Discovering the Secrets of Photosynthesis
van Helmont
Perhaps the first experiment
designed to explore the nature of
photosynthesis was that reported
by the Dutch physician van
Helmont in 1648. Some years
earlier, van Helmont had placed
in a large pot exactly 200 pounds
(91 kg) of soil that had been
thoroughly dried in an oven.
Then he moistened the soil with
rain water and planted a 5-pound
(2.3 kg) willow shoot in it. He
then placed the pot in the ground
and covered its rim with a perforated iron plate. The perforations allowed
water and air to reach the soil but lessened the chance that dirt or other debris
would be blown into the pot from the outside.
For five years, van Helmont kept his plant watered with rain water or distilled
water. At the end of that time, he carefully removed the young tree and found
that it had gained 164 pound, 3 ounces (74.5 kg). (This figure did not include
the weight of the leaves that had been shed during the previous four autumns.)
He then redried the soil and found that it weighed only 2 ounces (57 g) less
that the original 200 pounds (91 kg). Faced with these experimental facts, van
Helmont theorized that the increase in weight of the willow arose from the
water alone. He did not consider the possibility that gases in the air might be
involved.
Joseph Priestley
The first evidence that gases participate in photosynthesis was reported by
Joseph Priestley in 1772. He knew that if a burning candle is placed in a
sealed chamber, the candle soon goes out. If a mouse is then placed in the
chamber, it soon suffocates because the process of combustion has used up all
the oxygen in the air — the gas on which animal respiration depends.
However, Priestley discovered that if a plant is placed in an atmosphere
lacking oxygen, it soon replenishes the oxygen, and a mouse can survive in
the resulting mixture. Priestley thought (erroneously) that it was simply the
growth of the plant that accounted for this.
Ingen-Housz
It was another Dutch physician, Ingen-Housz, who discovered in 1778 that the
effect observed by Priestley occurred only when the plant was illuminated. A
plant kept in the dark in a sealed chamber consumes oxygen just as a mouse
(or candle) does.
Ingen-Housz also demonstrated that only green parts of plants liberated
oxygen during photosynthesis. Nongreen plant structure, such as woody
stems, roots, flowers, and fruits actually consume oxygen in the process of
respiration. We now know that this is because photosynthesis can go on only
in the presence of the green pigment chlorophyll.
Jean Senebier
The growth of plants is accompanied by an increase in their carbon content. A
Swiss minister, Jean Senebier, discovered that the source of this carbon is
carbon dioxide and that the release of oxygen during photosynthesis
accompanies the uptake of carbon dioxide. Senebier concluded (erroneously
as it turned out) that in photosynthesis carbon dioxide is decomposed, with the
carbon becoming incorporated in the organic matter of the plant and the
oxygen being released.
CO2 + H2O → (CH2O) + O2
(The parentheses around the CH2O signify that no specific molecule is being
indicated but, instead, the ratio of atoms in some carbohydrate, e.g., glucose,
C6H12O6.) The equation also indicates that the ratio of carbon dioxide
consumed to oxygen release is 1:1, a finding that was carefully demonstrated
in the years following Senebier's work. Using glucose as the carbohydrate
product, we can write the equation for photosynthesis as
6CO2 + 6H2O → C6H12O6 + 6O2
F. F. Blackman
The above equation shows the relationship between the substances used in and
produced by the process. It tells us nothing about the intermediate steps. That
photosynthesis does involve at least two quite distinct processes became
apparent from the experiments of the British plant physiologist F. F.
Blackman. His results can easily be duplicated by using the setup on the left.
The green water plant Elodea (available wherever aquarium supplies are sold)
is the test organism. When a sprig is placed upside down in a dilute solution of
NaHCO3 (which serves as a source of CO2) and illuminated with a flood lamp,
oxygen bubbles are soon given off from the cut portion of the stem. One then
counts the number of bubbles given off in a fixed interval of time at each of
several light intensities. Plotting these data produces a graph like the one
below.
Since the rate of photosynthesis does not continue to increase indefinitely with
increased illumination, Blackman concluded that at least two distinct
processes are involved: one, a reaction that requires light and the other, a
reaction that does not. This latter is called a "dark" reaction although it can go
on in the light. Blackman theorized that at moderate light intensities, the
"light" reaction limits or "paces" the entire process. In other words, at these
intensities the dark reaction is capable of handling all the intermediate
substances produced by the light reaction. With increasing light intensities,
however, a point is eventually reached when the dark reaction is working at
maximum capacity. Any further illumination is ineffective, and the process
reaches a steady rate.
