Inhibiting Metabolic Pathways in the Chloroplast PDF

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Young 3/21/2017

Inhibiting Metabolic Pathways in the Chloroplast

Introduction The U.S Census Bureau currently estimates the world population to be well over 7 billion. Therefore, increasing agricultural productivity is of great importance (Varejao et al, 2015). This is a difficult task when preserving the worlds ecosystems is also of dire importance. Agricultural business need to increase the yield of crops and decrease losses due to pests such as weeds (Varejao et al). Crops can be regulated by manipulating the rate of photosynthesis that occurs in the plant. Photosynthesis takes place in the chloroplasts of plants and converts light energy to chemical energy and stores it as sugar. This process begins with the Hill reaction which splits water to donate electrons to the next step: the electron transport chain (Liu et al, 2015). The hill reaction drives the rate of photosynthesis and can be measured by using an artificial electron acceptor which changes color as it becomes reduced. For this experiment, DCIP (2,6dichlorophenolindophenol) is used as the electron acceptor and it turns blue when oxidized and clear when reduced (Ammerman, n.d). The amount of fluorescence is influenced by events in photosynthesis, therefore, the rate of photosynthesis can be measured using a spectrophotometer (Liu et al). During this reaction, an artificial electron acceptor intercepts electrons before they enter the electron transport chain during photosynthesis. (Ammerman) The purpose of this experiment is to compare the rate of the normal Hill reaction to its rate in the presence of two inhibitors. If an inhibitor which is an uncoupler is used, there will be an increase in electron flow. This will, in turn, increase the rate of photosynthesis and increase the yield of the crop. If an inhibitor is used, there will be a decrease in electron flow through the Hill reaction and ETC.

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Substance which decrease photosynthesis and result in the death of the plant are known as herbicides and are used to kill weeds which interfere with crop growth. DCMU (3-(3,4dichlorophenyl)-1,1-dimethylurea) is a common commercial herbicide which will be used for comparison to the normal Hill reaction (Jodieh et al). Ammonia (NH3), a commonly used fertilizer, contains a lone pair of electrons on the nitrogen atom (Maharjan et al, 2014). It can easily donate these electrons making it a good reducing agent. I hypothesize that the rate of photosynthesis will be less than the normal Hill reaction when in the presence of DCMU and will be higher than the Hill reaction when in the presence of ammonia.

Methods Isolation of chloroplasts The major veins were removed from 4 grams of spinach leaves and cut into small pieces. The pieces were ground for two minutes in a chilled mortar with 15ml of ice cold Tris NaCl buffer and a dash of purified sand. The suspension was then filtered through 4 layers of cheesecloth into a chilled beaker and then transferred into a 15ml centrifuge tube. This tube was centrifuged at 200g for 1 minute. Afterwards, the supernatant was decanted into a clean, chilled tube and spun at 1300g for 5 minutes. The supernatant was discarded and the pellet was used to make a chloroplast concentrate once 10ml of ice cold Tris NaCl buffer was added to it. The mixture was gently stirred with a transfer pipet until the chloroplasts were thoroughly mixed. The total time for this may vary. A chloroplast suspension was made once 2ml of chloroplast concentrate was diluted with 5 ml of ice cold Tris NaCl buffer and then kept on ice for all experiments.

Testing the chloroplast activity A 250ml beaker was filled with 150ml of water and placed 25 cm under a lamp bulb. A thermometer was placed in the water and the temperature of the water was kept around 20°C by adding

