The Rate of Photosynthesis via Hill Reaction in Different Light Intensities - Research Paper

Abstract

The purpose of this experiment was to observe the rate of Hill Reaction under different types of lights with different intensities. Chloroplasts derived from fresh spinach leaves were isolated in the laboratory before subjecting the supernatant to visible spectra. Green and red light were used to investigate the differential Hill Reaction of chloroplasts under different types of light. The study found that the rates of Hill Reaction in both green and red lights were different. The study concludes that for optimal photosynthesis to occur, both red and infrared spectra must be available for the plant with photosynthesis being more efficient under the green light because it is slower.

Introduction

During the light-dependent reaction of photosynthesis, plants oxidize water to provide electrons for the chlorophyll pigment. An artificial electron receptor i.e. 2,6 dichlorophenolindophenol is used to accept electrons in the Hill Reaction. 2,6 dichlorophenolindophenol loses its color when it is reduced as follows:

H2O + 2,6 dichlorophenolindophenol (oxid.) 2,6 dichlorophenolindophenol (red.) + O2

BLUE COLOR COLORLESS

The aim of this experiment was to observe the rate of Hill Reaction under different types of lights with different intensities.

Methods

All glassware and the mortar and pestle were pre-cooled in ice bath. Fresh spinach leaves were selected and large veins removed by ripping out the leaf parts. The large veins were discarded and 6.0 grams of the deveined leaf tissue weighed. The leaves were chopped finely with a razor blade. The tissue was added to the previously precooled mortar containing 15ml of ice-cold Tris-NaCl buffer and ground to a fine paste for at least 3 minutes. A piece of cheesecloth about 4 inches wide was folded in half and placed over a cooled beaker upon which the solution was filtered by squeezing the tissue pulp to recover most of the liquid. 6ml of this fluid was transferred to into six Eppendorf tubes (each containing 1 ml) and the tubes labeled appropriately. The tubes were centrifuged at 200xg (2000 rpm) for 1 minute to pellet the unbroken cells and cell fragments. The supernatant was decanted into six clean tubes and the pellets discarded. The supernatant was centrifuged once more for 7 minutes at 10000 rpm. The resulting supernatant was discarded as waste. 0.75ml of ice-cold Tris-NaCl buffer was added to each pellet and then resuspended by pipetting up and down. All the six pellets were put together into one glass test tube and then placed in an ice bucket to keep the chloroplast cold. The spectra of the samples were then taken under green light and red light and the results recorded in a table.

Results

Table 1

Hill reaction in green light

Time (seconds) Hill Reaction in Bright Light (A600) Hill Reaction in Medium Light (A600) Hill Reaction in Low Light (A600)

0 0.581 0.761 0.721

20 0.423 0.669 0.683

40 0.300 0.610 0.646

60 0.184 0.544 0.599

80 0.095 0.493 0.561

100 0.043 0.444 0.541

120 0.001 0.405 0.481

Table 1 shows the hill reaction under green light from zero seconds to until the 120th second. A quick scan through the table shows that the reaction reduces with time all light intensities with the fast reaction occurring under the bright green light.

Figure 1: Hill reaction under green light of varying brightness

Figure 1 shows the hill reaction trends with time. According to the graph, hill reaction under bright light is almost zero (0) by the end of 120 minutes. Although the reaction decreases for the medium and low light respectively, the graph shows the least Hill reaction activity when chloroplasts are subjected to green light of low intensity.

A change in absorbance (DA) was calculated by subtracting each number from the absorbance of that tube at 0 seconds to represent the rate of photosynthesis. Table 2 shows the resultant data from the subtraction process. It is worth noting that LED bulbs were not used in this experiment and heat may have affected the results.

Table 2

The rate of Photosynthesis via Hill Reaction in Different Light Intensities:

Time (seconds) Rate of Photosynthesis in Bright Light (DA600) Rate of Photosynthesis in Medium Light (DA600) Rate of Photosynthesis in Low Light (DA600)

0 0 0 0

20 -0.158 -0.092 -0.038

40 -0.281 -0.151 -0.075

60 -0.397 -0.217 -0.122

80 -0.486 -0.268 -0.16

100 -0.538 -0.317 -0.18

120 -0.58 -0.356 -0.24

According to Table 2, the rate of photosynthesis is zero at in the absence of any form of light. However, the rate increases with time, the highest activity is observed at the end of 120 seconds for all the green lights. Figure 2 shows vividly the trends and differences in the individual rates of photosynthesis under different light intensities.

Figure 2. The different photosynthesis rates in different light intensities. The graphs show that the rate increases with time, the hill reaction in bright light being the fastest and that in low light the slowest.

Table 3

Hill reaction under red light

Time (seconds) Hill Reaction in Bright Light (A600) Hill Reaction in Medium Light (A600) Hill Reaction in Low Light (A600)

0 0.547 0.696 0.415

20 0.379 0.576 0.376

40 0.220 0.517 0.318

60 0.085 0.406 0.268

80 0.019 0.326 0.225

100 -0.005 0.236 0.152

120 -0.019 0.214 0.135

Table 3 shows the same Hill reaction trends as in Table 1. However, the reaction is more intense under the red light than the green light. A better representation of this data is shown in Figure 3 as follows:

Figure 3: Hill reaction under red light. The graphs show that the reaction decreases with time. As compared with the reaction under green light, the slopes appear steeper implying that the underlying processes are hastened by the red light.

A change in absorbance (DA) was calculated as before by subtracting each number from the absorbance of that tube at 0 seconds to represent the rate of photosynthesis. Table 4 shows the resultant data from the subtraction process. Figure 4 was derived from this data.

Table 4

Rate of Photosynthesis via Hill Reaction in Different Light Intensities

Time (seconds) Rate of Photosynthesis in Bright Light (DA600) Rate of Photosynthesis in Medium Light (DA600) Rate of Photosynthesis in Low Light (DA600)

0 0 0 0

20 -0.168 -0.12 -0.039

40 -0.327 -0.179 -0.097

60 -0.462 -0.29 -0.147

80 -0.528 -0.37 -0.19

100 -0.552 -0.46 -0.263

120 -0.566 -0.482 -0.28

Figure 4. The rate of Photosynthesis via Hill Reaction in Different Light Intensities.According to the data in Table 4 and the resulting graphs in Figure 4, the rate of photosynthesis is fastest under bright red light and slowest under the low red light. The slopes of these graphs are steeper indicating that the rate of photosynthesis is faster in red light that in green light.

Discussion

After a successful experiment, the results show that the Hill Reaction is light dependent. This experiment showed that in the absence of light, the first step of photosynthesis is practically impossible. In other words, plants need light in order to activate the photosynthetic cycle, a phenomenon that was observed at zero seconds when the reaction was zero but started taking place upon introduction of light. This data also presented another interesting observation that photosynthesis in the chloroplasts is affected by the specific wavelength of the light. For example, red light has a longer wavelength than the green light. In this experiment, the rate of Hill Reaction was higher under red light than under green light. This suggests that for optimal photosynthesis to occur, both red and infrared spectra must be available for the plant. However, photosynthesis is more likely to be more efficient under green light than red light because it is slower (Terashima et al., 2009).

References

Bio207- Cell Biology Lab Manual, 2012.

Terashima I, Fujita T, Inoue T, Chow WS, Oguchi R (2009) Green light drives leaf photosynthesis more efficiently than red light in strong white light: revisiting the enigmatic question of why leaves are green. Plant Cell Physiol 50: 684697