Investigating how light intensity will affect the rate of photosynthesis, with the help of DCPIP, in Spinach leaves.
The aim of this experiment is to find out how the rate of change of light intensity affect the photosynthesis in spinach leaves.
Successful farming of food crops is very essential for both the nutritional and financial health of a nation. Governments of almost every nation invest land and labour to crop farming, as much as is possible, keeping in mind the other needs of a country. But, however, due to many reasons the maximum crop yield possible is not always achieved.
For the production of a healthy crop certain conditions are necessary. The primary among these conditions are soil quality, pH balance of the soil, humidity and precipitation of the area, exposure to sunlight etc. The intensity of light available to the plant is also another important parameter. Plants undergo photosynthesis for their survival, where in the presence of sunlight ATP (Adenosine tri- phosphate) and NADPH (reduced Nicotinamide Adenine Dinucleotide phosphate) are produced from Carbon dioxide and water in the first phase. These products helps in carbon fixation in the second phase, as a result of which sugars are produced in the plant. These sugar molecules provide the nutrition required by the plant to grow.
If we go into a little more detail of this phenomena we will find that, it all starts with a chlorophyll molecule which absorbs and gets excited. It releases an electron which is passed through electron carriers to water(terminal electron carrier), bringing about the photolysis of water, producing H+ and oxygen. This H+ brings about the reduction of NADP (Nicotinamide Adenine Dinucleotide phosphate) to NADPH.
The intensity of light can be defined as the number of photon available to unit area of a surface per unit time. Thus as the intensity of light, meaning the number of photon available, increases more number of chlorophyll molecules can be excited. Until it reaches a saturation point where all the available chlorophyll molecule are already excited. If the intensity of light is increased beyond this saturation point no change in the number of excited chlorophyll molecules occur as all the available molecules were already excited. Maximum number of chlorophyll molecules excited means the plant is undergoing maximum possible rate of photosynthesis. Which, basically, means that the plant is producing maximum amount of food possible, for itself which finally amounts to maximum growth and thus yield of the food crop.
Thus if we can optimize the intensity of sunlight to its saturating level we can assure maximum yield of the crop, keeping other conditions appropriate. It is possible to achieve different intensity of light by changing the distance of the light source from the absorbing surface. As the energy of photons emitted by a light source decreases the more distance it travels, so decreasing the distance of the light source from the absorbing surface increases the intensity and vice versa.
DCPIP (Dichlorophenolindophenol) is a redox dye, and the rate of change of its colour amounts to the rate of photosynthesis. Thus by changing the distance of light source from the absorbing surface and recording the rate of change colour of DCPIP, we can find out the distance at which the rate of photosynthesis is the highest.
Photosynthesis is the process by which plants and other organisms convert sunlight energy into chemical energy in the presence of chlorophyll, a green photosynthetic pigment present in chloroplasts (Rastogi 40). Chloroplasts are double membraned cellular machinery (Thorpe 719) Plants are autotrophs, i.e. they can synthesise their own food from raw inorganic materials using sunlight's energy. Since light energy is utilized for synthesis of biomolecules, it is called as photosynthesis. Carbon dioxide and water are examples of inorganic materials used, which with a help of a chain reaction helps to ultimately produce organic molecules like glucose and ATP (also called as energy currency of the cell) which are then used to synthesise structural and functional biomolecules such as fats and proteins.
Photolysis occurs in which oxygen is produced as a by-product while the splitting of water in the presence of light. Plants are autotrophs, i.e. they can produce their own food (Singh 69). Autotrophs are also called as producers and they form the first trophic level. Thus this biological process is essential and vital to all life processes. Without producers, the whole ecosystem will collapse as organisms require oxygen in order to carry out respiration. Organisms that cannot synthesise their own food are known as heterotrophs. Such Organisms depend on other organisms in order to sustain their life. Herbivores rely on plants for food and carnivores rely on herbivores, in order to carry out metabolic activities. Herbivores are the primary consumers and carnivores are thus secondary consumers.
