SEARCH

SEARCH BY CITATION

Keywords:

  • Ammonium chloride;
  • isolated chloroplasts;
  • uncoupling;
  • dichlorophenolindophenol

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. Conclusion
  6. REFERENCES

The rate of flow of electrons from water to an artificial electron acceptor, dichlorophenolindophenol (DCPIP), through photosystem II in the thylakoid membranes of isolated chloroplasts is greatly enhanced in the presence of 10 mM ammonium chloride. Rate enhancement depends on irradiance levels. Uncoupling reagents like ammonium chloride prevent the formation of a proton gradient across the thylakoid membrane and consequently remove a constraint on the rate of electron transport. The mode of action of ammonium chloride is explained. Evidence obtained using an oxygen electrode that DCPIP itself also partially uncouples the system is presented as background information for instructors. Suggestions on how this reaction may be used in laboratory classes for students from high school to the senior undergraduate level are included.

We teach a section of a second year undergraduate laboratory course entitled “Scientific Methods in Biology.” The course encompasses experimental design, instrumentation, the evaluation of experimental data, and how to communicate results to the scientific community. Because students write about some of their work in the form of a scientific paper, we try to find a variety of experimental systems that exemplify similar principles. This allows different groups of students to write on different topics. The measurement of light-dependent rates of electron transport in the thylakoid membranes of isolated chloroplasts has been particularly useful in generating different questions. In this assay, the decline in absorbance of oxidized dichlorophenolindophenol (DCPIP)11 is measured over time. DCPIP becomes colorless as it is reduced by intercepting electrons from the electron transport chains in the thylakoid membranes. It has been shown previously that rates of electron transport change with irradiance levels [1], in response to pH and in the presence of inhibitory reagents [2]. In this paper we demonstrate that rates of electron transport in the thylakoid membrane of illuminated chloroplasts can be dramatically increased in the presence of millimolar concentrations of ammonium chloride. Reagents that act like ammonium chloride are called uncouplers because, by a variety of mechanisms, they prevent the formation of a proton gradient across the thylakoid membrane and consequently uncouple electron transport from ATP synthesis (photophosphorylation).

We believe that the information presented could be useful for teaching students from grade 10 to the senior undergraduate level. For younger students, the assay can be simply used as a demonstration to correct a common misconception [3] that light quantity limits the rate of photosynthetic reactions when the process is light-saturated. The more detailed studies are appropriate for second and third year undergraduates. We have, in addition, included information from studies using oxygen electrodes that we regard as unsuitable for the majority of undergraduates but which provide background information that may be of use to their instructors.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. Conclusion
  6. REFERENCES

Equipment

  • Visible range spectrophotometers (e.g. Spectronic 20) and suitable cuvettes or tubes.

  • 5- and 10-ml-capacity glass pipettes.

  • Variable volume mechanical pipettes (10–100-μl range) with tips.

  • Domestic blender.

  • Clinical centrifuge with graduated tubes.

  • Lamps with 100-W frosted bulbs.

  • (A Li-Cor LI-189 quantum/radiometer/photometer with an attached LI-190 SA quantum sensor is very useful but not essential.)

  • Cheese cloth, Parafilm.

Stock Solutions

  • Chloroplast isolation buffer: 50 mM Tricine, 400 mM sorbitol, 10 mM NaCl, 2.5 mM MgCl2·6H2O, 1.25 mM MnCl2, 0.3 mM Na2EDTA adjusted to pH 7.8 with saturated NaOH.

  • Chloroplast reaction buffer (5 × concentrated): 250 mM Tricine, 500 mM sorbitol, 100 mM KCl, 25 mM MgCl2·6H20 adjusted to pH 7.0 with saturated NaOH.

  • 90% (v/v) aqueous acetone.

  • 2-mercaptoethanol.

  • 100 mM NH4Cl.

Chloroplast Isolation—

Weigh 25 g of spinach leaves from which the petioles and major veins have been removed. Cut the leaves into smaller fragments and place them in a blender cup that has been stored in the freezer. Add 100 ml of cold chloroplast isolation buffer (with or without 0.25 μl of 2-mercaptoethanol/ml of buffer. If used, the 2-mercaptoethanol should be added to the buffer just prior to use). Blend the leaves with 3 × 5-s bursts at full speed. Filter the blended mixture (called a brei) through four layers of cheesecloth into a beaker on ice. Transfer 10-ml aliquots of the filtrate into 15-ml-capacity centrifuge tubes. Centrifuge the filtrate for 5 min at 1300 × g (this is achieved using setting 7 on a standard clinical centrifuge containing swinging buckets with a 15-cm radius from the axis of rotation). Discard the supernatants and add 2.0–2.5 ml of cold isolation buffer to each pellet. Resuspend the pellets with a paint brush and pool them in a single tube. This is the chloroplast preparation. It also contains other cellular components, but as the chloroplasts are the only photoactive constituents, further purification does not improve the assay. The resulting 12–15 ml of chloroplast preparation is enough for a minimum of 300 assays and can be stored on ice for hours with minimal loss of activity.

