A high photosynthetic rate can be interpreted by a fast rate of change from blue to colourless of the DCPIP as more electrons are flowing through the transport chain and reducing the DCPIP. The colour of DCPIP is measured using a spectrophotometer at 605nm. The rate of photosynthesis is dependent on many factors, in particular light quantity (Johkan et al. 2012). A greater quantity of light received by the chloroplasts equates to a greater amount of solar energy potentially converted into ATP. In turn this causes a higher flow of electrons in the transport chain.
The importance of light quantity for photosynthesis is relevant to the growth and harnessing of energy by plants. This can be useful for understanding ecosystems, such as rainforests where the amount of light received by plants is greatly reduced further down the canopy (Lee 1987), and for agriculture as optimum growth conditions can increase production of crops. The hypothesis was that the greater the quantity of light the higher the rate of photosynthesis and hence the faster reduction of DCPIP, as more electrons travel through the transport chain.
The effect of light quantity was answered using isolated chloroplasts exposed to differing intensities of light, with photosynthetic activity measured using DCPIP and a spectrophotometer at 605nm. Method Isolating chloroplast: Approximately 4g of spinach leaf was torn into pieces, removing major veins. The leaf pieces were then placed into a cooled mortar and pestle. 15mL of cold isolation medium was then dispensed into the same mortar and pestle, and the leaf pieces were ground into a smooth paste. Using a funnel and two pieces of gauze, the ground leaf paste was then filtered into a cold 15mL plastic centrifuge sitting on ice.
The plant material was then squeezed to recover as much filtrate as possible. While the filtrate was being centrifuged the mortar and pestle were washed, whilst the funnel and gauze were placed on the bench to dry. Tables were then set up for record keeping. The supernatant was carefully decanted by pouring it into a new, cold 15mL centrifuge tube and stored on ice, being careful not to lose the dark green pellet at the bottom. A sufficient amount of ice-cold isolation medium was added to the tube containing the pellet so that the final volume was approximately 1mL.
If 1mL isolate was already present, no more isolation medium was added. Very carefully, the pellet was re-suspended by swirling and gently flicking the tube. The chloroplast isolate was then stored on ice. Measuring absorbance: The spectrophotometer was blanked at 605nm using a phosphate buffer. Three cuvettes were labeled and 5mL DCPIP was dispensed into each cuvette. Using a pipette, 20uL chloroplast isolate was added to each cuvette, which were then covered with Parafilm and inverted to mix, measuring their absorbance at 605nm (A605) immediately.
Cuvette 1 was placed in a dark cupboard, cuvette 2 in front of a 25W unfiltered light source, and cuvette 3 in front of a 125W unfiltered light source. The A605 was measured every two minutes for a total of 8 minutes for all cuvettes. Results were recorded in a table and the experiment repeated three times. Results Photosynthetic activity of isolated chloroplasts was highest in the samples exposed to the 125W unfiltered light source and lowest in samples kept in darkness (Figure 1). No photosynthetic activity occurred in the chloroplasts which were not exposed to light (Figure 1).
The chloroplasts exposed to the 125W light source increased in photosynthetic activity at an increasing rate for the first four minutes and then steadied (Figure 1). The samples exposed to 25W light recorded a small increase in photosynthetic activity over time (Figure 1). Photosynthetic activity was 50% higher in samples exposed to 125W light source than those exposed to 25W (Figure 1). Figure 1: The effect of light quantity on photosynthesis of isolated chloroplasts of spinach leaf. The absorbance of chloroplasts mixed with DCPIP exposed to differing light quantities was measured using a spectrophotometer at 605nm.
Discussion The results show that photosynthetic activity peaked in samples exposed to 125W light source, and was not present in samples unexposed to light. This shows that light quantity does affect photosynthetic activity as the DCPIP was reduced at a faster rate in samples exposed to high light intensity, suggesting more electron transfer in the isolated chloroplasts. The higher amount of electrons de-colourised the DCPIP at a faster rate, accounting for its decreased absorbance. Hence, greater light quantity results in greater photosynthetic activity.
These results are confirmed by Turnball showing that greater plant growth occurred in plants exposed to the most light (1991). This suggests that photosynthetic activity was higher than in the plants exposed to low light intensities, allowing the production of more ATP and subsequent growth of the plant. Although, plants inhabiting the lower canopy region displayed better growth in low light conditions than those adapted to mid or high canopy regions (Turnball 1991). This infers that photosynthetic level varies between species and habitat as species adapt to different light intensities.