Photosynthesis has two phases. The first phase is light dependent called Light reaction and the other is independent of the light, known as Dark reaction.
Photosynthesis occurs in the chloroplasts of plants which contain chlorophylls that absorb light energy to synthesise carbon dioxide and water into glucose and oxygen. Chlorophyll is found within the thylakoid membranes which are stacked up to form granum. We can see that stacks of granum are joined by a structure called lamella and surrounded by a fluid called the stroma (see Fig. 1).
There are two stages of photosynthesis: The light dependent stage and the light independent stage. The thylakoid membrane is the site of the light dependent stage in which light energy is required in order for photosynthesis to occur. The stroma is the site of the light independent stage, in which light energy is required indirectly. The thylakoid membranes are made up of photosystems I and II which are protein complexes that absorb light energy (6). Photosystems contain a reaction center which consists of primary pigments: forms of chlorophyll a molecules and accessory pigments such as chlorophyll b, cartenoids and xanthophyll in different amounts. Accessory pigment captures light energy and passes it to the primary pigment (Bryne 1994). Photosystem I, also known as P700, contains chlorophyll and other pigments which absorbs red light at a wavelength of 700 nanometers (nm) and with photosystem two, also known as P680, absorbs blue-green light at a wavelength of 680nm (Ibim 133).
As light energy is absorbed by the chlorophylls, energy levels of electrons in photosystems one and two increases which triggers them to become ‘excited’ and accepted by the first electron carrier, Plastoquinone, of the electron transport chain. Here, electrons reduce Plastoquinone to Plastocyanin, catalysed by cytochrome b6f, an enzyme present in the thylakoid membranes (Singh 69). At the same time, photolysis occurs in which water is split into electron, H+ ion and oxygen is released as by product (see Fig. 2). The electrons from the photosystems are replaced by the electrons from photolysis. Thus, the complex is returned to the original state for the next cycle of events.
As electrons are passed down the electron transfer chain, a series of redox reaction occurs in which energy is released at each transfer. Electrons are accepted by cytochrome b6f, which is an electron carrier protein that pumps protons from the stroma across the thylakoid membrane and sets up a proton gradient. This creates a ‘proton-motive force’ (7) which is harvested to generate Adenosine tri- phosphate (ATP). ATP is generated when the protons are transported back to the stromal compartment down the proton gradient through ATP synthase. The process is called chemiosmotic synthesis of ATP. Chemiosmotic hypothesis was first postulated by Peter Mitchell in 1961. ATP synthase catalyzes the production of Adenosine triphosphate (ATP), by the addition of inorganic phosphate with ADP. This is known as photophosphorylation.
Electrons from Plastocyanin are accepted by photosystem I which are then accepted by the electron carrier, Ferredoxin, which carries electrons ‘to the enzyme Ferredoxin NADP+ oxidoreductase (FNR)’ (5). The electrons then combine with protons released from photolysis to reduce the co-enzyme, Nicotinamide Adenine Dinucleotide Phosphate (NADP) to form reduced NADP (NADPH). During cyclic photophosphorylation, electrons from Ferrodoxin are transferred to Plastoquinone, back to cytochrome b6f and returned to Plastocyanin to photosystem I. This cycle is continues on as electrons are constantly cycled in which ATP is synthesised from ADP and Pi (Byrne 1994). The light dependent stage within the thylakoid membranes in the chloroplast can be demonstrated by Figure 3:
The products of light reaction are NADPH and ATP which are then utilized in the light independent reaction to synthesize simple sugars. The thylakoid membrane electron transport is coupled to the dark reactions (Calvin cycle). NADPH provides the reducing power in the dark reaction. Calvin cycle cannot function without the products of Light dependent reaction and thus is dependent on the light dependent reaction which thus is rate limiting. Measuring the rate of light dependent reaction will provide the rate of photosynthesis.
Calvin cycle is a series of biochemical redox reactions that takes place in the stroma of the chloroplasts. Calvin cycle is the light independent reaction or dark reaction. Calvin cycle uses the energy of transient electronically excited charge carriers to synthesize organic compounds (simple sugars) from carbon dioxide and water.