This interpretation is strengthened by repeating the experiment as a somewhat
higher temperature. Most chemical reactions proceed more rapidly at higher
temperatures (up to a point). At 35°C, the rate of photosynthesis does not level
off until greater light intensities are present. This suggest that the dark
reaction is now working faster. The fact that at low light intensities the rate of
photosynthesis is no greater at 35°C than at 20°C also supports the idea that it
is a light reaction that is limiting the process in this range. Light reactions
depend, not on temperature, but simply on the intensity of illumination.
The increased rate of photosynthesis with increased temperature does not
occur if the supply of CO2 is limited. As the figure shows, the overall rate of
photosynthesis reaches a steady value at lower light intensities if the amount
of CO2 available is limited. Thus CO2 concentration must be added as a third
factor regulating the rate at which photosynthesis occurs. As a practical
matter, however, the concentration available to terrestrial plants is simply that
found in the atmosphere: 0.035%.
Van Niel
It was the American microbiologist Van Niel who first glimpsed the role that
light plays in photosynthesis. He studied photosynthesis in purple sulfur
bacteria. These microorganisms synthesize glucose from CO2 as do green
plants, and they need light to do so. Water, however, is not the starting
material. Instead they use hydrogen sulfide (H2S). Furthermore, no oxygen is
liberated during this photosynthesis but rather elemental sulfur. Van Niel
reasoned that the action of light caused a decomposition of H2S into hydrogen
and sulfur atoms. Then, in a series of dark reactions, the hydrogen atoms were
used to reduce CO2 to carbohydrate:
CO2 + 2H2S → (CH2O) + H2O + 2S
Van Niel envisioned a parallel to the process of photosynthesis as it occurs in
green plants. There the energy of light causes water to break up into hydrogen
and oxygen. The hydrogen atoms are then used to reduce CO2 in a series of
dark reactions:
CO2 + 2H2O → (CH2O) + H2O + O2
If this theory is correct, then it follows that all of the oxygen released during
photosynthesis comes from water just as all the sulfur produced by the purple
sulfur bacteria comes from H2S. This conclusion directly contradicts
Senebier's theory that the oxygen liberated in photosynthesis comes from the
carbon dioxide. If Van Niel's theory is correct, then the equation for
photosynthesis would have to be rewritten:
6CO2 + 12H2O → C6H12O6 + 6 H2O + 6O2
In science, a theory should be testable. By deduction, one can make a
prediction of how a particular experiment will come out if the theory is sound.
In this case, the crucial experiments needed to test the two theories had to
await the time when the growth of atomic research made it possible to
produce isotopes other than those found naturally or in greater concentrations
than are found naturally.
Samuel Ruben
In air, water and other natural materials containing oxygen, 99.76% of the
oxygen atoms are 16O and only 0.20% of them are the heavier isotope 18O. In
1941, Samuel Ruben and his coworkers at the University of California were
able to prepare specially "labeled" water in which the 0.85% of the molecules
contained 18O atoms. When this water was supplied to a suspension of
photosynthesizing algae, the proportion of 18O in the oxygen gas that was
evolved was 0.85%, the same as that of the water supplied, and not simply the
0.20% found in all natural samples of oxygen (and its compounds like CO2).
EXPERIMENT
1.
2.
START
FINISH
START
FINISH
% 18O FOUND IN
H2O CO2 O2
0.85 0.20 —
0.85 0.61* 0.86
0.20 0.68 —
0.20 0.57 0.20
* A non-biochemical exchange of oxygen atoms between the water and the
bicarbonate ions used as a source of CO2 explains the uptake of the isotope by
CO2 in the first experiment.
These results clearly demonstrated that Senebier's interpretation was in error.
If all the oxygen liberated during photosynthesis comes from the carbon
dioxide, we would expect the oxygen evolved in Ruben's experiment to
contain simply the 0.20% found naturally. If, on the other hand, both the
carbon dioxide and the water contribute to the oxygen released, we would
expect its isotopic composition to have been some intermediate figure. In fact,
the isotopic composition of the evolved oxygen was the same as that of the
water used.
Ruben and his colleagues also prepared a source of carbon dioxide that was
enriched in 18O atoms. When algae carried out photosynthesis using this
material and natural water, the oxygen that was given off was not enriched
in 18O. It contained simply the 0.20% 18O found in the natural water used. The
heavy atoms presumably became incorporated in the other two products
(carbohydrate and by-product water).
These experiments lent great support to Van Niel's idea that one function of
light in photosynthesis was the separation of the hydrogen and oxygen atoms
of water molecules. But there remained to work out just how the hydrogen
atoms were made available to the dark reactions. The process is described
in Photosynthesis: The Role of Light.
The details of the dark reactions of photosynthesis are described
in Photosynthesis: Pathway of Carbon Fixation
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Photosynthesis_history.html