Estimation of the Total Chlorophyll Concentration in the Chloroplast Suspension—

To compare results obtained using different chloroplast preparations or different quantities of the same chloroplast preparation, it is necessary to know the chlorophyll concentration in the chloroplast suspension. The following is not the most accurate method to determine chlorophyll concentration, but it is rapid and reproducible.

Add 50 μl of the chloroplast suspension to 5.0 ml of 90% acetone. Cover the tube with Parafilm and shake to dissolve the chlorophyll. Centrifuge the solution at 1300 × g for 3 min to pellet any particulate material. Read the absorbance of the supernatant at 652 nm (A652) using a blank containing 5.0 ml of 90% acetone and 50 μl of isolation buffer. The chlorophyll concentration is calculated from Equation 1.

  • equation image(1)

For example, if A652 = 0.175, then the following is true.

  • equation image(2)

If, for example, you choose to add a volume of chloroplast suspension containing 20 μg of chlorophyll to a reaction mixture, that volume is given by the following.

  • equation image(3)

Assay Protocol—

The composition of the reaction mixtures and the reference blank are shown in Table I. When several reaction mixtures containing identical components are to be used, we encourage our students to prepare the solutions in bulk and pipette 5.0 ml to each reaction tube. This eliminates systematic pipetting error, and each tube contains exactly the same concentrations of the reagents.

Reactions can be run at different distances from the lamp by fixing a piece of masking tape to the bench top, ruling it in measured increments from the light source, and placing the reaction tubes in a 100-ml beaker astride one of the distance markers. A quantum sensor can be used to measure the photon fluence rate (in μmol of photons/m2/s) at each distance from the lamp.

Add a volume of chloroplast preparation containing a known amount of chlorophyll to the reference blank. Cap the tube with Parafilm and invert three times to mix the contents. Use the blank to set the spectrophotometer to an absorbance of zero.

The remainder of the work is performed in a darkened room. We turn off the room lights and shine spotlights onto the ceiling in the four corners of the lab. This provides enough light to do the work but not enough to affect the reactions.

For each reaction, add the chosen volume of chloroplast preparation, cap with Parafilm, and invert the tube three times. Take the initial absorbance reading at 600 nm (A600). Place the tube in the beaker at the selected distance from the lamp and start timing when the lamp is turned on. Take A600 readings at 1, 2, and 3 min from the time that the lamp was turned on. Return the tube to the beaker between readings. A dark control is treated identically except that it is wrapped in aluminum foil between A600 readings.

Rate Calculations—

Because the rate of these reactions decays exponentially with time and because we cannot continuously monitor the decline in absorbance of the DCPIP while simultaneously exposing the reaction tubes to light, it is not possible to obtain an accurate estimate of the initial rate of the reaction. Instead, we have found empirically that an average of the rates during the first 2 min of the reaction provides satisfactory data.

  • Calculate the cumulative change in absorbance during the first 2 min (ΔA600/2 min) for each reaction by subtracting the A600 at 2 min from the A600 at zero time.

  • If the dark control shows any activity, subtract the ΔA600/2 min for the dark control from the A600/2 min for each of the reactions. This is the corrected ΔA600/2 min for each reaction and reflects only the decline in absorbance due to the presence of illuminated chloroplasts.

  • This step requires a standard curve of absorbance at 600 nm against the concentration of DCPIP. The buffer must be the same as that used in the reactions because the absorbance of DCPIP is pH-sensitive. Determine the molar absorption coefficient (ϵ) from the slope of the standard curve where

    • equation image(4)

    and divide the ΔA600/2 min by the absorption coefficient (ϵ) as follows.

    • equation image(5)
  • Divide Δc/2 min by 2, which equals Δc/min.

  • Correct for the volume of the reaction mixture.

    • equation image(6)
  • Divide the rate by the number of μg of chlorophyll added to the reaction mixture in the chloroplast preparation to derive the final rate in mol of DCPIP photoreduced/min/μg of chlorophyll.

