Expected Student Lab Report

Abstract:
The process of photosynthesis has been known to exist for a very long time. When in the
presence of light, the leaves and green parts of plants will produce organic compounds, mainly
sugars, and oxygen gas. In a sense, photosynthesis is the complement of cellular respiration.
Instead of breaking down organic molecules into waste products, gases, and energy,
photosynthesis uses the suns, or any other light sources, energy and other compounds to store
chemical energy into organic molecules. However, photosynthesis is not the complete reversal of
the complex reactions in cellular respiration. Thus, the stages and processes of photosynthesis
have been obscure for quite some time.
general equation: 6CO2 + 12H2O + light energy C6H12O6 + 6O2 + 6H2O
In the 1930s, Dr. C. B. van Niel of Stanford University investigated photosynthesis in
bacteria. Using radioactive labeling of heavy oxygen isotopes, he was able to deduce the origin
and destination of oxygen atoms throughout the process of photosynthesis. With these
radioactive tracers, modern scientists have been able to understand the steps of photosynthesis.
In addition, the origins and fates of carbon and hydrogen atoms have also been able to be
deduced. This investigation will model the radioactive tracing of reactant substances using a
chemical modeling set. Students will hypothesize, based on prior knowledge of photosynthesis,
the origins and final destinations of carbon, hydrogen, and oxygen in the production of G3P, a
three carbon sugar used to form glucose.
Hypothesis:
- If CO2 and H2O are the precursor reactants for the process of photosynthesis and G3P is
composed of carbon, hydrogen, and oxygen, then the carbon and oxygen atoms present in
G3P come from CO2 only because the oxygen atom in H2O is released as gas in the light
reactions.
- If CO2 and H2O are the precursor reactants for the process of photosynthesis and G3P is
composed of carbon, hydrogen, and oxygen, then the hydrogen atoms present in G3P
come from H+ ions present in the chloroplast stroma because excess H+ ions from the
light reactions diffuse into the stroma. Here, H+ reacts with G3P and is phosphorylated in
a chain of complex organic reactions to become RuBP, an important molecule for carbon
fixation and ultimately sugar production.
Variables:
- Independent Variable: radioactive isotope
- Dependent Variable: origin, locations, and destinations of C, H, and O atoms, respectively
- Controls: 1 radioactively labeled isotope per trial, color of radioactive isotope, process of
light reactions, process of Calvin cycle, chemical modeling set
Procedures:
1 Build the models for CO2 and H2O. Use black atoms for carbon, red atoms for oxygen,
and white atoms for hydrogen.
2 Pick the carbon atom in CO2, the hydrogen atoms in H2O, the oxygen atoms in CO2, or
the oxygen atom in H2O. Remove the selected atom(s) and replace it with a radioactive

4
5
6
7
8
9

isotope. Radioactive hydrogen atoms are orange and radioactive carbon/oxygen are
green.
Begin with the light reactions. Split the two hydrogen atoms off of H2O and replicate the
binding of H+ to NADP+ to form NADPH by placing one atom of hydrogen on the
NADP+ card. Simulate the production of ATP by passing a hydrogen atom through the
ETC card.
Now, begin the Calvin cycle. Use one molecule of CO2 and with Rubisco, create 2
molecules of 3-phosphoglycerate.
Using a molecule of ATP, phosphorylate 3-phosphoglycerate by removing the first carbon
and attaching a phosphate group.
Using a molecule of NADPH, reduce the newly formed 1,3-biphosphoglycerate into G3P.
G3P will be phosphorylated by ATP and react with hydrogen to become RuBP again.
Repeat steps 1-7 twice, radioactively labeling the other two atoms not chosen yet.
In steps 1-7, record the locations of radioisotopes in the given data table. Only enter in
data in the column of the atom that is currently being traced; this is done in order to
emulate the nature of radioactive labeling since only that one atom will be focused on.

