Metabolism in an Isolated Organelle


  1. To demonstrate the light requirement for generating reducing power by isolated chloroplasts.
  2. Show the site of action of two herbicides which inhibit electron transport
  3. To become familiar with and use phase microscopy and a spectrophotometer to examine the integrity of isolated chloroplasts and to measure absorption levels.
  4. Background Knowledge:

    There are several forms of chlorophyll, differing slightly in colour, chemical structure and absorption peaks. Carotenoids are hydrocarbons situated close to the chlorophyll. The table below shows the nature and occurrence of these pigments.

    The absorption spectrum is a graph that shows how much light a particular pigment absorbs at each wavelength. It is made by subjecting selections of each pigment to different wavelengths of light and measuring how much light is absorbed.


    This practical was carried out with the use of chloroplasts that were obtained from pea leaves. These were provided to us in the laboratory.

    Chloroplastsareorganelles that arefound inplant cellsand othereukaryotic organisms that conductphotosynthesis. Chloroplasts captureUV light energy to conservefree energywhich is in the form ofATPthrough photosynthesis in different stages.

    1. The Light dependent stage, this occurs on the thylakoid membranes. Photosensitive pigments are organized into Photosystems on the thylakoid membranes.

    a. There are two Photosystems:

    1. Photosystem I is smaller and is found on the stromal thylakoids ( intergranal thylakoid \ Lamellae )
    2. Photosystem II is larger and associated with granal thylakoids. Both Photosystems are visible as particles on the thylakoid membranes.

    When the chlorophyll-a molecule at the reaction centre receives light energy, the electrons within the molecule gets excited to form high energy electrons. These electrons are then emitted by the chlorophyll molecule which are then are then taken up by electron carriers and passed onto other molecules.

    This is the first step in the conversion of light energy into chemical energy; the energy from these high energy electrons is then used to synthesize ATP.

    This process is called photophosphorylation. There are two different ways in which ATP can be synthesized by phosphorylation:

    1. Non-cyclic photophosphorylation
    2. Cyclic photophosphorylation

    Non-cyclic photophosphorylation.

    1. Light is absorbed by Photosystem II and passed onto chlorophyll a (P680).
    2. The energized chlorophyll a (P680) molecule emits two electrons. These high energy electrons are raised to a higher energy level and are picked up by an electron acceptor.
    3. The electron acceptor passes the electrons along a chain of electron carriers to Photosystem I. The energy released from the electrons is used to make ATP from ADP + Pi.
    4. Light is also absorbed by Photosystem I and passed on to chlorophyll a (P700). Chlorophyll a (P700) also emits 2 electrons.
    5. The energized electron rise to a higher level and are picked up by a second electron acceptor.
    6. Since both chlorophylls (P680 and P700) have now lost electrons, they will both be positive and unstable.
    7. The 2 electrons released from chlorophyll a (P680) of PS II go to replace the two that have been lost by chlorophyll a (P700) of PS1.
    8. P680 of PSII receives its replacement electrons from the splitting of water (Photolysis). During photolysis, the water molecule dissociates into electrons, hydrogen ions and oxygen. The electrons go to Photosystem II. The oxygen is released as a waste gas.
    9. The hydrogen ions combine with electrons held by the second electron acceptor to give NADPH. This passes to the reactions of the light independent stage.
    10. So. The products of the light dependent stage are NADPH, ATP and waste oxygen.

    Cyclic Photophosphorylation

    1. This process only involves PSI
    2. Light is absorbed by PSI, The chlorophyll-a molecule at the reaction centre receives light and passed onto chlorophyll a (P700).
    3. This causes the chlorophyll molecule to emit one electron.
    4. The 'energized' electron is raised to a higher energy level and is picked up by an electron acceptor.
    5. The electron is then passed along a chain of electron carriers before it is returned to the chlorophyll a molecule.
    6. As the electron passes along the electron carrier chain, enough energy is released to make ATP from ADP and Pi.
    7. This ATP is used in the light independent stage. No NADPH is produced in cyclic photophosphorylation.

    The light independent stage (Takes place in the stroma of chloroplast).

    This is a cyclic pathway often referred to as the Calvin cycle. CO2 combines with a fine carbon compound RUBP. This reaction is catalysed by the enzyme RUBP carboxylase (RUBISCO), which is the most abundant enzyme on earth. An unstable six-carbon compound is formed which breaks up to form two molecules of GP.

    ATP ( From light dependent stage ) is used to phosphorylate the two molecules of GP to produce two molecules of glycerate biphosphate. NADPH is then used to reduce each molecule of glycerate biphosphate to glyceraldehyde-3-phosohate (GALP).

    For every six molecules of GALP formed, five and converted into RUBP, through a series of reactions. One of the six GALP molecules is converted into glucose, other carbohydrates and lipids.

    Herbicides Used:


    Paraquat(N,N'-dimethyl-4,4'-bipyridinium dichloride) is one of the most widely used herbicides.