Range Finding: What Concentration of Ammonium Chloride Is Needed to Maximize the Rate of Electron Transport in the Thylakoid Membrane?—

Reaction mixtures contained 30 μM DCPIP, 50 mM Tricine buffer containing 100 mM sorbitol, 10 mM KCl, 5 mM MgCl2·6H2O at pH 7.0, and enough 100 mM NH4Cl and distilled water to produce concentrations of 0, 0.5, 1.0, 5.0, 10.0, 20.0, 30.0, 40.0, or 50.0 mM NH4Cl in a total volume of 5.0 ml. A volume of chloroplast preparation containing 20 μg of chlorophyll was added to each reaction mixture, and reactions were run at 20 cm from the lamp at a photon fluence rate of 110 μmol of photons/m2/s.

Does 10 mM NH4Cl Influence the Rate of Reduction of DCPIP by Isolated Chloroplasts When the Reaction Is Light-saturated?—

Reactions were run with or without 10 mM NH4Cl at 5 and 10 cm from a 100-W lamp at photon fluence rates of 597 and 307 μmol of photons/m2/s, respectively. The volume of chloroplast preparation used contained 20 μg of chlorophyll.

Does the Ratio of Rates of DCPIP Reduction by NH4Cl-treated Chloroplasts to Those by Untreated Chloroplasts Change with Declining Photon Fluence Rates?—

Reactions with and without 10 mM NH4Cl were run at 10, 20, 30, 40, and 50 cm from the lamp at photon fluence rates of 226, 85, 43, 26, and 18 μmol of photons/m2/s, respectively. The volume of chloroplast preparation used contained 5 μg of chlorophyll.

For a statistical comparison, the experiment was repeated twice more using four replicates/treatment. In the first trial, reactions were run at 5, 10, 20, 30, and 40 cm from the lamp at photon fluence rates of 368, 208, 83, 45, and 28 μmol of photons/m2/s using a volume of chloroplast preparation containing 10 μg of chlorophyll. The second set of reactions was run at 40, 50, 60, and 70 cm from the lamp at photon fluence rates of 36, 25, 18, and l4 μmol of photons/m2/s. The volume of chloroplast preparation used contained 30 μg of chlorophyll at 40, 50, and 60 cm and 40 μg of chlorophyll at 70 cm from the lamp.

DCPIP Is an Uncoupling Reagent. To What Extent Does 30 μM DCPIP Affect the Light-dependent Reactions in Isolated Chloroplasts?—

DCPIP, the reagent we use to measure rates of electron transport in the thylakoid membrane of isolated chloroplasts, is also an uncoupler. It causes very rapid electron transport in the membrane that is uncoupled from photophosphorylation (ATP synthesis) at concentrations of 50–100 μM [4, 5]. In our assay we use DCPIP at a concentration of 30 μM. This concentration clearly does not completely uncouple electron transport from ATP synthesis because higher rates of DCPIP reduction are observed when ammonium chloride is present. To determine whether, or to what extent, 30 μM DCPIP uncouples the thylakoid membrane, we used an oxygen electrode. The oxygen electrode measures the increase in dissolved oxygen resulting from the oxidation of water by the oxygen-evolving complex associated with photosystem II (PS II, Fig. 3). As Reaction 1 shows under “Results and Discussion,” the rate of oxygen evolution is stoichiometrically related to the rate at which electrons are transported through the series of carriers in the thylakoid membrane.

Measurements of Oxygen Evolution—

Oxygen evolution was measured polarographically using a thermostatted, aqueous phase Clarke-type oxygen electrode (Hansatech Ltd., King's Lynn, UK). Measurements were made using a 2.0-ml reaction mixture containing 50 mM Tricine-NaOH buffer (pH 7.0), 100 mM sorbitol, 20 mM KCl, 5 mM MgCl2, 30 μM DCPIP, 2 mM potassium ferricyanide as the electron acceptor and a volume of chloroplast preparation containing 40 μg of chlorophyll. The reactions were run at 20 °C at photon fluence rates of 50, 130, 200, 420, and 680 μmol of photons/m2/s. Actinic light was supplied by a 150-W halogen lamp using a fiber optic cable (Fiber-Lite, Dolan-Jenner Industries, Lawrence, MA). The photon fluence rate was measured in the middle of the electrode cuvette using a photodetector (Hamamatsu G1125–02, Hamamatsu Corp., Bridgewater, NJ) connected to a calibrated Li-Cor 185A quantum sensor (Li-Cor Inc., Lincoln, NE). Electrode signals were recorded on an Omnigraphic 2000 chart recorder (Bausch & Lomb Inc., Rochester, NY).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. Conclusion
  6. REFERENCES

Range Finding: What Concentration of Ammonium Chloride Maximizes the Rate of Electron Transport in the Thylakoid Membrane?—

The rate of photoreduction of DCPIP increased with increasing NH4Cl concentration up to ∼5–10 mM. Further increases in NH4Cl concentration produced no greater rate increases (Fig. 1). Consequently, a concentration of 10 mM NH4Cl was used for the remainder of this study.

