Photosynthesis I – Light-Dependent Reactions Name: Course ID: Introduction: Photosynthesis literally means “synthesis” using light. The net reaction for photosynthesis involves the synthesis of carbohydrates from carbon dioxide and water in association with the evolution of oxygen gas (Equation 1) Light, chlorophyll Eq. 1 6 CO2 + 6 H2O C6H12O6 + 6 O2 The process of photosynthesis is generally thought to consist of two distinct sets of reactions: a series of light-dependent reactions, termed the thylakoid reactions, based on their location of the internal (thylakoid) membranes of chloroplasts and a series of light independent reactions, termed then the stroma reactions, based on their location in the interior fluid (stroma) of the chloroplasts. Today’s lab will focus specifically on the light-dependent reactions. In the light dependent reactions, light absorption is used to drive the synthesis of ATP and NADPH (Equation 2) that are used as sources of energy and reducing power, respectively, for carbon fixation in the light-independent reactions. Light, chlorophyll Eq. 2 H2O + ADP + Pi + NADP+ ½ O2 + ATP + NADPH + H+ The light reactions originate with the coordinate loss of “excited state” electrons from a pair of specialized chlorophyll a molecules, termed the “reaction center” chlorophyll molecules (see Part A, below). Excited-state electrons are electrons that have absorbed energy and moved into a higher energy orbital (typically an oribital located further from the nucleus of an atom). In the case of the reaction center chlorophyll a molecules, electrons may be excited through direct light absorption or via energy transfer from adjacent photosynthetic pigments following their light absorption. The electrons lost from chlorophyll a are transferred through a series of electron carriers located in the thylakoid membranes in association with the generation of a pH gradient across the membranes that is used to drive ATP synthesis via chemiosmosis. Ultimately, the transported electrons are used for the reduction of NADP+ and the oxidized chlorophyll a molecules of the reaction center are reduced via the splitting of water by means of a process termed photolysis. Aside from generating electrons, photolysis also produces protons that contribute to the development of a pH gradient across the thylakoids and oxygen gas. Supplies: Equipment: Flood lights, ring stands and clamps, forceps, 250 ml beakers, #4 cork borers, 60 cc syringes, Coupland jars (used chromatography chambers), rulers, pencils, meter sticks, petri plates Plant Material: Tobacco (Nicotiana tabacum) and English Ivy (Hedera helix) stockplants Reagents: Boiled distilled water, 0.02% and 0.2% sodium bicarbonate, tapwater with detergent (Tween-20, 3 drops per 200 ml), chromatography solvent (petroleum ether:acetone:propanol [84:15:1] ), chloroplast extract in petroleum ether Consumables: Glass capillaries, disposable gloves, Baker-flex® Silica Gel 1B TLC sheets, foil Biol 219, Lab 6 -2- A) Separation and Identification of the Photosynthetic Pigments In theory, the only pigment molecule that is essential for photosynthesis in plants is chlorophyll a, whose photooxidation represents the source of electrons for NADP+ reduction and ATP synthesis via chemiosmosis. However, the thylakoid membranes of the chloroplasts typically contain several additional pigments, including chlorophyll b and the carotenoids (carotenes and xanthophylls), termed accessory pigments (Fig. 1). The photosynthetic pigments and associated thylakoid proteins are organized so as form larger protein-pigment complexes termed photosystems. Each photosystem consists of a more or less centrally position pair of reaction center chlorophyll a molecules surrounded by several hundred accessory pigments including additional chlorophyll a molecules that are not directly involved in electron transfer. In contrast with chlorophyll pigments which are green to blue-green in color the carotenoid pigments of chloroplasts are typically yellow, orange, or pick in color; however, they are normally masked by the green color of the more abundant chlorophyll molecules. Figure 1. Structures of several common photosynthetic pigments. When conditions are favorable for photosynthesis the primary function of the accessory pigments appears to be as antenna pigments that transfer light energy to the reaction center chlorophyll molecules by resonance energy transfer. Since the absorption spectra of the accessory pigments are distinct from that of chlorophyll a, the accessory pigments increase the percentage of visible light that is able to be used in photosynthesis. However, even if the absorption spectra of the accessory pigments did not differ from chlorophyll a, they are necessary to maintained more less continuous electron flow, i.e., the rate of light absorption by the reaction center chlorophyll molecules is insufficient to maintain a stable level of electron carrier activity. In contrast, during periods of excess illumination the accessory pigments protect against photoinhibition. Photoinhibition is defined as the inhibition of photosynthesis by excess light. The basis for photoinhibition appears to be the reaction between excited state chlorophyll molecules and molecular oxygen. This reaction generates active