311243134 INVESTIGATING THE EFFECT OF LIGHT QUANTITY ON THE PHOTOSYNTHESIS OF GREEN LAND PLANTS.

INTRODUCTION Photosynthesis is fundamental towards the maintenance of life. This process, summarized by the equation 6H2O + 6CO2 + light energy C6H12O6 + 6O2, essentially harnesses light energy from the sun and converts it to a form of chemical energy through a complex 2 step reaction procedure. This experiment deals primarily with the first step, known as the light reaction step. Pigments in chloroplast absorb electromagnetic radiation thereby entering into an excited state (Halliwell 1984). Once in this state, their excited electrons are passed down an electron transport chain and are utilised to produce NADPH and ATP with its reducing power. The eukaryotic organelle at the basis of this reaction, on which photosynthesis is wholly dependant, is the chloroplast. The chloroplast can be likened to a factory that houses all the different photosynthetic reactions; hence its subsequent isolation from live tissue and subjection to scientific manipulation is logical in the study of photosynthesis (Paterson and Arntzen 1982). Numerous studies have shown light quantity to be a critical factor affecting the rate of photosynthesis. Lowman (1986) found that reduced light exposure of smaller plants under canopies that shielded incoming light in forests had hindered rates of photosynthesis, consequently resulting in decreased growth. Lowmans experiment however was performed in an environmental setting where variables were less tightly controlled and regimented. This experiment will seek to reproduce a similar investigation under stricter lab conditions by isolating chloroplasts from spinach leaves and exposing the mixture under different intensities of light, a variable which is easily controlled by using lamps of different power readings (Paterson and Arntzen 1982). Following scientific logic and past research, it is expected that photosynthetic activity will be proportional to light intensity as more electromagnetic radiation will be available to excite electrons to drive the electron transport chain. In order to test this hypothesis a 125 W and a 25 W lamp will be utilised along with a 125 W lamp containing a neutral density filter (ND) of 0.6. Photosynthetic activity is proportional to the amount of electrons flowing through the electron transport chain; a way of monitoring this factor is by mixing a blue dye (2,6-dichlorophenol-indophenol, or DCPIP) with the live chloroplast. DCPIP in effect replaces NADPH in the reaction and is reduced in its place. When DCPIP is reduced, its colour changes from blue to clear. It is this property of DCPIP that is monitored in the reaction via measurement with a spectrophotometer.

311243134 METHOD Chloroplast isolate was prepared by grinding fresh spinach mixed with 15mL of cold isolation medium. The paste was filtered through gauze and centrifuged for 5 minutes at 1300xg at 40C and the resulting supernatant was extracted. Ice-cold isolation medium was added to the pellet containing tube until the final volume of 1 ml was achieved, the pellet was resuspended. Both tubes containing supernatant and isolated chloroplast was stored on ice. Microscopic examination of the chloroplast isolate and supernatant was performed. A 5uL of chloroplast was dropped onto a clean glass slide and viewed under a microscope at x40 objective. Chlorophylls were counted under a number of different fields of view and a rough average number of chlorophylls calculated. This process was repeated but with supernatant as the sample the second time. 4 test tubes were set up each containing 5 mL of DCPIP and 20 uL of live chloroplast isolate. Each test tube was exposed to a different light condition. Tube 1 placed in front of a 125 W unfiltered lamp, tube 2 in front of a 25 W lamp, tube 3 in front of a 125 W lamp with a 0.6 neutral density filter and tube 4 in a dark place exposed to no light. Each tube was placed at an equal distance of 30 cm from the light source. Once placed, a timer was started and the absorbance of each test tube was measured every 2 minutes using a spectrophotometer that was readily set to measure absorbance at 605nm, blanked with distilled water. Once 4 readings had been obtained (8 minutes in total) the process was repeated 4 times. Once done, a test was performed with 20 uL of supernatant instead of live chloroplast in the test tubes to act as a control.

RESULTS Photosynthetic activity was shown as a general decrease in absorbance over time as the blue colour of the DCPIP fades to clear as it is oxidised. Figure 1 shows that photosynthetic activity was present in all cases with chlorophyll isolate and exposure to light, the exception being supernatant showing activity under 125 W light. The gradient of the line for 125 W, chloroplast isolate was the steepest showing the most dramatic change, indicating that it was the condition producing most photosynthetic activity followed by 75% reduced light, 25 W and then finally no light producing no marked change in absorbance. The results for the supernatant showed no changes under all conditions excluding 125 W where a decrease in absorbance was seen.