Ammonium Chloride Increases the Rate of Reduction of DCPIP by Chloroplasts When the Reaction Is Light-saturated—

Reactions that were run without NH4Cl showed light saturation. No difference in reaction rates was seen for reactions run at 5 and 10 cm from the lamp even though the photon fluence rate at the former distance was approximately double that at the latter (Fig. 2). In the presence of 10 mM NH4Cl, the mean reaction rates at 5 and 10 cm from the lamp were more than twice those seen in reactions with no NH4Cl but not significantly different from each other (p >0.05) (Fig. 2). The ratios of rates of DCPIP reduction by NH4Cl-treated chloroplasts to those by untreated chloroplasts were about 2.7 at both distances from the lamp.

The concept that light is not the limiting factor when photosynthesis is light-saturated is, according to studies by educational psychologists [3], misunderstood or poorly understood by a large proportion of grade 11 and 12 students specializing in biology. Data in Fig. 2 unequivocally show that light is not the limiting factor when photosynthetic reactions are light-saturated because the reaction rates can be increased at a given light level by adding NH4Cl. The NH4Cl is apparently lifting some constraint on the rate that electrons flow through the photosynthetic complexes in the thylakoid membrane. This constraint must be a process that lies between the origin of the electron transport chain and the site at which DCPIP intercepts the electrons.

Why Does Ammonium Chloride Increase the Rate of Electron Transport in the Thylakoid Membrane of Isolated, Illuminated Chloroplasts?—

To answer this question, a review of some of the light-dependent steps of photosynthesis seems appropriate. The internal thylakoid membranes of chloroplasts separate two compartments, the outer stroma and the inner lumen. A number of molecular complexes are embedded in, or associated with, the thylakoid membranes (Fig. 3). Photons in the wavelength range from 400 to 700 nm are absorbed by pigments in the antenna complexes (AC) associated with photosystems II and I (PS II and PS I). Some of the absorbed energy is transferred to the reaction centers (RC) in each photosystem. This energy is expended to initiate a flow of electrons that starts with the oxidation of water by the oxygen-evolving complex on the lumenal surface of PS II (Reaction 1).

  • equation image(7)

The electrons are transported in a linked series of redox reactions through PS II, plastoquinones that diffuse within the thylakoid membrane, the cytochrome b6f complex, plastocyanin, and PS I and finally are accepted by NADP+, which is consequently reduced. As a result of the transfer of electrons from water to NADP+, a proton gradient is generated across the thylakoid membrane by two mechanisms. First, protons are generated in the lumen by the oxidation of water (Reaction 1), and second, the plastoquinones “pump” protons from the stroma to the lumen. The latter occurs because plastoquinones accept protons from the stroma as well as electrons from PS II when they become reduced. When reduced plastoquinones become oxidized by donating electrons to the cytochrome b6f complex (or to DCPIP in our assay), they discharge the protons into the lumen (Reaction 2).

  • equation image(8)

In intact chloroplasts, the potential energy in the proton gradient is used to convert ADP and Pi to ATP as the protons return to the stroma through the CF0CF1 complexes. Consequently, photophosphorylation (ATP synthesis) is said to be coupled to electron transport in the thylakoid membrane. Reagents such as ammonium chloride are called uncouplers because they dissipate the proton gradient leaving no energy for ATP synthesis. In solution, the NH4+ ion dissociates to NH3 (ammonia) and H+. Because it is not charged, the ammonia freely crosses the membrane to the lumen, where it reassociates with protons to again form an ammonium ion. As a result, free protons are sequestered in the lumen, and no gradient is generated. When a steep proton gradient is present, it is more difficult for the reduced plastoquinones within the membrane to become oxidized because they have to give up the protons against the concentration gradient. As the proportion of reduced plastoquinones in the membrane increases, there are fewer oxidized plastoquinones to accept electrons from PS II, so the rate of electron efflux from PS II declines. This is probably the step that limits the rate of electron transport through the redox pathway at light saturation because when the proton gradient is abolished, the rate of electron transport can increase dramatically.

The preceding explanation suggests a testable hypothesis. The magnitude of the proton gradient is directly related to the rate of electron transport through the thylakoid membrane. The rate of electron transport also depends on the amount of irradiance to which the chloroplasts are exposed [1]. We would consequently predict that the ratio of the rate of photoreduction of DCPIP by NH4+-treated chloroplasts to that by untreated chloroplasts will decline with a decreasing photon fluence rate. At very low photon fluence rates we would further expect this ratio to reach 1:1.

For the remainder of this paper, rates of electron transport (i.e. rates of DCPIP reduction) in chloroplasts treated with NH4Cl will be called uncoupled, and those in untreated chloroplasts will be described as coupled.