oxygen species, especially single oxygen, which damages the proteins of the photosystem II reaction center. Reactions between excited chlorophyll molecules and oxygen are typically only observed when the chlorophyll molecules are delayed from returning to a “ground state” via oxidation (electron loss) or resonance energy transfer. In such Biol 219, Lab 6 -3- instances, carotenoid pigments, which do not contain sufficient energy in their excited state to produce single oxygen can serve a photoprotective role by removing excited state electrons from chlorophyll by resonance transfer. In addition, the carotenoid zeaxanthin is also thought to protect against photoinhibition by binding to the protein component of the photosystems so as to cause conformational changes that appears to lead to chlorophyll quenching via heat dissipation. Aside from accessory pigments, chloroplasts also contain an additional pigment type termed pheophytin. Pheophytin, is equivalent to chlorophyll, but lacks a magnesium atom. Pheophytin is not involved in light absorption during photosynthesis but rather functions as an initial component of the thylakoid electron transport chain. In this exercise we will use chromatographic techniques to separate the pigments of a chloroplast extract. In chromatography, molecules are separated based on their differing affinity for mobile and stationary phases. The specific type of chromatography that we will employ is termed thin layer chromatography (TLC). In TLC the stationary phase is adfixed to a sheet of glass or plastic. Although the composition of the stationary phase may vary, in general, polar silica-based stationary phases are employed as is this case here (Baker-flex® Silica 1B gel plates). In contrast the solvent (mobile phase) that we will employ is non-polar consisting of a mix of petroleum ether, acetone, and propanol (84:15:1, v:v:v) In TLC, movement of the mobile phase is mediated by capillary action, with the solvent being pulled across the stationary phase. Depending upon their differing affinities for the mobile and stationary phases the chloroplast pigments in the extract will travel different distances on the chromatograph with the most non-polar pigments traveling the farthest. The distance traveled is used to calculate ratio-to-front (Rf) values which are defined as the quotient of the distance traveled by the compound in question relative to the total distance traveled by the liquid phase. Protocol: 1) Working in pairs and wearing gloves, obtain a TLC plate and use a pencil to draw a line approximately 2 cm from one end of the plate. 2) Using the glass capillaries provided apply two spots of chloroplast extract to the “starting line”. To ensure that the concentration of pigments is sufficient for a good chromatographic separation, multiple aliquots of the extract (n=6-10) should be applied to each spot with the spots being allowed to air dry between applications. 3) Transfer your TLC plate to a chromatography chamber containing solvent. NOTE: To ensure that the solvent moves through the plate at an equal rate the side of the TLC plate should not contact the walls of the chromatography chamber. 4) Start the chromatography run, by placing the lid back onto the chromatography chamber. 5) After the solvent has climbed to within 1-2 cm of the far end of the TLC plate the plate should be removed from the chromatography chamber and the position of the solvent front should be marked with a pencil. How far did the solvent travel (from the “starting line”)? 6) After the plates have air-dried they can be removed from the exhause hood and be examined for evidence of multiple pigment types. Since the pigments are susceptible for rapid fading, you should circle the position of each distinct pigment. The pigments should be numbered sequentially starting from the base of each plate. Using the table provided below indicate the color and relative yield of each pigment. The Rf values for each pigment should also be calculated. Biol 219, Lab 6 -4- Table 1. TLC chromatography data. Pigment (#)1 Color Relative Yield2 Distance Migrated (cm) Rf 1 The pigment spots should be numbered beginning with those closest to the starting line. 2 Rank the quantity of pigment on a scale from 1-3 (+ - +++) based on the size of the pigment spot. However, you should beware that chlorophyll spontaneously degrades to pheophytin under acidic conditions. Since specific steps were not taken to control the pH during extract preparation it is possible that the proportions of pheophytin to chlorophyll on the plates may not be indicative of the in vivo proportions. 