BIOS 100 - Investigating Form and Function in Photosynthesis

Determining the Absorption Spectrum of Spinach Extract

For this portion of the laboratory, you used a spectrophotometer to measure the absorption spectrum of an extract prepared from spinach leaves. The absorption spectrum of the spinach extract reflects the wavelengths of light most useful by the plant in photosynthesis.

To prepare the absorption spectrum, measure the absorption of the extract from 400 to 650 nm at 25nm intervals. Then, plot the absorption values versus wavelength. When completed, the graph should look like this:

Be sure you indicate which wavelenghts of light are most conducive to photosynthesis.


Measuring Photosynthesis Efficiency as a Function of Light Intensity

For this experiment, you used O2 production as an indicator of photosynthetic activity. The photosynthetic process utilizes CO2 as a reactant and produces O2 as a waste product. You measured the photosynthetic rate of Elodea, an aquatic plant, in a solution saturated with HCO3-. The HCO3- can serve as a source of CO2, but since this is dissolved in solution, it's consumption does not appreciably alter the volume of the solution. The gaseous waste product of photosynthesis, O2, does not dissolve in the solution. Instead, the O2 bubbles to the top of the system, forming a large air bubble. The air bubble forces the solution out of the pipette, which you measured as an indicator of photosynthetic activity.

Intensity of light is a measure of the amount of photons striking a given area over a given period of time. The most common unit of measurement of intensity used by plant physiologists is umol m-2 s-1 (read: micromoles of photons per meter squared per second - one mole = 6 x 1023).

In this experiment, we are measuring how increasing the amount of photons striking the leaf will affect the photosynthetic rate. 

Data Analysis

The first step in analyzing the data is to prepare a graph of the volume of O2 produced versus time for the three intensities. When you are done, your graph should look like this:

From this graph, you can determine the photosynthetic rate for each of the three intensities by taking the slope of each line. Once you have done this, prepare a second graph plotting photosynthetic rate versus light intensity. When you are done, your graph should look like this:

You should note that this line does not pass through the origin. Instead, it crosses the x-axis at what is called the photosynthetic compensation point. The photosynthetic compensation point is the intensity of light required for no net carbon assimilation, meaning that the CO2 consumed by photosynthesis is exactly the same amount produced by respiration. At light intensities above the photosynthetic compensation point, the plant is taking in more carbon that it is producing as a waste product by its metabolism. At light intensities below the photosynthetic compensation point, the plant is actually losing carbon!

To mathematically determine the photosynthetic compensation point, use the equation of the line, set y=0 and solve for x

        0 = 0.00001 x - 0.0003

        x = 30 umol / m2 s

Note:  There is no such thing as a negative compensation point (this would imply that the plant is gaining energy from photosynthesis in total darkness).  This lab sometimes doesn't work for one reason or another and when you solve for x in the above equation, you may find that the compensation point is negative.  You should interpret this as an illogical result and simply state that you cannot determine the compensation point with your dataset and recommend that you perform the experiment again

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Frequently Asked Questions

1. If I took two plants and grew one under green light and the other one under blue light, would the blue-light plant grow better?

Yes, the plant grown under the blue light would grow better. The absorption spectrum that you generated indicates that the chloroplasts absorb blue light much more readily than green light, meaning that more photons will be available to excite the photosystems.


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