Light intensity is one of the abiotic factors that affect the rate of photosynthesis. Light intensity is defined as energy per unit area per time. Thus in plants it is number of photons of certain energy hitting a leaf surface in unit time. Increasing Light intensity means increasing the photons that can excite chlorophyll molecule as light is used to excite chlorophyll molecules in the light reaction. This reaction initiates the electron transport which generates Adenosine Tri-Phosphate (ATP) and reduced Nicotinamide Adenine Dinucleotide phosphate (NADPH). These products of light reaction are then used in the Dark reaction to synthesize simple sugars. Rate of photosynthesis is directly dependent on the amount of chlorophyll molecules that are excited which then depends upon the intensity of light. Higher the intensity, more the number of excited chlorophyll molecule and thus higher the rate of photosynthesis. At lower light intensities, the rate of photosynthesis increases linearly with the increase in light intensity until a maximum rate is achieved. As it reaches a plateau, the rate of photosynthesis will not increase further. At this point all the chlorophyll molecules are saturated with the photons. Therefore there is no effect of increasing light intensity on the rate of photosynthesis. This is known as the ‘light saturation point’. Saturation is achieved also because other limiting factors such as carbon dioxide will be limiting and very high light intensity can cause sunburns in the plants.
The aim of the experiment as already stated is to find out the optimum light intensity that can maximise the rate of photosynthesis for maximum crop yield and faster production of spinach. To optimize that we need an efficient system to measure the rate of photosynthesis. Here, we use DCPIP (Dichlorophenolindophenol) to measure the rate of photosynthesis.
Dichlorophenolindophenol is a redox dye which exist in both reduced & oxidised state in two different colors. When oxidised it is blue in color with absorbance maxima at 600nm but it is colorless when reduced. It is a part of Hill's reagents family i.e. the artificial dyes that absorb differently when reduced or oxidised. These reagents helped in deciphering the electron transport chain (ETC) in plants.
Oxidised DCPIP reduced DCPIP
Dichlorophenolindophenol has higher reduction potential or higher affinity for electrons than ferrodoxin. Therefore it will get reduced by the electron instead of ferrodoxin (part of the electron transport chain). Reduction of Dichlorophenolindophenol by the electrons excited by chlorophyll molecules will change its color from blue to colorless. Change in color is directly related to the amount of electrons released from the chlorophyll molecules, or the amount of photosynthesis. The change in colour can be measured using spectrophotometer. Faster the depletion of color, greater is the reduction, i.e. greater electron transport and photosynthesis. This rate of change in absorbance at 600nm can be used to correlate the rate of photosynthesis with light intensity. DCPIP is also used to determine the level of Ascorbate in the plasma membrane (Vander Jagt 1986).
Thus measuring rate of change in the absorbance of DCPIP with varying light intensities can be used to observe the effect of light intensity on the rate of photosynthesis.
In this investigation, spinach has been chosen as the experimental model. Spinach is an edible flowering plant belonging to the Amaranthaceae family which is specifically grown in central Asia. It is a small plant ‘having a large, oval-shaped leaves’ (11) categorised in 3 main types: savoy (wrinkled), semi-savoy and smooth (Small 2009). Spinach is highly nutritious, being rich in vitamins A, antioxidants such as vitamins E and X, which are important in reducing cancer and heart disease (11) It also contains high levels of beta carotene, ‘the inactive stage of vitamin A’ (11). The main reasons behind choosing spinach as the experimental model are its easy availability and the big leaf surface which ensures the presence of a good quantity of easily extractable chlorophyll molecules.
Null hypothesis: There will be no significant correlation between varying the light intensity and the absorbance level of DCPIP.
Experimental hypothesis: There will be a significant correlation between varying the light intensity and the absorbance level of DCPIP.
