Lactate Fermentation In mammals, the pyruvate is removed by converting it to lactate Hexose sugar (glucose) 2 x triose phosphate Reduced NAD NAD 2 x pyruvate 2 x lactate * The lactate that is produced (usually in muscles) diffuses into the blood and is carried in solution in the blood plasma to the liver. * Liver cells convert the lactate back to pyruvate. This requires oxygen, so extra oxygen is required after exercise has finished. * This extra oxygen is known as the oxygen debt. It is also known as EPOC (extra post-exercise oxygen consumption). * Later, when the exercise has finished and oxygen is available again, some of the pyruvate in the liver cells is oxidised through the link reaction, the Krebs cycle and the electron transport chain. Some of the pyruvate is reconverted to glucose in the liver cells. The glucose may be released into the blood or converted to glycogen and stored. ### Ethanol Fermentation In yeast and in plants the pyruvate is removed by converting it to ethanol Hexose sugar (glucose) 2 x triose phosphate Reduced NAD NAD 2 x pyruvate 2 x ethanal 2 x ethanol Ethanol dehydrogenase ### Key Terms Oxygen debt is the extra oxygen that is taken in after a period in which anaerobic respiration has taken place, in order to break down the lactic acid that was formed. ### Now Test Yourself 6 Explain why the Krebs cycle and the link reaction stop if oxygen is not present. ### Adaptations of Rice for Wet Conditions Rice is often grown in fields that are flooded with water during the growing season. The roots therefore have little oxygen available to them. Rice has adaptations that allow it to grow successfully even when respiration in root cells takes place in these anaerobic conditions. | Feature | How it helps the plant to survive when roots are submerged | |--------------------------------------------------------------|--------------------------------------------------------------| | Cells are tolerant of high concentrations of ethanol | When roots are submerged in water, less oxygen is available than when the soil contains air spaces; cells therefore respire anaerobically, producing ethanol. | | Root cells produce large quantities of ethanol dehydrogenase | This reduces the concentration of ethanol in the root cells. | | Stems and roots have tissue called aerenchyma, containing large air spaces | Aerenchyma allows oxygen from the air to diffuse down to the roots. | | Some types of rice are able to grow elongated stems to keep their leaves above the water as its level rises | The leaves remain exposed to the air, which facilitates gas exchange for photosynthesis and respiration. | ### ATP Yield in Aerobic and Anaerobic Respiration - Only small amounts of ATP are produced when one glucose molecule undergoes anaerobic respiration. This is because only glycolysis is completed. The Krebs cycle and oxidative phosphorylation, which produce most ATP, do not take place. - The precise number of molecules of ATP produced in aerobic respiration of one glucose molecule varies between different organisms and different cells, but is usually between 30 and 32 molecules. #### Figure 12.10 A summary of the four stages of aerobic respiration, and shows the ATP yields of each stage. > Anaerobic Respiration >> Glucose >>> Glycolysis >>>> 2 ATP produced directly >>>> 2 reduced NAD >> Pyruvate >>> Link Reaction >>>> 2 reduced NAD >> Acetyl CoA > Aerobic Respiration >>> Krebs Cycle >>>> 6 reduced NAD >>>> 2 reduced FAD >>>> 5 ATP from oxidative phosphorylation >>>> 5 ATP from oxidative phosphorylation >>>> 15 ATP from oxidative phosphorylation >>>> 3 ATP from oxidative phosphorylation >>>> 2 ATP produced directly >>> Total: 32 ATP ### Respiratory Substrates Glucose is not the only respiratory substrate. All carbohydrates, lipids and proteins can also be used as respiratory substrates. | Respiratory substrate | Energy releasedkJg-1 | |-----------------------|--------------------| | Carbohydrate | 16 | | Lipid | 39 | | Protein | 17 | - You can see from Table 12.2 that lipid provides more than twice as much energy per gram as carbohydrate or protein. - A lipid molecule contains relatively more hydrogen atoms (in comparison with carbon or oxygen atoms) than carbohydrate or protein molecules do. It is hydrogen atoms that provide the protons that are used to generate ATP via the electron transport chain. - Many cells in the human body are able to use a range of different respiratory substrates. However, brain