Young 3/21/2017 a few ice chips as needed. A blank was made with 3.5ml Tris NaCl buffer, 0.5ml DCIP, 1.0ml distilled water, and 0.5 ml of the chloroplast suspension all added in this order. Before the cuvette was inserted into the spectrophotometer, the opening was covered with a gloved hand and inverted. It was then used to zero the spectrophotometer at a 600nm wavelength. Four tubes were prepared by adding the following solutions in the order they are listed. Tube 1 -- 3.5 ml Tris NaCl buffer, 0.5ml DCIP, 0.5ml distilled water, then 0.5ml chloroplast suspension. Tube 2 -- 3.5 ml Tris NaCl buffer, 0.5ml DCIP, 0.5ml distilled water, then 0.5ml chloroplast suspension. Tube 3 -- 3.5 ml Tris NaCl buffer, 0.5ml DCIP, 0.5ml ammonia, then 0.5ml chloroplast suspension. Tube 4 -- 3.5 ml Tris NaCl buffer, 0.5ml DCIP, 0.5ml DCMU, then 0.5ml chloroplast suspension. Tube 2 was prepared first, covered with a gloved hand, inverted, and the 0-minute absorbance was immediately recorded. It was then placed in the water under the lamp. After one minute the tube was removed from the water, wiped with a kimwipe, and inverted. The absorbance was immediately taken and the sample was quickly returned to the light since the DCIP will become more blue as it becomes oxidized once it is out of the light. Each tube was then immediately returned to the water bath and the absorbance was taken at one minute intervals for ten minutes. The absorbance dropped too quickly and after three minutes was almost zero. Therefore, the chloroplast suspension was diluted with 7ml Tris NaCl buffer. This new suspension was used to prepare a new blank per the procedure above. Tube 2 and the remaining samples were prepared using the new suspension. Tube 1 was wrapped in aluminum foil, prepared with the solutions above and capped. It was then placed in the water bath and the absorbance was recorded once after ten minutes. During this time tubes 3 and 4 were then prepared using the same procedure for tube 2. Incubation was staggered at 30 second intervals, so that the experiment could be run for both tubes simultaneously.

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Results Tube 2 was the positive control of the experiment. It represented the normal rate of the Hill reaction which decreased 0.02 absorbance of DCIP per minute. Treatment with DCMU caused a decrease of 0.0018 absorbance of DCIP per minute. The rate of the Hill reaction in the presence of ammonia caused a decrease 0.0258 absorbance of DCIP per minute (Figure 1). The absorbance of the negative control in the absence of light was 0.179.

Discussion Tube 1 was a control that illustrated chloroplast activity in the absence of light. A trendline was applied to the curved lines representing the change in absorbance over time for the positive control, ammonia treatment and DCMU treatment. The slope of each line is the reaction rate of the Hill reaction for that treatment. Ammonia showed the fastest reaction rate (Figure 1). This agrees with my hypothesis since ammonia is a strong reducing agent and readily donates electrons to the Hill reaction which can be used for the rest of photosynthesis. A study by R. U. Berrum and A. A. Benson on the effect of ammonia concentrations in symbiotic algae concluded that ammonia concentrations between 10 and 300µM significantly increase the rate of photosynthesis. Treatment with DCMU showed the slowest reaction rate which was almost zero. This also shows my hypothesis was correct. However, I did not expect the rate of the reaction to be so close to zero. That means that photosynthesis is hardly occurring and the death of the plant is inevitable. Francoeur et al (2007) found DCMU to rapidly inhibit photosynthesis in algae. They also cited studies which determined the time required to halt photosynthetic activity. According to these studies, it can take anywhere from less than five minutes to more than a day for DCMU to inhibit photosynthesis (Paerl et al 1993, Espeland and Wetzel 2001 in Francoeur et al, 2007).

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Figures and Tables

Figure 1: The effects of different inhibitors on the Hill reaction. The ‘positive control’ represents the normal Hill reaction.

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References Ammerman, L. “Inhibiting metabolic pathways in the chloroplast”. Biology 3403. Angelo State University. San Angelo, Tx. N.d. Print. Byerrum, R. U. and Benson, A. A. (1975). Effect of ammonia on photosynthetic rate and photosynthate release by Amphidinium carterae (dinophyceae). J. Phycol. 11: 449-452. Francoeur, N. S., Johnson, C. A., Kuehn, K. A., and Neely, R. K. (2007). Evaluation of the photosystem II inhibitor DCMU in periphyton and its effects on nontarget microorganisms and extracellular enzymatic reactions. J. N. Am. Benthol. Soc. 26: 633-641. Liu, H., Zhang, S., Zhang, X., Chen, C. (2015). Growth inhibition and effect on photosystem by three imidazolium chloride ionic liquids in rice seedlings. J. Hazard. Mater 286, 440-448. Maharjan, B. and Venterea, R.T. (2014). Anhydrous ammonia injection depth does not affect nitrous oxide emissions in a slit loam over two growing seasons. J. Environ. Qual. 43: 1527–1535. Varejao, J.O.S., Barbosa, C.A.L., Ramos, G.A., Varejao, E.V.V., King-Diaz, B., Lotina-Hensen, B. (2015). New rubrolide analogues as inhibitors of photosynthesis light reactions. J. Photochem. Photobiol. 1118....