This conclusion is supported by Stroop and Boyer who showed that the rate of ATP synthesis was significantly lower in plants exposed to low light (1987). At 2% light intensity the rate of photophosphorylation, the production of ATP by photosynthesis, was approximately 97% lower than in chloroplasts exposed to 100% light intensity (Stroop and Boyer 1987), supporting the hypothesis. Plant species are adapted to different light intensities depending on their habitat. Species adapted to the shade contain higher chlorophyll content than those residing in sun environments (Grumbach and Lichtenthaler 1982).
Grumbach and Lichtenthaler showed that plants were able to adapt to different light intensities through changing their pigment composition, being chlorophyll and carotenoids, as well as the thickness of the thylakoid membrane (1982). Chloroplasts adapted to high light intensities exhibited few small thylakoid systems, however chloroplasts adapted to low light intensity contained large grana stacks and enlarged photosystems within the thylakoid membrane (Grumbach and Lichtenthaler 1982).
Hence, there are multiple factors, such as habitat, acting on the photosynthetic activity of chloroplasts when exposed to varying light intensities. The experiment conducted only investigated the photosynthetic activity of spinach, which is adapted to relatively high light intensity, accounting for its significantly lowered photosynthetic activity at 25W light in comparison to 125W. To extend research, species adapted to low light should be included in the method to account for the effects of light quantity on a broader range of species and habitat.
To achieve this, species from low regions of the rainforest canopy should be researched and compared to plants from the high and mid canopy. Furthermore, the effects of the entire plant could be observed rather than only the activity of the isolated chloroplasts. More specifically, the growth of the plant exposed to a certain light intensity over a period of time could provide a broader analysis of the effects of light quantity on photosynthesis, such as in Turnball’s experiment (1991).
To further improve methodology, controls are essential. In particular the supernatant, which contained few chloroplasts, could be compared to the pellet to confirm whether chloroplast density has an effect on photosynthetic activity when exposed to different light intensities. Sources of error existed primarily due to timing, as cuvettes containing the solution were exposed to the light for differing periods before measurement by the spectrophotometer. This was due to a delay as only one cuvette could be measured at a time.
To improve this aspect of the method, measurement of the absorbance of the samples could be completed in increments to ensure all cuvettes have exactly the same light exposure before analysis. This research shows the significance of light quantity on the photosynthesis and development of plants. This enhances understanding of the rainforest environment and the photosynthetic levels of plants at different levels of the canopy. It can also be applied to the harvesting of crops, as greater understanding of the requirements for optimum growth is essential for commercial production.
To conclude, light quantity has a significant effect on the photosynthetic activity of isolated chloroplasts of the spinach leaf. High light intensity results in higher photosynthetic activity than in chloroplasts exposed to low light intensity, suggesting greater electron transfer and ultimate reduction of DCPIP. References Dean, R. , Miskiewicz, E. (2006) Rates of electron transport in the thylakoid membranes of isolated, illuminated chloroplasts are enhanced in the presence of ammonium chloride. Biochemistry and molecular biology education, vol. 31: pp. 10 – 417. Grumbach, K. , Lichtenthaler, H. (1982) Chloroplast pigments and their biosynthesis in relation to light intensity. Photochemistry and Photobiology, vol. 35: pp. 209 – 212. Hoober, J. K. (1984) The process of photosynthesis: the light reactions. Chloroplasts. Plenum Press, New York, pp. 79 – 110. Johkan, M. , Shoji, K. , Goto, F. , Hahida, S. , Yoshihara, T. (2012) Effect of green light wavelength and intensity on photomorphogenesis and photosynthesis in Lactuca sativa. Environmental and experimental botany, vol. 75: pp. 128 – 133. Ladiges, P. , Evans, B. Saint, R. , Knox, B. (2010) Biology: an Australian focus, McGraw Hill, North Ryde, NSW. Lee, D. W. (1987) The spectral distribution of radiation in two neotropical rainforests. Biotropica, vol. 19: pp. 161 – 166. Stroop, S. , Boyer, P. (1987) Catalytic and regulatory effects of light intensity on chloroplast ATP synthase. Biochemistry, vol. 26: pp. 1479 – 1484. Turnball, M. (1991) The effect of light quantity and quality during development on the photosynthetic characteristics of six Australian rainforest tree species. Oecologia, vol. 87: pp. 110 – 117.