cells can only use glucose. Heart muscle preferentially uses fatty acids. ### Respiratory Quotients It is possible to get a good idea of which respiratory substrate the cells in an organism are using by measuring the volume of oxygen it is taking in and the volume of carbon dioxide it is giving out. Respiratory quotient, RQ = volume of CO2 given out / volume of O2 taken in - We can calculate the RQ from the balanced equation for the respiration of the substrate. The number of molecules of oxygen used and carbon dioxide produced are in the same ratio as the volumes of these two gases. For example: 2C18H36O2+5002 → 36CO2 + 34H2O RQ = 36 / 50 = 0.7 - The RQ values in Table 12.3 are for aerobic respiration. If a cell or an organism is respiring in anaerobic conditions, then no oxygen is being used. The RQ is therefore Infinity (∞). ### Key Terms Respiratory quotient, RQ is the volume of carbon dioxide given out divided by the volume of oxygen taken in. | Respiratory Substrate | RQ | |-----------------------|---| | Carbohydrate | 1.0| | Lipid | 0.7| | Protein | 0.9| ### Now Test Yourself 7 A locust produces 3.5 cm³ of carbon dioxide and uses 5 cm³ of oxygen. a. Calculate the RQ and suggest what substrate is being used. b. Suggest what would happen to the RQ if no oxygen was present. ### Skills Focus #### Using Respirometers A respirometer is a piece of equipment that is used to measure the rate of oxygen absorption by respiring organisms. There are different types of respirometer. One type is shown in Figure 12.11. #### Using a Respirometer to Measure the Rate of Uptake of Oxygen - The organisms to be investigated are placed in one tube, and non-living material of the same mass in the other tube. - Soda lime is placed in each tube, to absorb all carbon dioxide. Cotton wool prevents contact of the soda lime with the organisms. - Coloured fluid is poured into the reservoir of each manometer and allowed to flow into the capillary tube. It is essential that there are no air bubbles. You must end up with exactly the same quantity of fluid in the two manometers. - Two rubber bungs are now fitted with tubes, as shown in Figure 12.11. Close the spring clips. - Attach the manometers to the bent glass tubing, ensuring an airtight connection. Next, place the bungs into the tops of the tubes. - Open the spring clips (this allows the pressure throughout the apparatus to equilibrate with atmospheric pressure). Note the level of the manometer fluid in each tube. - Close the clips. - Each minute, record the level of the fluid in each tube. #### How the Respirometer Measures the Rate - As the organisms respire, they take oxygen from the air around them and give out carbon dioxide. - The removal of oxygen from the air inside the tube reduces the volume and pressure, causing the manometer fluid to move towards the organisms. - The carbon dioxide given out is absorbed by the soda lime. The distance moved by the fluid is therefore affected only by the oxygen taken up and not by the carbon dioxide given out. #### Now Test Yourself 8 Predict and explain what would happen to the levels of fluid in the manometers if no soda lime was used. #### The Control Tube - You would not expect the manometer fluid in the tube with no organisms to move, but it may do so because of temperature changes. - This allows you to control for this variable, by subtracting the distance moved by the fluid in the control manometer from the distance moved in the experimental manometer (connected to the living organisms), to give you an adjusted distance moved. #### Calculating the Oxygen Consumption - Calculate the mean (adjusted) distance moved by the manometer fluid per minute. - If you know the diameter of the capillary tube, you can convert the distance moved to a volume: volume of liquid in a tube = length × πr² - This gives you a value for the volume of oxygen absorbed by the organisms per minute. #### Using a Respirometer to Investigate the Effect of Temperature on the Rate of Respiration - The respirometer can be placed in water baths at different temperatures. As living organisms are being used, you should not expose them to temperatures that are high enough to harm them. You can use the same respirometer for the whole experiment. Or you could have different ones for each temperature. - At each temperature, you need a control respirometer with no organisms in it. - If you are simply comparing the rates of respiration at