The Ratio of Uncoupled to Coupled Rates of DCPIP Reduction Declines with Declining Photon Fluence Rates—

As the photon fluence rates were reduced from 226 to 18 μmol of photons/m2/s, the ratio of uncoupled to coupled rates of photoreduction of DCPIP declined from 3.43 to 0.8 (Table II). Although consistently observed in additional experiments, a ratio lower than 1:1 at low irradiance levels was unexpected, so statistical studies using 4 replicates/treatment at a variety of photon fluence rates were carried out. Over the range of 5, 10, 20, 30, and 40 cm from the lamp (at photon fluence rates of 368, 208, 83, 45, and 28 μmol of photons/m2/s) the ratios of uncoupled to coupled mean rates of DCPIP reduction were 2.69, 2.25, 1.97, 1.24, and 1.11, respectively (Fig. 4). Differences between treatments at each distance from the lamp were significant (p >0.05) except for those at 40 cm. When assayed at distances of 40, 50, 60, and 70 cm from the lamp (36, 25, 18, and 14 μmol of photons/m2/s) the ratios were 1.19, 1.02, 0.91, and 0.88, respectively (Table III). Although the ratios of uncoupled to coupled rates fell below 1:1, differences between treatments at 50, 60, and 70 cm from the lamp were not significant (p >0.05).

We interpret these data as follows. When the reaction is light-saturated, electrons flow rapidly in the thylakoid membrane generating a steep proton gradient, which in turn places a limit on the rate of electron transport. Abolition of the gradient in the presence of ammonium ion removes the constraint, and the rate of electron transport increases. As the photon fluence rate is reduced, electrons flow at a lower rate, and a smaller proton gradient is generated. Consequently, abolishing the proton gradient has less effect on the rate of electron flow. At a photon fluence rate of ∼20–30 μmol of photons/m2/s, little or no proton gradient is generated, so the uncoupled and coupled rates of electron transport are not significantly different. The rate-limiting step therefore depends on the irradiance level. When light-saturated, the rate-limiting step is the oxidation of plastoquinone. At lower photon fluence levels the rate-limiting step is light absorption.

A Note on the Condition of the Isolated Chloroplasts—

Chloroplasts have an outer limiting envelope that would normally prevent the DCPIP in the reaction mixture from coming into contact with the internal thylakoid membranes. Chloroplasts isolated using gentler procedures than we use do not reduce DCPIP. If the chloroplasts are immersed briefly in distilled water the outer envelope bursts; however, the thylakoid membranes remain intact, and in this condition, they reduce DCPIP. We conclude that our isolation protocol ruptures the outer envelope in all of the chloroplasts because no difference in rates of DCPIP reduction is seen from reactions using non-shocked chloroplasts compared with those exposed briefly to distilled water (data not shown).

DCPIP Partially Uncouples the Thylakoid Membrane—

At concentrations between 50 and 100 μM, DCPIP completely uncouples photophosphorylation (ATP synthesis) from electron transport in the thylakoid membrane [4, 5]. These concentrations are too high for use in our assay as they would produce absorbances of 1.0 and 2.0, respectively. We use 30 μM DCPIP, which has an absorbance of ∼0.6, a value that lies within the useful absorbance range for the Spectronic 20. It has been suggested that values for concentrations of DCPIP that are reported to uncouple electron flow from ATP synthesis are to be viewed with caution because lower concentrations are required at lower pH values [5]. Moreover, Good notes that “uncouplers tend to be strongly absorbed by the chloroplasts, in which case the amount of uncoupler relative to the chloroplast volume is more significant than the overall concentration in the reaction mixture” [5]. Our question then becomes the following. Does the concentration of DCPIP as used in the conditions of our assay affect the rate of electron transport through the thylakoid membrane? Samples of traces obtained using an oxygen electrode are shown in Fig. 5. The rate of oxygen evolution is determined from the slopes of the lines. Oxygen production is enhanced in the presence of ammonium chloride (Fig. 5A) or DCPIP (Fig. 5B). When ammonium chloride is added after the addition of DCPIP the rate of oxygen evolution is additive (Fig. 5B). Evidently, 30 μM DCPIP partially uncouples the thylakoid membrane, which can be further uncoupled by the addition of ammonium chloride.

Three independent determinations of the rates of oxygen evolution at different photon fluence rates in the presence and absence of ammonium chloride without (Table IV) and with (Table V) 30 μM DCPIP demonstrate the following points.