Planning: This experiment aims to investigate how light intensity will affect the light dependent stage and at what point the light intensity will no longer be the limiting factor during photosynthesis. Thylakoid membranes will be isolated from chloroplasts in spinach leaves and combining it with DCPIP. During the light dependent stage, redox reactions occur in which elections are reduced and oxidized as it passes down the electron transport chain. Hence, a reducing agent is produced which results in the reduction of NADP. This is known as the Hill reaction, demonstrated by Robert Hill in 1939. He implied that light dependent reaction is a redox reaction series where an electron is shuttled from donor to final acceptor. The final acceptor of electron in the light dependent reaction is water. On accepting electron water is split into water and oxygen. This oxygen is released forming bubbles in aquatic autotrophs. The release of bubbles can also be used as an indicator of photosynthesis.
Here we have used thylakoid membranes which only have membrane bound proteins, rest of the soluble components is lost which limits the electron transport till the final acceptor molecule that is water. Thus we have used DCPIP molecule which will indicate the rate of photosynthesis because of its change in color by reduction.
The in vitro experiment of isolating thylakoid membranes in spinach leaves will provide us a further understanding of the light dependent stage by measuring the absorbance level of DCPIP as it slowly decolourises. DCPIP will be used as an artificial electron carrier of NADP+ that is normally present in the thylakoid membranes as it changes from blue to colourless when reduced. The effect of light intensity will be investigated by placing the cuvette filled with the chloroplast suspensions at placing it at different light intensities for 1 minute. Between each set, the cuvettes will be placed in a colorimeter and the absorbance level of the DCPIP will be measured. All other factors that is not measured or changed in this experiment will be controlled, which will allow the relationship between absorbance level and light intensity to be determined.
In my trial experiment, I will be testing to see if the values that I have used for my distance away from the cuvette will be suitable. Here, a bench lamp will be placed 15cm away from a test tube with each lasting a minute. The absorbance level of DCPIP will be measured every minute for 8 minutes using a colorimeter set at a wavelength of 620nm.
Independent variable: Light intensity by varying the distance of the bench lamp from test tubes containing the test solution.
Dependent variable: The absorbance level of DCPIP will be measured every minute for 8 minutes.
Control: One of the test tubes will be wrapped with aluminium foil to prevent light from entering as light energy is required to excite the electrons in the light dependent reaction.
Remove the midribs and stalk from spinach leaves, measure 30g of leaves using a balance and cut it into small pieces using scissors.
Place the cut spinach leaves in a blender and add 120 ml of isolation buffer to it. Blend for 10 seconds
Filter the extract into a beaker using a funnel and 6 layers of muslin cloth.
Pour the same volume of the filtrated extract into 6 pre-cooled 1.5ml centrifuge tubes placed in an ice-water-salt bath.
Place the tubes into a centrifuge and spin it at 300xg for 3 minutes to isolate the chloroplasts.
Only pour the supernatant (a liquid that contains free chloroplasts) into a separate centrifuge tubes. The pellet contains unbroken cells and fragments.
Centrifuge the supernatant again but this time at 1000xg for 7 minutes. A green pellet will then be formed which contains chloroplasts.
Pour the supernatants into a boiling tube and keep the green pellet.
Pipette 0.09ml of pre-chilled isolation buffer into the centrifuge tube with the green pellet and resuspend it by pipetting up and down to mix them together. This will be the chloroplast suspension and place back in ice-water-salt bath.
Using a marker pen, label 6 cuvette tubes 1 to 6.
Set the colorimeter at a wavelength of 620nm. Tube 1 will be used to set blank for the colorimeter containing 0.06ml of chloroplast suspension, 0.09ml of isolation buffer and 0.03ml of cold distilled water. Transfer the content in a cuvette. Calibrate the colorimeter to read Tube 1 as baseline/blank.
Tube 2 will be used as a control which will contain 0.06ml of chloroplast suspension, 0.09ml of isolation buffer, 0.03ml of cold distilled water and 0.03ml of DCPIP. Transfer the content in a cuvette, place in the colorimeter and record the absorbance level. Then wrap aluminium foil around tube 2 to prevent light from entering. Keep it wrapped in aluminium foil for the rest of the experiment.