    Paraquat is quick-acting and non-selective, killing green plant tissue on contact. It is also toxic to human beings when swallowed. Paraquat also more importantly inhibits photosynthesis which then in turn leads to the death of the plant species it is being used on.

    How it Works:

    1. In light-exposed plants, it accepts electrons fromphotosystem Iand transfers them to molecular oxygen.
    2. In this manner, destructivereactive oxygen speciesare produced. In forming these reactive oxygen species, the oxidized form of paraquat is regenerated, and is again available to shunt electrons fromphotosystem Ito start this cycle again.
    3. DCMU

      Another herbicide that is quite well known is DCMU, a herbicide known as diuron, blocks electron transport on the acceptor side of PSII

      How it Works:

      DCMU is a very specific and sensitive inhibitor of photosynthesis. It blocks theplastoquinone binding site ofphotosystemII, disallowing the electron flow from where it is generated, in photosystem II, to plastoquinone interrupts the photosyntheticelectron transport chainin photosynthesis and thus blocks the ability of the plant to turn light energy into chemical energy (ATPandreductant potential).

      However, because it absorbs electronsoxidizedfrom water inPS II, the electron "hole" ofPS I cannot be satisfied, effectively shutting down photosynthesis by blocking the reduction of NADP+ to NADPH, and the cyclic photosynthetic pathway since electron shuttling is associated with proton pumping across the membrane into thelumen.

      Structures of the Herbicides

      The use of herbicides to kill unwanted plants is widespread in modern agriculture. Many different classes of herbicides have been developed, and they act by blocking amino acid, carotenoid, or lipid biosynthesis or by disrupting cell division.

      Data Collected:


      When analysing graph 1 which is for the light dependent reaction, we are easily able to tell that as time in minutes increases the nMol reduced DCPIP also increases showing that a positive correlation in effect. Graph 2 for the light independent stage shows a similar positive correlation. However, from graph 2 we can see that the nMol of reduced DCPIP is significantly less when compared to the light dependent stage.

      This now allows us to make the statement that photosynthesis occurs much more efficiently in the light dependent stage as there more UV light energy present for the chloroplasts to absorb.

      The results obtained were taken from a suspension constitution that was 60% intact and therefore there was more than sufficient amounts of chloroplasts available to photosynthesize. When analysing the electron micrograph of the suspension that was going to be used in the investigation stroma and thylakoids were both able to be seen, this allows us to say that both the stages of photosynthesis was able to take place. After looking at the results gained and plotting the graphs we can clearly see that the rate of photosynthesis was much greater in the light dependent stage.

      When looking at graph 3 which contained the paraquat. This is a common herbicide. The only way for the DCPIP to have been reduced was only if the Paraquat released electrons by Photosystem II.

      Paraquat effects only photosystem I- it prevents electrons which in turn stops NADPH from being reduced, therefore the final stage of the light dependent reaction also known as the Calvin cycle cannot occur..

      You can notice a slight plateau between 7 and 10 minutes this can be for a number of different reasons mainly due to the law of limiting factors which states that when a process is influenced by several factors, the rate at which the process proceeds is determined by the factor in the shortest supply.

      CO2 is needed in the light independent stage of photosynthesis (Calvin cycle).

      The CO2 concentration of the atmosphere is about 0.035% or 350ppm (parts per million) by volume. This is far less than the optimum CO2 concentration for photosynthesis. Thus CO2 acts as a limiting factor. It has been shown that by increasing the CO2 concentration in greenhouses has increased the yield of tomatoes and lettuces.

      However it is important to note that, prolonged exposure to CO2 concentration of above 0.5% can cause closure of stomata.

      Temperature affects the enzymes involve in the Calvin cycle, thus it influences the rate of photosynthesis. If other factors are not limiting then a 10C temperature (Within the range 10-35C) will lead to a doubling of the rate of photosynthesis.

      Usually a temperature of about 25C is considered as the optimum temperature for photosynthesis.

      The results from the herbicides and photosynthesis reactions show that as time increases the reduced DCPIP for both Diuron and paraquat increases. The herbicide DCMU as previously discussed works by blocking theplastoquinone binding site ofphotosystemII, disallowing the electron flow from where it is generated, in photosystem II, to plastoquinone interrupts the photosyntheticelectron transport chainin photosynthesis and thus blocks the ability of the plant to turn light energy into chemical energy.

      No reduction is taking place this can be seen in graph 4, where the rate of DCPIP reduction is less than the reduction which occurred in darkness. The chloroplasts are being prevented from receiving any ATP. This in turn inhibits the light independent stage of the reaction therefore preventing photosynthesis.

      Sources Used

      4. Edexcel biology A2 Level the Complete Study Guide
      5. Lecture notes
      6. Life the Science of Biology - Sadava et al
      7. Other notes and learning materials

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