  • As would be predicted from the data obtained using the DCPIP reduction assay (Tables II and III and Fig. 4), in all cases, the ratio of uncoupled to coupled rates of oxygen evolution declines with decreasing photon fluence rate. Ratios from each determination at each photon fluence rate are remarkably similar (Tables IV and V). Ratios of rates of DCPIP reduction by NH4Cl-treated chloroplasts to those by untreated chloroplasts fall below 1:1 at 50 and 130 μmol of photons/m2/s in the presence of DCPIP (Table V) but not in its absence (Table IV).

  • At the highest photon fluence rate used (680 μmol of photons/m2/s), rates are similar (ranging from 80.91 to 111.81 μmol of oxygen/mg of chlorophyll/h) in the presence of 10 mM NH4Cl regardless of whether DCPIP is present or not (Tables IV and V).

  • When no NH4Cl is present, rates of oxygen evolution are much greater in the presence of 30 μM DCPIP than in its absence.

We believe that electron transport is completely uncoupled from photophosphorylation by 10 mM NH4Cl because further increases in its concentration do not generate greater rates of photoreduction of DCPIP (Fig. 1). If this is the case, we can estimate the degree of uncoupling in the presence of 30 μM DCPIP. For example, when no DCPIP is present, at 680 μmol of photons/m2/s, the average rate of oxygen evolution with no NH4Cl is ∼14.4% of the average rate in the presence of NH4Cl (Table IV). At the same photon fluence rate and in the presence of 30 μM DCPIP, the average rate of oxygen evolution with no NH4Cl is ∼64.3% of the average rate in the presence of NH4Cl (Table V). We consequently conclude that in these conditions, 30 μM DCPIP uncouples the membrane by ∼50%.

Comparison of Rates of Oxygen Evolution and DCPIP Photoreduction by Isolated Chloroplasts—

The oxygen electrode and the DCPIP reduction assay both measure products of the same reaction, the oxidation of water (Reaction 1). The reduction of one DCPIP molecule requires two electrons and two protons. The oxidation of two molecules of water produces one molecule of oxygen and enough electrons and protons to reduce two molecules of DCPIP. We should therefore be able to predict rates of DCPIP reduction from rates of oxygen evolution in trials run under similar conditions. The predicted rate of DCPIP reduction is obtained by multiplying the rate of oxygen evolution by 2 (2 DCPIP reduced/oxygen produced), dividing by 1000 (converting mg of chlorophyll to μg of chlorophyll), and dividing by 60 (converting h to min).

We tested this prediction by calculating the rates of reduction of DCPIP that corresponded to the rates of oxygen evolution at 680 μmol of photons/m2/s in the presence and absence of 10 mM NH4Cl (Table V) for comparison with rates of DCPIP reduction measured in the presence and absence of NH4Cl at 597 μmol of photons/m2/s (Fig. 2). The results showed good correspondence (Table VI). Although the photon fluence rates were not identical, this should not matter because in both cases the reaction was light-saturated.

Should 2-Mercaptoethanol Be Included in the Chloroplast Isolation Buffer?—

Under “Experimental Procedures” we indicated that 2-mercaptoethanol could be omitted from the chloroplast isolation buffer. As one of us argued in an earlier paper [1], 2-mercaptoethanol serves a useful pedagogical purpose because when this reducing agent is present in the chloroplast suspension that is added to the reaction mixture, some DCPIP reduction occurs even in the dark. This shows our students that appropriate controls are necessary and that controls do not always show a lack of activity. However, in most of the data presented here, 2-mercaptoethanol has been omitted from the chloroplast isolation buffer because it increases the variance in the data. For example, reactions run in quadruplicate at 17l μmol of photons/m2/s in the presence of 10 mM NH4Cl with chloroplast suspension in the absence and presence of 2-mercaptoethanol produced mean reduction rates of 3.35 ± 0.04 × 10−9 and 3.15 ± 0.60 × 10−9 mol/min/μg of chlorophyll, respectively (± 2 × S.E.). We do not known why, but 2-mercaptoethanol greatly increases the variance in the data. Therefore, to increase the probability that students will generate more consistent results, we recommend that 2-mercaptoethanol should be omitted from the chloroplast isolation buffer when using NH4Cl for studies on membrane uncoupling.

Conclusion

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. Conclusion
  6. REFERENCES

The work described in our paper can be used for laboratory education at a variety of levels. For high school students it can be used to demonstrate the concept of light saturation. A reasonable hypothesis is that photosynthesis becomes light-saturated because the pigments of the antenna complexes have reached the limit at which they can absorb light. Fig. 2 clearly shows that this is not the case. The rate-limiting step at light saturation must be at some stage between light absorption and the reduction of DCPIP. A deep understanding of the information in Fig. 3 is not needed to appreciate this point. As long as it is understood that chloroplasts transduce light energy to chemical energy at a rate that can be measured by the loss of color of DCPIP over time, no further description of the mechanism is required. A quantum sensor is not required to measure photon fluence rates; simply find two distances from the lamp that produce identical rates of DCPIP reduction. (Five and ten cm from a 100-W lamp are usually satisfactory.) Moreover, younger students need not calculate reduction rates as molar values. Rates can be expressed as changes in absorbance/min.