different temperatures, then you do not need to convert the distance moved by the manometer fluid to a volume. You could just plot distance moved on the y-axis of your graph and time on the x-axis. - The rate of respiration is represented by the gradient of the graph. #### Figure 12.12 Comparing rates of respiration at different temperatures - Distance moved by manometer fluid/mm - 50°C - 30°C - 20°C - Time/s - At 50°C the manometer fluid travelled 47 mm in 50 s: rate of respiration = 47/50 = 0.94 mm s-1 - At 30°C the manometer fluid travelled 40 mm in 60s: rate of respiration = 40/60 = 0.67 mm s-1 - At 20°C the manometer fluid travelled 21 mm in 70 s: rate of respiration = 21/70 = 0.30 mm s-¹ #### Using a Respirometer to Measure RQ - To measure RQ using a respirometer we need to know both how much oxygen is taken in, and how much carbon dioxide is given out. - Set up two respirometers. - The second respirometer should also contain the same mass of live maggots (or whatever organism you are investigating) but should not contain soda lime. - You could put some inert material into the tube (for example, beads) instead of soda lime. The mass and volume of the inert material should be the same as the mass and volume of the soda lime. - The second tube is just like the first one except that it does not contain soda lime. The carbon dioxide given out by the respiring organisms is therefore not absorbed. - The difference between the distance moved by the manometer fluid in the experimental tube and the distance moved in the control tube is due to the carbon dioxide given out. distance moved by fluid in experimental tube = x mm distance moved by fluid in control tube = ymm xmm represents the oxygen taken up. x -y represents the carbon dioxide given out. Therefore: RQ = (x−y) / X - For example, if the respiratory substrate is carbohydrate, then the amount of carbon dioxide given out will equal the amount of oxygen taken in. The fluid in the control tube will not move, so y = 0. In this case: RQ = (x-0) / x = 1 #### Skills Focus #### Using a Redox Indicator to Investigate Rate of Respiration in Yeast - Respiration involves stages in which hydrogen is removed from a substrate, a process called dehydrogenation. - We can investigate the rate of dehydrogenase reactions using an indicator that can accept the hydrogens removed in respiration, and that changes colour when it is reduced (has hydrogens added) or oxidised (has hydrogens removed). - Suitable indicators include methylene blue and DCPIP. Both of these indicators are blue when oxidised, and become colourless when they are reduced. - Place a suspension of yeast in glucose solution into two boiling tubes - To one of the tubes, add your indicator. - Note the time taken for the indicator to change colour. As the yeast suspension is not colourless, it is helpful to compare the colour of the tube with the indicator with the tube to which you did not add indicator. - You can use this technique to investigate the effect of temperature or substrate concentration on the rate of respiration. - To vary temperature, use water baths. - To vary substrate concentration, change the amount of glucose in the yeast suspension. - Measure the time taken for the indicator to become colourless. #### Key Terms A redox indicator is a substance that changes colour when it is oxidised or reduced. #### Now Test Yourself 9 Suggest what factors will need to be kept constant when measuring the effect of temperature on the rate of decolourisation of DCPIP by yeast. ## 13 Photosynthesis ### Now Test Yourself 1 Where in a chloroplast do the light-dependent and light-independent stages take place? ### Revision Activity Compare the structures of a mitochondrion and a chloroplast. ### Chloroplast Pigments A pigment is a substance that absorbs light of some wavelengths but not others. The wavelengths that it does not absorb are reflected from it. Chloroplasts contain several different pigments. - Chlorophyll is the main pigment contained in chloroplasts. It looks green because it reflects green light. Other wavelengths (colours) of light are absorbed. There are two types of chlorophyll, called chlorophyll a and chlorophyll b. - Figure 13.3 shows the wavelengths of light absorbed by three of the pigments found in chloroplasts. These graphs are called absorption spectra. > Amount of light absorbed > Key >> Chlorophyll a >> Chlorophyll b >> Carotene > 400 500 600 700 > Wavelength of light/nm > Figure 13.3 Absorption spectra for chloroplast pigments - If we shine light of various wavelengths on chloroplasts, we can measure the rate at which they give off oxygen. The resulting graph is called an action spectrum. #### Now Test Yourself 2 Explain the similarities between an absorption spectrum and an action spectrum. ### Skills Focus #### Separating Chlorophyll Pigments by Chromatography Chromatography is a method of separation that relies on the different solubilities of different solutes in a solvent. #### Method - A mixture of chlorophyll pigments is dissolved in a solvent. - A small spot is placed onto chromatography paper. - The solvent gradually moves up the paper, carrying the solutes with it. The more soluble the solvent, the further up the paper it is carried. - There are various methods. The one described in Figure 13.5 uses thin-layer chromatography on specially prepared strips instead of paper. Only an outline of the procedure is given here, so you cannot use these instructions to actually carry out the experiment. You can find more details about this technique on the Science and Plants for Schools website, student sheet 10: - https://tinyurl.com/SAPS-chromatography #### Figure 13.5 Thin-layer chromatography - Cut a TLC plate into narrow strips, about 1.25 cm wide, so they fit into a test tube. Do not put your fingers on the powdery surface. - Put 2 or 3 grass leaves on a slide. Using another slide scrape the leaves to extract cell contents. - Add 6 drops of propanone to the extract and mix. - Transfer the mixture to a watch glass. Allow this to dry out almost completely a warm air flow will speed this up. - Transfer tiny amounts of the concentrated extract onto a spot 1 cm from one end of the TLC strip. Touch very briefly with the fine tip of the brush and let that spot dry before adding more. Keep the spot to 1 mm diameter if you can. The final spot, called the origin, should be small but dark green. - Put the TLC strip in a test tube. Mark the tube below the pigment spot and remove the TLC strip. - Add the running solvent to the depth of the mark, then return the TLC strip to the tube and seal it. #### Calculating Rf Values - Measure the distance from the start line to the solvent front. - Measure the distance of each pigment spot from the start line. - For each spot, calculate the Rf value: Rf = distance from start line to pigment spot / distance from start line to solvent front - You can use the Rf values to help you to identify the pigments. Rf values differ depending on the solvent you have used, but typical values might be: > Chlorophyll a >> 0.60 > Chlorophyll b >> 0.50 > Carotene >> 0.95 > Xanthophyll >> 0.35 - You may also see a small grey spot with an Rf value of about 0.8. This is phaeophytin, which is not really a chlorophyll pigment, but is a breakdown product generated during the extraction process. #### Now Test Yourself 3 Red light does not penetrate far through water. Suggest why deep-sea seaweeds have additional accessory pigments. ### The Light-Dependent Stage The light-dependent stage of photosynthesis is the series of events that only occurs when light is present. It involves the use of energy from light to split water molecules and produce ATP and reduced NADP. There are two reaction pathways that occur as part of the light-dependent reaction, called non-cyclic photophosphorylation and cyclic photophosphorylation. #### Non-Cyclic Photophosphorylation - Chlorophyll molecules in PSI and PSII absorb light energy. The energy excites electrons, raising their energy level so that they leave the chlorophyll. The chlorophyll is said to be photoactivated. - PSII contains an enzyme in an oxygen-evolving complex that splits water when activated by light. This reaction is called photolysis ('splitting by light'). - The water molecules are split into oxygen and hydrogen atoms. - Each hydrogen atom then loses its electron to become a positively charged hydrogen ion (proton), H+. - The electrons from the hydrogen are picked up by the chlorophyll in PSII, to replace the electrons they lost. - The oxygen atoms from the water join together to form oxygen molecules, which diffuse out of the chloroplast and into the air around the leaf. 2H₂O light →4H+ + 4e + O2 - The electrons emitted from PSII are picked up by electron carriers (electron transport chain) in the membranes of the thylakoids. - Similarly to oxidative phosphorylation in mitochondria, as the