Analysis of Photosynthetic Membranes & Energy Production in Plants
Basis in Organelle Isolation, Photosynthesis Controls, and Membrane Energy Analysis with 2,4-Dinitrophenol Inhibitor
Photosynthesis is a vital chemical reaction for plants. The Hill reaction plays an important role in the overall scheme, transferring electrons, cleaving water, and consequently freeing oxygen. Effects of the inhibitor molecule 2,4-dinitrophenol (DNP) on multiple isolated chloroplast concentrations were assayed using a redox dye, 2,6-dichlorophenolindophenol (DCPIP). Suspected results proved true, with the expected outcomes showing the inhibitor to hinder photosynthetic reactions. The reduction of DCPIP was shown to cease throughout each sample where the inhibitor was added, showing a significant decrease in the reaction rate for chloroplasts.
Phototrophic organisms are able to capture photons, or light, in order to acquire energy. (Stern, 2008) The energy obtained from light allows them to operate and function at a cellular level metabolically, enabling cellular reactions through, primarily, ATP production. One of the important pathways sustained by this enables the photosynthetic organisms to store chemical energy in the form of sugars and carbohydrates. Carbon dioxide and water is utilized as the building blocks for these materials, and supports a process that is fundamental for all life on earth to thrive, supplying and maintaining the atmospheres level of oxygen. Such carbon fixation provides the energy and organic foundation for biomes globally.
Photosynthesis occurs in specialized double-membrane intracellular organelles known as chloroplasts. (Collins, 2014) Performed differently by different species, photosynthesis contains a multitude of variables concerning reaction centers, differences in photo-reactionary plastids (dependent upon proplastid differentiation), and carbon fixation cycles, to name a few. (Blankenship, 2014) The overall reaction is typically split into two stages, one light-dependent, the light reaction, and one light-independent, or the dark reaction. (Voet, 2013) The first harnessing the light energy in order to oxidize water, and the second using those free electrons to reduce carbon dioxide.
The overall reaction is seen in equation 1 below, depicting the general formula for photosynthesis in plants.
6CO2 + 6H2O + Energy –> C6H12O6 + 6O2
This all occurs across the thylakoid membrane within the chloroplast organelle, where the principal photoreceptors, chlorophyll, are found in abundance throughout the membrane. These green pigments primary functions are to gather light (Voet, 2013) Once absorbed, photons migrate across the photosynthetic antenna complexes until they happen upon a reaction center, and can enter into photosystem II (PSII), and then photosystem I (PSI).
These are large protein complexes. The reaction begins by PSII absorbing a photon thereby energizing an electron. At this higher energy level, the electron is then transferred by plastoquinone to PSI which absorbs more light and further increases the electron energy state. (Collins,2014) Photolysis follows, oxygen is freed along with hydrogen ions (H+), which create a chemical-electrical gradient capable of driving ATP synthesis. While the electrons produced ultimately reduce nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH. (Voet,2013)
2,6-dichlorophenollindophenol (DCPIP), is a chemical commonly used as a redox dye, appearing blue before being reduced, and changing to a colorless solution after reaction. This is depicted in the chemical equation below. DCPIP acts as a Hills Reagent when placed into a photosynthetic reaction (Stern,2008). These are molecules which aided in the discovery of photosynthesis, accepting electrons off the electron transport chains on the thylakoid membranes, due to their higher affinity in comparison to competing molecules such as ferredoxin, (Blankenship, 2014) reducing it as a substitute for NADP+ at the end of photosynthesis.
pH gradients are the driving forces of ATP synthesis. Electrochemical gradients across membranes make photosynthesis and cellular respiration possible in the chloroplast and mitochondria of eukaryotic cells. Prokaryotes, though lacking organelles, can use their cell membrane folds to create the gradients they need to sustain life, operating essentially the same type of systems.
Certain chemicals can disrupt this charged gradient. 2,4-dinitrophenol (DNP) is a derivative of 2,4-dinitrochlorobenzene and when in cells acts as an ionophore, binding and shuttling hydrogen ions across biological membranes.(Blankenship,2014) This effectively destroys the proton gradient which chloroplasts, and mitochondria, require in order to produce ATP. The energy taken by DNP becomes lost as heat, and the ATP synthesis is essentially stalled. For these reasons it, and molecules like it (DCMU, etc) are commonly used to explore chemiosmotic issues in biochemical labs.
With this in mind, Photosynthesis activity was measured using isolated chloroplasts at varying concentrations and varying distances from a static light source. Measurements were done in vitro with and without the presence of inhibitors within the chloroplast suspension. The activity was chemically indicated using DCPIP by measuring its reduction through absorbance. Effects of an inhibiting agent were calculated to give an indication of membrane quality, highlighting the need for an intact membrane for photosynthesis to occur (Collins,2014).
Materials & Methods
The published procedure (Collins, 2014) was modified, with the chloroplast isolation and master mix made up as a group versus individual chloroplast solutions, during absorbency readings in weeks 1 and 2 of the experiment. The lab had to be dimmed at times to prevent photosynthesis from occurring in the chloroplast suspension. Distances of 30cm, 60cm, 90cm and 120cm were used for first week measurements. With all data following being collected at 60cm from a static light source.
Data was recorded and analyzed using vernier units with two attached colorimeters to expedite data uptake and help nullify possible error found in time differences between sample measurements. Wavelengths for all these measurements were made at 565nm. The inhibitor used for membrane gradient disruption was 2,4-dinitrophenol (DNP) at concentrations of 0.05mM, 0.07mM, 0.15mM, and 0.20mM.
Measurements of DCPIP reduction via photosynthesis through absorbency readings reveal membrane energy trends within the isolated chloroplasts. Over two-minute intervals these readings depict variations in the membranes ability to create a gradient suitable to energy production, highlighting the membrane efficiencies. In the case of controls and samples with the addition of the inhibitor, the measurements highlighted deficiencies in the photosynthetic membranes when effected by outside variables.