Tube 3 will contain 0.06ml of chloroplast suspension, 0.09ml of isolation buffer, 0.03ml of cold distilled water and 0.03ml of DCPIP. Transfer the contents in a cuvette and place in the colorimeter and record the absorbance level. Place Tube 3 ,10cm away from the bench lamp using a meter ruler. After 1 minute is up, place tube 3 in the colorimeter and record absorbance level. Zero the colorimeter. Repeat this step every minutes for 8 times.
Tube 4 will also contain 0.06ml of chloroplast suspension, 0.09ml of isolation buffer, 0.03ml of cold distilled water and 0.03ml of DCPIP. Transfer the contents in a cuvette, place the tube in the colorimeter and record the absorbance level. Place tube 4 25cm away from the bench lamp. After 1 minute is up, place tube 4 in the colorimeter and record absorbance level. Zero the colorimeter. Repeat this step every minutes for 8 times.
Tube 5 will also contain 0.06ml of chloroplast suspension, 0.09ml of isolation buffer, 0.03ml of cold distilled water and 0.03ml of DCPIP. Transfer the content in a cuvette, place the tube in the colorimeter and record the absorbance level. Place tube 4 40cm away from the bench lamp. After 1 minute is up, place tube 5 in the colorimeter and record absorbance level. Zero the colorimeter. Repeat this step every minutes for 8 times.
Tube 6 will also contain 0.06ml of chloroplast suspension, 0.09ml of isolation buffer, 0.03ml of cold distilled water and 0.03ml of DCPIP. Transfer the content in a cuvette, place the tube in the colorimeter and record the absorbance level. Place tube 6 55cm away from the bench lamp. After 1 minute is up, place tube 6 in the colorimeter and record absorbance level. Zero the colorimeter .Repeat this step every minutes for 8 times.
Set the baseline of the colorimeter using the tube 1.
Unwrap aluminium foil from tube 2, transfer the content in a cuvette and measure the absorbance level. Repeat this step every minutes for 8 minutes.
Repeat the experiment two more times to calculate the average and then plot a graph of the average results obtained.
Trial experiment results
Effect of varying the distance of light intensity on the absorbance level of DCPIP
We can see, there were huge fluctuations between the different trials, suggesting that the chloroplast suspension may not have been properly mixed. The heterogenous solution thus obtained showed different absorbances at different time intervals.
Colorimeters, in general, more sensitive when used in the absorbance range of 0.2-0.9. but my results show higher reading. Thus suggesting that the concentration of the chloroplast suspension was too high.
However, absorbance versus distance plot generated from the above table has been shown in figure 4.
The plot shows the effect of light intensity on the absorbance level of DCPIP. The absorbance level of DCPIP was calculated by averaging the absorbance level of the average trial results. Figure 6. shows huge fluctuations and thus a correlation between the variables of distance against absorbance level cannot be established.
Evaluation of the trial experiment
As mentioned earlier, the rate of photosynthesis vs. intensity(in this case the distance-1 ) plot is a hyperbola, initially increasing linearly and becoming a straight line parallel to X- axis at saturation.(see figure 4.)
Rate of photosynthesis is indicated by the decolorisation of the DCPIP, meaning more the absorbance of DCPIP lesser is the photosynthesis. Thus plotting absorbance vs. distance plot should give us a similar hyperbolic curve.
During the conduction of my trial experiment, I found that the amount of spinach I used for the chloroplast suspension was too much, which was 30g, as the concentration I had included was too concentrated. Therefore I will be planning to reduce the amount of spinach I used, to 15g.
The fluctuation of the readings could be due to improper mixing of the solutions before setting the experiment. Thus the mixture should be properly vortexed after adding DCPIP & chloroplast suspension.
Planning for the final experiment
The following modification will be made:
1. The spinach will be weighed at 15g instead of 30g.
2. The solution will be properly mixed by vortexing.
3. I will be conducting a statistical test using spearman’s rank on the curve between 0-10cm to calculate a critical value for the correlation coefficient.
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