electrons move along the chain they release energy. - This energy is used to actively transport protons (hydrogen ions) across the thylakoid membrane into the space between the membranes. - A high concentration of protons builds up in the thylakoid space. - The protons are allowed to move back by facilitated diffusion into the stroma from the thylakoid space through ATP synthases, by chemiosmosis. This provides energy to cause ADP and inorganic phosphate to combine to make ATP. This is called photophosphorylation. - At the end of the electron carrier chain the electrons are picked up by PSI to replace the electrons the chlorophyll in PSI had lost. - The electrons from PSI are passed along a different chain of carriers to NADP. - The NADP also picks up the protons (hydrogen ions) from the split water molecules. The NADP becomes reduced NADP. #### Key Terms The light-dependent stage of photosynthesis is the series of events that only occurs when light is present. It involves the use of energy from light to split water molecules and produce ATP and reduced NADP. #### Figure 13.6 Summary of the light-dependent stage of photosynthesis - the Z-scheme > Photosystem >> High energy electron >>> e >>> Chain of electron carriers > Photosystem >> || >>> ADP + Pi >>> e >>> ATP > Energy level >> H2O >>> e >>> O2 >>> H+ >> Light >>> e >>> Chain of electron carriers >> Oxidised NADP >>> + H+ >>> e >>> Light >>> Reduced NADP - At the end of this process, two new substances have been made. These are ATP and reduced NADP. Both of these will now be used in the next stage of photosynthesis - the light-independent stage. #### Now Test Yourself 4 The Z-scheme shows that electrons lose energy as they pass along the chain of electron carriers. Where does this energy go? 5 List the products of the light-dependent stage of photosynthesis. ### Cyclic Photophosphorylation There is an alternative pathway for the electron that is emitted from PSI. - The electron can simply be passed along the electron transport chain, then back to PSI again. - ATP is produced as it moves along the electron transport chain (photophosphorylation). - However, no reduced NADP is produced. This is called cyclic photophosphorylation. ### The Light-Independent Stage The light-independent stage of photosynthesis is a series of reactions that can take place even when light is not present. It uses ATP and NADP from the light-dependent stage to synthesise carbohydrates from carbon dioxide. - The light-independent stage is made up a cycle of reactions known as the Calvin cycle. - It takes place in the stroma of the chloroplast, where the enzyme ribulose bisphosphate carboxylase, usually known as rubisco, is found. #### Study Tip Do not call the light-independent stage 'the dark stage'. Although this stage does not need light, it can take place in the light. - Carbon dioxide diffuses into the stroma from the air spaces within the leaf. - The carbon dioxide enters the active site of rubisco, which combines it with a 5C compound called ribulose bisphosphate, RuBP. The products of this reaction are two 3C molecules of glycerate 3-phosphate, GP. - The combination of carbon dioxide with RuBP is called carbon fixation. #### Figure 13.7 The Calvin cycle. > Ribulose bisphosphate (RuBP) >> ADP >> ATP >>> 2 x Triose phosphate (TP) >>>> 000 > CO2 >> Rubisco >>> Intermediate compound >>>> 00000 >>> 2 x Glycerate 3-phosphate (GP) >>>> 000 > Triose phosphate >> can be used to make >>> glucose, other >>>> carbohydrates, >>>>> lipids or amino acids. > ADP + Pi >> ATP > NADP > Reduced NADP #### Now Test Yourself 6 What are the substrate and product of rubisco? 7 What are the roles of ATP and reduced NADP in the Calvin cycle? ### 13.2 Limiting Factors in Photosynthesis The rate at which photosynthesis takes place is directly affected by several environmental factors. - **Light intensity.** This affects the rate of the light-dependent stage, because this is driven by energy transferred in light rays. - **Temperature.** This affects the rate of the light-independent stage. At higher temperatures, molecules have more kinetic energy and so collide more often and are more likely to react when they do collide. (The rate of the light-dependent stage is not affected by temperature, because the energy that drives this process is light energy, not heat energy.) - **Carbon dioxide concentration in the atmosphere.** Carbon dioxide is a reactant in photosynthesis. Normal air contains only about 0.04% carbon dioxide. - **Availability of water.