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1 0.9 0.8 0.7 0.6 A605 nm 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6 TIME (minutes) 8 10 125 W 75% Reduced (31 W) 25 W No Light

0.9 0.8 0.7 0.6 A605 nm 0.5 0.4 125 W 0.3 0.2 0.1 0 0 2 4 6 TIME (minutes) 8 10 75% Reduced (31 W) 25 W No Light

Figure 1. Photosynthetic activity of spinach chloroplast isolate (top) and supernatant (bottom) under different intensities of light. Activity was determined by measuring the decrease in DCPIP absorbance at 605 nm using a spectrophotometer.

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0.07 0.06 Rate of Decrease (A605/minute) 0.05 0.04 0.03 0.02 0.01 0 125 W -0.01 75% Reduced 25 W No Light 125W 75% Reduced 25 W No Light

CHLOROPLAST ISOLATE

SUPERNATANT

Figure 2. A comparison of rate of decrease in DCPIP absorbance indicating relative photosynthetic activity in spinach chloroplast isolate and supernatant. Rate was calculated using the results obtained from Figure 1

311243134 Figure 3. Microscopic image of chloroplast isolate (left) and supernatant (right) examined on the x40 objective. Average density was estimated by counting number of chloroplasts (dark green) under different fields of view

A comparison of the average rate of decrease in absorbance and hence photosynthetic activity in figure 2 clearly shows that chloroplast isolate under the brightest light of 125 W resulted in the most activity. The results in figure 2 supports those established in figure 1 however shows great variability with the size of the error bars. For the instances regarding chloroplast isolate under 75% reduced light and 25 W, the error was larger than the calculated result itself. Figure 3 gives us an idea of the amount of chloroplast we were dealing with and confirmed their presence. The chloroplast isolate showed a huge confluent number of viable chloroplasts and a small amount of chloroplasts and other organelles were seen in the supernatant also despite after centrifuging and separation.

DISCUSSION A decrease in absorption of the solution over the observation period is indicative of photosynthesis taking place, as the reduction of DCPIP turns the solution steadily clearer. It also shows that the electron transport chain is working efficiently because DCPIP substitutes as an electron acceptor (NADP+) within the light reaction process (Paterson and Arntzen, 1982). Because of this we can conclude that no change in absorption indicates photosynthesis is not functioning properly or even at all. Regarding this experiment, the case would be a lack of electromagnetic radiation to excite electrons to contribute to the electron transport chain within the chloroplasts. The results clearly indicate that the presence of light and its intensity does indeed impact upon the level of photosynthetic activity in chloroplast containing green spinach. This concept is supported by the control test tube that contained chloroplast but not subjected to light exposure. This test showed no corresponding change in absorbance at 605nm, indicating no photosynthetic activity; this allowed us to conclude that light plays a vital role in driving photosynthesis. Testing with supernatant that should not have contained chloroplasts also highlighted the vital role of the organelle in the process of photosynthesis. The exposure of supernatant to light did not result in any obvious changes in absorbance, however there was slight activity when exposed to the strongest light intensity of 125 W. A subsequent microscopic analysis of the supernatant showed that it was not entirely devoid of chloroplasts and contained many other organelles. We can conclude from this that the absorbance reading

311243134 for 125 W exposed supernatant was a result of the little chloroplast present. Because it was exposed to such a bright intensity of light it produced notable photosynthetic activity compared to the dimmer lights of 75% reduced and 25 W. The results showed steadily increasing rate of decrease of absorbance with increased light brightness when chloroplasts were present. In other words, photosynthetic activity is proportional to light quantity. This confirms the hypothesis made at the start of the experiment and is synonymous to the results of past studies which investigated the effect of light quantity on the photosynthesis of different plants under varying degrees of rainforest canopy and their ability to utilise available light energy (Lowman 1986;Turnbull 1991). Understanding the role that light quantity plays in photosynthesis is vital to improving techniques of agriculture in attempt to yield better crops or ensuring plant survival in reforestation. This experiment showed prominent errors. This could be due to human error or wrong experimental technique. In order to improve the experiment, perform more repetitions or standardise the temperature at which the experiment is conducted.

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REFERENCES:

Halliwell, B. (1984) Structure, function and isolation of chloroplasts. In: Chloroplast metabolism, revised edition (Ed.: B. Halliwell). Oxford University Press, Oxford, 1-30. Hoober, J. K. (1984) The process of photosynthesis: the light reactions. In: Chloroplasts (Ed.: J. K. Hoober). Plenum Press, New York, 79-110.

Lowman, M. D. (1986) Light interception and its relation to structural differences in three Australian rainforest canopies. Australian Journal of Ecology.

Paterson, D. R. and Arntzen, C. J. (1982) Detection of altered inhibition of photosystem II reactions in herbicide-resistant plants. In: Methods in Chloroplast Molecular Biology (Eds. Edelman et al.). Elsevier Biomedical Press, New York.

Turnbull, M. H. (1991) The effect of light quantity and quality during development on the photosynthetic characteristics of six Australian rainforest tree species. Oecologia.