In Graph 1a, DCPIP reduction was measured by absorbance in regards to non-inhibited chloroplast suspension at various distances from the light source. Reduction of DCPIP from the blue colored state to the colorless state occurred rapidly in the samples closest to the light, here being the R1 and R2-30 and R1 and R2-60 assays. Each assay throughout the experiment was duplicated, hence the R1 and R2 readings. Graph 1a is the standard depiction of chloroplast reactions and subsequent DCPIP reduction.
Graph 1a Data Table
In Graph 1b, Membrane efficiency was measured by pH in regards to non-inhibited chloroplast suspension at various distances from the light source. Creation of free hydrogen from photosynthesis occurred at a steady state across the time frame, maintaining a healthy pH gradient along the chloroplast thylakoid membrane. This graph acts as the standard for the fluctuation of pH in accordance to chloroplast reactivity over time at different lengths to a static light source.
Graph 1b Data Table
In Graph 2, The chloroplast reactivity was measured in the presence of the inhibitor molecule, DNP. The reduction of DCPIP which correlated with chloroplast reactivity was present at the initial time but by the second or third minute into each assay the dye reduction plateaus due to the inhibitor’s actions. This is seen in each assay ran regardless of concentration, though the lesser concentrations were able to show some decreases in absorbance at a very small rate of change. All measurements here were done at 60cm
In Graph 3a, Membrane efficiency was again measured in the presence of DNP, differences in DCPIP reduction were noted for the initial time readings, but all assays again plateaued off as they did in Graph 2. The lower inhibitor concentrations, as expected, were the only ones to show slight DCPIP reduction.
Graph 3a Data Table
In Graph 3b, Membrane efficiency was measured by pH in regards to inhibited chloroplast suspensions at 60cm from the light source. Creation of free hydrogen from photosynthesis occurred at a steady state across the time frame, maintaining a fairly stable pH gradient along the chloroplast thylakoid with minimal decrease for the membranes in each control. The one twin assays tested with inhibitor, Series 1 and Series 2 in Graph 3b, show drastic drops in hydrogen ion content outside the chloroplast. Though initial readings at four minutes show a wide variation, by six minutes the pH for each sample had met around 6.1 and continued on, significantly lower than any of the control groups.
Graph 3b Data Table
The reactions rates for assays were graphed in column graphs 1-3. For each column graph, series 1 depicts the concentration of the membrane disruptor, 2,4-dinitrophenol, while series 2 in these assays depict the measured concentration of DCPIP in the solution. These provide good visualizations in regard to the association between inhibitor concentration and DCPIP reduction via photosynthesis.
Table 1- Shows the calculated DCPIP reduction rates (M/min)
Table 2- Shows the calculated rates of mol/min/g of chlorophyll in each assay.
Depicted in Tables 1 and 2 are the calculated reduction rates of DCPIP using the standardized slopes from Graph 1a, along with the M/min/g of chlorophyll seen in Table 2.
***All example calculations are attached via Appendix 1.
It was stated that throughout this experiment, it would be apparent that DNP acted as an inhibiting agent. This was shown to be correct, with DNP acting as an inhibitor during each assay that was ran. There were multiple variables to consider to properly gauge the effects of DNP. Distance from the light source was important, since optimal photoreactivity would be preferred so as to show the greatest effect of the inhibiting agent used. Here 30 and 60cm was found to be the best suited for photosynthesis.
DNP acting as a membrane disruptor is an ionophore that shuttles ions across the electrochemical gradient, neutralizing the environment the chloroplasts require to achieve photosynthetic reactions and produce ATP. The effects of DNP are seen by the difference in the reduction rates of DCPIP in treated samples. This effect would relate to a chloroplast whose membrane integrity was compromised, effectively destroying the chance to create an electrochemical gradient capable of turning ATP synthase.
The reduction rates calculated in M/min are much smaller in comparison to the M/min/g of chlorophyll. Table 2 reduction rates however do not differ in relation to table 1 in respect to the higher reaction rates involving the lower inhibitor concentrations and the lower reaction rates involving the higher inhibitor concentration. These reaction rates could be manipulated with additional treatments. ADP and Pi would allow for a higher reduction rate, though most likely it would be slight since the electrochemical gradient would still be hindered, while addition of electron donors such as ascorbate and β-mercaptoethanol would help overwhelm the influence of the DNP disruption, shifting the electrochemical gradient back to normal, and normalizing the reduction rates to the standard.
Chloroplasts must balance multiple variables and need intact membranes if they are to utilize the electrochemical gradient required for photosynthetic reactions. Therefore DNP and other inhibitors can be applicable in commercial use concerning crop and foliage management. Plant selectivity, long-term environmental chemical degradation rates, and effects on terrestrial organisms and mankind would need to be fully understood should it be used as a herbicidal agent for food production.
- Collins, J., Membranes and Energy Production, Biochemistry I Laboratory Experiments [Online] 2014, 13th edition, week #3. Blackboard. http://blackboard.usi.edu/ (October, 1st, 2014).
- Voet, Donald., Voet, J. G., Pratt, C. W., Fundamentals of Biochemistry, 4th 2013. John Wiley and Sons, Ltd., 268-290,614, 624-634p.
- Stern, Kingsley R., Bidlack, J. E., Jansky, S. H., Introductory Plant Biology, 11th 2008. McGraw-Hill Company, Inc., 40-41p.
- Blankenship, Robert E., Molecular Mechanisms of Photosynthesis, 2nd 2014. John Wiley and Sons, Ltd., 32-101p.
Date: 2 October 2014
CHM431 Section 002