** Water is a reactant in photosynthesis, but there is usually far more water available than carbon dioxide, so even if water supplies are low this is not usually a problem. However, water supply can affect the rate of photosynthesis indirectly, because a plant that is short of water will close its stomata, preventing carbon dioxide from diffusing into the leaf. #### Figure 13.8 Limiting factors for photosynthesis > Rate of photosynthesis >> Over this range of the graphs, >>> light intensity is the limiting >>>>> factor. If light intensity >>>>>>> increases, then the rate of >>>>>>>> photosynthesis increases. >> Over this range of the graph, light >>> intensity is not a limiting factor. If light >>>>> intensity increases, there is no effect >>>>>>> on the rate of photosynthesis. Some >>>>>>>> other factor is limiting the rate. >> X >> High CO2 >>> concentration >> Lower CO2 >>> concentration >>> Here, carbon dioxide is the >>>> limiting factor. We can tell >>>>> this is so because when the >>>>>>> concentration of carbon >>>>>>>> dioxide is increased, the >>>>>>>>> rate of photosynthesis >>>>>>>>>> increases (see top curve). >> Light intensity #### Key Terms A limiting factor is a factor that, when in short supply, limits the rate of a reaction or process. #### Now Test Yourself 8 What factors are limiting the rate of photosynthesis at the point labelled X in Figure 13.8? ### Skills Focus #### Investigating the Effect of Environmental Factors on the Rate of Photosynthesis of Whole Plants - One way to measure the rate of photosynthesis is to measure the rate at which oxygen is given off by an aquatic plant such as Elodea or Cabomba. There are various ways in which oxygen can be collected and measured. One method is shown in Figure 13.9. #### Figure 13.9 Apparatus for measuring the rate of photosynthesis - Oxygen - the length of this bubble, collected over a measured time, represents the rate of photosynthesis. - Capillary tube - If bubbles need to be cleared from the tube, this reservoir provides water to do this. - Oxygen collects in the flared end of the capillary tube over a measured length of time. - A healthy, photosynthesising water plant has its stem cleanly cut under water so that bubbles of oxygen can be released during photosynthesis. - The three-way tap is turned so that a connection is made between the syringe and the capillary tube (OFF up). The syringe is very carefully used to pull the oxygen, collected above the plant, into the capillary tube. The collection time is noted and the length of bubble is measured. - Alternatively, you can make calcium alginate balls containing green algae and place them in hydrogencarbonate indicator solution. - As the algae photosynthesise they take in carbon dioxide, which causes the pH around them to increase. The indicator changes from orange, through red to magenta. - You can find details of this technique on the Science and Plants for Schools website, student sheet 23: - https://tinyurl.com/SAPS-alginate - Whichever technique is used, you should change one factor (your independent variable) while keeping all others constant (the standardised variables). - The dependent variable will be the rate at which oxygen is given off (measured by the volume of oxygen collected per minute in the capillary tube) or the rate at which carbon dioxide is used (measured by the rate of change of colour of the hydrogencarbonate indicator solution). - You could analyse your results by carrying out a statistical test, such as Spearman's rank correlation, to see if there is a significant correlation between light intensity and rate of photosynthesis. This is outlined in the chapter on A Level experimental skills and investigations (pp. 222-223). - You could investigate the following independent variables: - **Light intensity.** You can vary this by using a lamp to shine light onto the plant or algae. The closer the lamp, the higher the light intensity. The relationship between light intensity and the distance of the lamp from the aquatic plant is not a simple, linear one. The light intensity is inversely proportional to the square of the distance. You can calculate the light intensity by using the formula: light intensity = 1/distance² - This means that you can plot a graph of photosynthesis rate against distance, or a graph of photosynthesis rate against the light intensity. The two graphs will have different shapes. - **Wavelength of light.