4) Based on a review of the chemical structures of the chemical structures of the photosynthetic pigments and your knowledge of factors affecting molecular polarity indicated which pigments spots represent each of the following pigment types. NOTE: The absence of Mg2+ in pheophytin serves to make the molecule less polar (more non-polar) the chlorophyll precurser. chlorophyll a chlorophyll b pheophytin carotenes xanthophylls Biol 219, Lab 6 -5- B) Effects of Light and Carbon Dioxide on the Thylakoid Reaction Rate During photolysis water is split to replace the electrons lost by the oxidation of the reaction center chlorophyll a molecules with oxygen (O2) being evolved in the process. In this experiment we will monitor the rate of oxygen production as an indirect measure of the rate of photosynthesis in leaf discs and will test for the effects of differences in light intensity and carbon dioxide (CO2) concentration on the rate of photosynthesis. However, since the reducing power and energy (ATP) generated via photosynthetic electron transport are not used exclusively for the light-independent reactions of photosynthesis (carbon fixation), the correlation between oxygen evolution rate and the rate of photosynthesis is not perfect. The process nitrite reduction, for example, occurs in the chloroplast at the expense of electrons generated via photolysis. In this experiment the intercellular spaces of the leaf disks will be infiltrated with water and oxygen production will be monitored based on changes in leaf disk buoyancy. Light will applied using flood lamps with the distance between the leaf disks and lamps being used to control the light intensity. Carbon dioxide will be supplied via sodium bicarbonate addition (NaHCO3 Equation 3). Eq. 3 CO2 + H2O H2CO3 H+ + HCO3- H+ + H+ CO3-2 Protocol: 1) Working in groups of 3-5 obtain several healthy leaves of tobacco or aspen and use a cork borer (#4) to isolate 50-60 leaf disks being sure to avoid the large veins of the leaves. 2) Transfer the leaf disks to a 60 cc syringe and draw in 10-15 ml of tap water with detergent (3 drops of Tween-20 per 200 ml). Place the thumb of one of your hands over the tip of the syringe and pull back the plunger with your other hand to draw a vacuum. Hold the vacuum for 5-10 seconds and repeat as necessary until the leaf discs have been successfully infiltrated and as evidence by a loss of buoyancy. NOTE: Be careful not to pull the plunger completely out of the syringe. Also, you may want to wear gloves and or to use paper towel sheets to alleviate the stress of sealing the syringe tip. 3) After the leaf discs have been successfully infiltrated remove the plunger and transfer the infiltration solution and leaf discs to a petri plate. 4) Using forceps assign eight leaf disks to each of five beakers representing the following experimental treatments. Each beaker should have 200 mls of the appropriate incubation solution and the leaf disks should be placed into the beakers edge first to avoid trapping air bubbles that will prevent them from sinking to the bottom of the beakers. The high light treatment will use of flood lamps positioned 55 cm above the surface of the lab bench. The low light treatment will involve the use of lamps set at a height of 80 cm (50% light intensity). 1) Distilled Water, High Light 2) 0.02% Sodium Bicarbonate, High Light 3) 0.2% Sodium Bicarbonate, High Light 4) 0.2% Sodium Bicarbonate, Low Light 5) 0.2% Sodium Bicarbonate, Dark [Wrapped in Foil] Biol 219, Lab 6 -6- 5) Record the time when the beakers are placed into the appropriate light environments as the start time for the experiment. Monitor the beakers in the light on a regular basis and record the time at which each leaf disk floats to the surface in the table provided. For the light disks assigned to the dark treatment data will be collected on the percentage of buoyant leaf disks at the end of the lab period. Experimental Treatment DI Water 0.02% NaHCO3 0.2% NaHCO3 Leaf Disk 100% Light 100% Light 100% Light 50% Light Dark Sum of times Reciprocal of average time 6) For all treatments where 100% of the leaf disks have floated to the surface of the incubation solution by the end of the lab period calculate the reciprocal of the average time required for the leaves to regain buoyancy. The reciprocal calculated is proportion to the rate of oxygen evolution, with larger reciprocals indicating more rapid rates of oxygen production. Biol 219, Lab 6 -7- Review Questions: 1) Would the rate of oxygen evolution be greater for cyclic or non-cyclic electron flow (see textbook, p. 133). Please explain you answer. 2) Explain how ammonium is able to uncouple the processes of electron transport and ATP synthesis (chemiosmosis). Please review Fig. 12.2 and explain the mechanism including the role of pH differences across cell membranes. 3) The carotenoid pigments of plant chloroplast are normally masked by the more abundant chlorophyll pigments. However, they frequently become apparent in senescent leaves and are primarily responsible for the fall color of trees with yellow, orange, or pink foliage. Remembering the plant growth is frequently limited by nitrogen and that chlorophyll contain nitrogen (Fig. 1) suggest a mechanism that accounts for the color changes observed in the leafs of these species in the Fall. 4) For several horticultural crops mutants with purple-colored leaves have been isolated. In most cases the purple color is due to anthocyanins that are localized in the epidermis as we have observed in previous labs. Anthocyanins, which absorb light from in the UV region of the spectrum, are also induced by high light intensities. In terms of the visible spectrum, anthocyanins absorb primarily between 500-580 nm. Would you expect the presence of anthocyanins to adversely affect the photosynthetic abilities of leaves to a significant degree? (See Fig. 7.7 in your textbook).
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