If the information presented in Fig. 3 and the mechanism by which ammonium chloride affects the proton gradient are presented, more experienced students should be able to formulate and test the hypothesis that the ratios of uncoupled to coupled rates will decrease with declining photon fluence rate. Instructors may choose not to reveal that DCPIP is also an uncoupling agent, but if they do, the data given in our paper could be presented in class as evidence.

Finally, upper level students taking courses in plant physiology, bioenergetics, or biochemistry who have access to oxygen electrodes could compare the relative contributions of different concentrations of ammonium chloride and DCPIP to uncoupling the thylakoid membrane.

thumbnail image

Figure FIGURE 1.. The rate of photoreduction of DCPIP by isolated chloroplasts increases with increasing ammonium chloride concentration up to 5–10 mM. Higher ammonium chloride concentrations do not cause further rate increases. A volume of chloroplast preparation (without 2-mercaptoethanol) containing 20 μg of chlorophyll was added to the reaction mixtures. Reactions were run at 20 cm from the lamp at a photon fluence rate of 110 μmol of photons/m2/s.

Download figure to PowerPoint

thumbnail image

Figure FIGURE 2.. Ammonium chloride (10 mM) more than doubles the mean rate of electron transport from water to DCPIP when the reaction is light-saturated. (Error bars = 2 × S.E., n = 4) A volume of chloroplast preparation (without 2-mercaptoethanol) containing 20 μg of chlorophyll was added to the reaction mixtures. Reactions were run at 5 and 10 cm from the lamp at photon fluence rates of 597 and 307 μmol of photons/m2/s, respectively.

Download figure to PowerPoint

thumbnail image

Figure FIGURE 3.. Schematic of electron transport and proton translocation in the thylakoid membrane. Electrons that are produced when water is oxidized in the lumen flow (unbroken line) to the final electron acceptor (NADP+) in the stroma via PS II, plastoquinones, cytochrome b6f (cyt b6/f), plastocyanin, and PS I. Protons (H+) are translocated from the stroma to the lumen (broken line) by plastoquinones. Translocated protons and those derived from the oxidation of water return to the stroma through the CF0CF1 complexes providing energy for ATP synthesis. In the DCPIP reduction assay, electrons are accepted from plastoquinones by DCPIP (dotted line) instead of the cytochrome b6f complex. (This figure has been adapted from Alberts et al. [6].)

Download figure to PowerPoint

thumbnail image

Figure FIGURE 4.. The ratio of ammonium chloride-treated to -untreated mean rates of photoreduction of DCPIP declines with decreasing photon fluence rates. A volume of chloroplast preparation (without 2-mercaptoethanol) containing 10 μg of chlorophyll was added to the reaction mixtures. Reactions were run at 5, 10, 20, 30, and 40 cm from the lamp at photon fluence rates of 368, 208, 83, 45, and 28 μmol of photons/m2/s, respectively. (Error bars = 2 × S.E., n = 4.)

Download figure to PowerPoint

thumbnail image

Figure FIGURE 5.. Oxygen electrode traces obtained during measurements of electron transport in thylakoid membranes of isolated chloroplasts in the absence (A) or presence (B) of 30 μ M DCPIP. Potassium ferricyanide (final concentration 2 mM) was present at the start of the reaction. DCPIP (6 μl of a 10 mM solution in 95% (v/v) ethanol) and NH4Cl (final concentration 10 mM) were added where shown. The ethanol did not affect the rate of oxygen evolution (A). The photon fluence rate was 420 μmol of photons/m2/s. Rates of oxygen evolution were determined from the slopes of the lines.

Download figure to PowerPoint

Table Table I. Composition of reaction mixtures (with or without ammonium chloride) and the reference blank prior to adding the chloroplast preparation
Mixture0.1 mM DCPIPDistilled water100 mM NH4ClReaction buffer
 mlmlmlml
No NH4Cl1.52.501.0
10 mM NH4Cl1.52.00.51.0
Blank04.001.0
Table Table II. The ratio of rates of DCPIP reduction by ammonium chloride-treated chloroplasts to those by untreated chloroplasts declines with declining photon fluence rates
Distance from lampPhoton fluence rateReaction ratesaRatio of treated to untreated rates
  • a

    a Mol of DCPIP reduced/min/μg of chlorophyll × 10−9.