** You can vary this by placing coloured filters between the light source and the plant. Each filter will allow only light of certain wavelengths to pass through. #### Study tip - It is not easy to vary light intensity without also varying temperature, because the light from a lamp also heats the water. You can try putting a piece of transparent plastic between the light and the water. - Filters of different colour can affect light intensity, so make sure that you consider this when changing the wavelength of light. - **Carbon dioxide concentration.** You can vary this by adding sodium hydrogencarbonate to the water around the aquatic plant. This contains hydrogencarbonate ions, which are used as a source of carbon dioxide by aquatic plants. - **Temperature.** The part of the apparatus containing the plant or algae can be placed in a water bath at a range of controlled temperatures. ### Skills Focus #### Investigating the Rate of Photosynthesis Using a Redox Indicator - Photosynthesis, like respiration, involves the acceptance of hydrogen by a coenzyme. This occurs during the light-dependent stage, when hydrogen is accepted by NADP. We can investigate the rate at which this occurs by adding a redox indicator, such as DCPIP, to a suspension of chloroplasts. - The indicator takes up the hydrogen ions that are produced as the light-dependent stage occurs in the chloroplasts, and loses its colour. This is called the Hill reaction. - The rate at which the colour is lost is determined by the rate of the light-dependent stage. - You can find a detailed protocol for carrying out this investigation on the Nuffield Foundation website: - https://tinyurl.com/Nuffield-photosynthesis - You can use this technique to investigate the effect of light intensity or light wavelength on the rate of photosynthesis. - See above for methods of altering these two independent variables. You will also need to consider how to standardise different factors and how to ensure that the chloroplasts are not damaged due to high temperatures and osmotic effects. #### Now Test Yourself 9 A student investigates the effect of light wavelength on photosynthesis in a piece of pond weed. What factors would need to be standardised and how could this be achieved? ### End of Chapter Check By the end of this chapter, you should be able to: - describe the relationship between the structure of chloroplasts, as shown in diagrams and electron micrographs, and their function - explain that energy transferred as ATP and reduced NADP from the light-dependent stage is used during the light-independent stage (Calvin cycle) of photosynthesis to produce complex organic molecules - state that, within a chloroplast, the thylakoids, which occur in stacks called grana, are the site of the light-dependent stage and the stroma is the site of the light-independent stage - describe the roles of chlorophyll a, chlorophyll b, carotene and xanthophyll in light absorption - interpret absorption spectra of chloroplast pigments and action spectra for photosynthesis - describe and use chromatography to separate and identify chloroplast pigments - state that cyclic photophosphorylation and non-cyclic photophosphorylation occur during the light-dependent stage of photosynthesis - explain how ATP and reduced NADP are synthesised in non-cyclic photophosphorylation and how ATP is synthesised in cyclic photophosphorylation, including the roles of PSI, PSII, the electron transport chain, the oxygen-evolving complex and the process of chemiosmosis - outline the main stages of the Calvin cycle, including the combination of RuBP with carbon dioxide to form GP, the reduction of GP to TP using reduced NADP and ATP, and the regeneration of RuBP from TP using ATP - state that Calvin cycle intermediates are used to produce other molecules, such as carbohydrates, lipids and amino acids - state that light intensity, carbon dioxide concentration and temperature are examples of limiting factors of photosynthesis - explain the effects of changes in light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis - describe and carry out investigations using redox indicators and a suspension of chloroplasts to determine the effects of light intensity and light wavelength on the rate of photosynthesis - describe and carry out investigations using whole plants, including aquatic plants, to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis