CBL Interfacing
Photosynthesis In Isolated Chloroplasts
Introduction
Imagine a world without photosynthesis. According to the heterotroph
hypothesis that is just what the world was like when life originated
on earth. First consider the food situation: an ocean of "organic
soup" which would eventually be eaten up by the first organisms.
The organic substance would only be replaced by the slow processes
that formed it in the first place. The earth's biota would have
to wait for the first chemosynthetic or photosynthetic organisms
before biosynthesis of organic food could take place.
Second, consider the atmosphere...no molecular oxygen to breathe
in. Many believe that the primitive atmosphere contained only
methane, ammonia, hydrogen and water; hardly the hospitable oxygen
laden air we breathe today.
But photosynthesis changed all that. It allowed the earth's early
life forms to use ever available light energy to synthesize food
( now 150,000,000,000,000 kg per year, globally) and permanently
modify the atmosphere to include oxygen.
Photosynthesis occurs in two major steps, the light reaction and
the dark reaction. The process is summarized below. (Click here if your browser does not support graphics.)
There are three investigations on photosynthesis in this manual.
This, the first involves measuring the light reaction.
The light reaction occurs in the grana membranes of the chloroplast.
It is a sequence of electron transport reactions. In the process
water is "split" resulting in oxygen gas and the hydrogens
being accepted by NADP to form NADPH2. In a cell the NADPH2 is
used in the dark reaction.
However, in today's investigation we are going to provide a substance
which will intercept the hydrogens and accept them instead of
NADP. This artificial hydrogen acceptor is called DPIP.
When DPIP accepts hydrogens it turns from blue to colorless. It
is this color change which we will monitor using a colorimeter.
The colorimeter contains a light source which emits three different
colors (wavelengths)-green (565 nm), red (635 nm) and blue (470
nm). For this experiment we will use the red light. When the red
light shines through our specimen, some of it will be absorbed.
The colorimeter measures how much of the light is absorbed. As
DPIP turns clear, less red light is absorbed. Using chloroplasts
isolated from spinach, we will compare the rate of photosynthesis
in three conditions:
- Unboiled Chloroplasts incubated in the light
- Unboiled Chloroplast incubated in the dark
- Boiled Chloroplast incubated in the light.
For each of these conditions, we will plot the Absorption (the
dependent variable) vs Time (the independent variable. Thus, the
slope of each plot will be a measure of the rate of photosynthesis.
In this investigation we will graphically find the solution to
the questions-How is the rate of photosynthesis affected by light
and the condition of the chloroplasts?
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