A volume of chloroplast preparation (including 2-mercaptoethanol) containing 5 μg of chlorophyll was added to the reaction mixtures.
  −NH4Cl+NH4Cl 
cmμmol of photons/m2/s   
50180.880.700.80
40260.951.131.18
30431.412.081.48
20851.913.511.84
102261.766.023.43
Table Table III. At low photon fluence rates, the ratio of rates of DCPIP reduction by ammonium chloride-treated chloroplasts to those by untreated chloroplasts falls below 1:1, but the differences are not significant
Distance from lampPhoton fluence rateReaction ratesaRatio of treated to untreated rates
  • b

    a Mol of DCPIP reduced/min/μg of chlorophyll ± 2 × S.E. × 10−9.

  • a

    b Difference between treatments not significant (p >0.05).

A volume of chlorophyll preparation (without 2-mercaptoethanol) containing 30 μg of chlorophyll was added to the reaction mixtures.
  −NH4Cl+NH4Cl 
cmμmol of photons/m2/s   
70140.41 ± 0.020.36 ± 0.01b0.88
60180.46 ± 0.020.42 ± 0.02b0.91
50250.64 ± 0.010.65 ± 0.02b1.02
40360.83 ± 0.040.99 ± 0.061.19
Table Table IV. Rates of oxygen evolution in the absence of DCPIP at various photon fluence rates
Photon fluence rateRate of oxygen evolutionaAverage ratio of treated to untreated rates
  • a

    a μmol of oxygen/mg of chlorophyll/h.

No 2-mercaptoethanol was present in the chloroplast preparation.
 Trial 1Trial 2Trial 3 
 −NH4Cl+NH4Cl−NH4Cl+NH4Cl−NH4Cl+NH4Cl 
μmol/m2/s       
506.7118.794.7013.407.9817.962.67
13010.0732.227.6525.2310.9734.003.20
20011.1939.178.8230.8711.9743.893.57
42013.4285.3510.5961.7412.9774.325.97
68015.11111.8111.7680.9113.9791.926.97
Table Table V. Rates of oxygen evolution in the presence of 30 μM DCPIP at various photon fluence rates
Photon fluence rateRate of oxygen evolutionaAverage ratio of treated to untreated rates
  • a

    a μmol of oxygen/mg of chlorophyll/h.

No 2-mercaptoethanol was present in the chloroplast preparation.
 Trial 1Trial 2Trial 3 
 −NH4Cl+NH4Cl−NH4Cl+NH4Cl−NH4Cl+NH4Cl 
μmol/m2/s       
5020.1417.3218.8216.4715.4613.000.86
13035.8132.9532.9331.6132.9228.970.92
20044.7648.7938.8141.9141.9038.971.04
42059.3175.9254.1070.8751.8759.651.27
68062.69104.0663.5199.0855.8781.571.60
Table Table VI. The equivalence of rates of oxygen evolution and rates of photoreduction of DCPIP by isolated chloroplasts in similar experimental conditions
[DCPIP][NH4Cl]Average rate of oxygen evolutionaPredicted rate of DCPIP reductionbMeasured rate of DCPIP reductionc
  • a

    a μmol of oxygen/mg of chlorophyll/h (from Table 5).

  • b

    b mol of DCPIP photoreduced/min/μg of chlorophyll (calculated).

  • c

    c mol of DCPIP photoreduced/min/μg of chlorophyll (from Fig. 2).

μMmM   
30060.72.02 × 10−91.80 × 10−9
301094.93.16 × 10−94.87 × 10−9
  • 1

    The abbreviations used are: DCPIP, dichlorophenolindophenol; PS I and II, photosystem I and II; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CF0CF1, coupling factor 0-coupling factor 1.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS AND DISCUSSION
  5. Conclusion
  6. REFERENCES
  • 1
    R. L. Dean (1996) The Hill reaction of photosynthesis in isolated chloroplasts: a quantitative approach, Am. Biol. Teach. 58, 303306.
  • 2
    R. L. Dean (2002) It's laboratory class time, do you know what your buffer is doing? Am. Biol. Teach. 64, 620627.
  • 3
    R. Amir, P. Tamir (1994) In-depth analysis of misconceptions as a basis for developing research-based remedial instruction: the case of photosynthesis, Am. Biol. Teach. 56, 94100.
  • 4
    S. Izawa, N. E. Good, in A.San Pietro, Ed. (1972) Methods in Enzymology, Vol. 24, Academic Press, New York, pp. 355377.
  • 5
    N. E. Good, in A.Trebst, M.Avron, Eds. (1977) Encyclopedia of Plant Physiology, Vol. 5, Springer-Verlag, Heidelberg, Germany, pp. 429436.
  • 6
    B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts, J. D. Watson (1994) Molecular Biology of the Cell, 3rd ed., Garland, New York.