Answers to Analysis Questions

1 Carbon and oxygen originate from CO2 gas, which enters the plant through leaf stomata.
Combined with RuBP by catalysis through rubisco, carbon fixation occurs, where this
CO2 can be used in the formation of sugars in the Calvin cycle.
Hydrogen originates from H2O, which is carried to the site of photosynthesis by vascular
tissue or direct contact with water by osmosis. When light strikes photosynthetic
pigments in the photosystems located in the thylakoid membranes, the splitting of water
occurs, which releases oxygen gas, hydrogen ions, and electrons. The electrons follow
through a cytochrome complex and eventually combine with NADP+ to become
NADPH. O2 is released as a waste product from the light reactions. Lastly, the hydrogen
ions are released into the thylakoid lumen. The H+ ions are used to generate ATP through
chemiosmosis by means of ATP synthase. This causes H+ to leave the thylakoid lumen
and enter the stroma of the chloroplast, where the Calvin cycle occurs. Hydrogen
becomes incorporated into sugars at the end of the Calvin cycle, where G3P is converted
back into RuBP. During the complex organic reactions of the Calvin cycle, G3P is
phosphorylated by ATP and reacts with the H+ ions in the stroma to eventually regenerate
RuBP. RuBP is then used to generate more sugars as it accepts CO2 for fixation.
2 NADPH serves to reduce the intermediates of the Calvin cycle by donating electrons.
ATP serves to phosphorylate the intermediates of the Calvin cycle by undergoing
hydrolysis to remove one its phosphate group so that an intermediate can be
phosphorylated. Reduction and phosphorylation are necessary processes for the Calvin
cycle, and ultimately the entire process of photosynthesis, to occur.
3 Reduction, or the receiving of electrons, adds potential energy to an organic compound.
In the Calvin cycle, the intermediates after carbon fixation undergo reduction by means
of the reducing agent NADPH. When NADPH oxidizes to become NADP+ + H+, the
intermediates gain potential energy by the addition of electrons. These are the same
food electrons that are used to reduce NAD+ to NADH in cellular respiration to
generate ATP.

Photosynthesis and cellular respiration are similar in that they use oxidation-reduction
reactions in order to transfer and move free energy. This transfer of electrons to store and
release free energy through organic compounds is key for cellular work and ultimately
the proliferation of life. Fundamentally, photosynthesis and cellular respiration are
different in that one stores energy in organic compounds and the other releases energy in
organic compounds, respectively.
From this investigation, it is known that carbon originates from CO2. On a hot day, plants
will close their stomata in an effort to prevent dehydration. In the process though, CO2
will not be able to enter the plant, and O2 cannot leave the plant as waste. During the
Calvin cycle, rubisco has an ambiguous affinity for CO2 and O2. Without new input of
CO2 and a continual production of O2 in the hot sun, rubisco will bind RuBP to O2
instead of CO2. This lowers the efficiency of photosynthesis in a process known as
photorespiration.
From this investigation, it is known that hydrogen originates from H2O in the splitting of
water in the light reactions. In the dark, plants do not have access to sufficient light
energy in order to split H2O into H+ and 1/2O. Without the production of H+ ions, ATP
cannot be produced because there are not enough ions to form a gradient (there also will
not be enough electrons to travel through the ETC). Additionally, no NADPH will be able
to form and the plant will lose its ability to regenerate ribulose biphosphate (RuBP), an
essential carbon acceptor in the Calvin cycle that is partly responsible for carbon fixation.
From this investigation, it is known that oxygen originates from CO2, which enters the
plant through the stomata. Growing a plant with carbon monoxide (CO) would not be a
sufficient means for the plant to incorporate oxygen into sugars. Although CO differs
from CO2 by just one oxygen atom, the production of sugars would not even occur
because RuBP carboxylase (rubisco) catalyzes the reaction between rubisco and CO2.
Enzymes are proteins that have specific active sites in which specific substrates must
bind to during catalysis. Since CO2 and CO have different shapes, carbon fixation will
not occur and sugar production will stop.

Data and Results:

Stage of Photosynthesis
Hydrolysis of H2O

Location of Carbon
N/A

Location of Hydrogen
bonded in H2O at

Location of Oxygen
bonded in H2O at

first; enters thylakoid

first; leaves through

stomata
N/A

Chloroplast

N/A

lumen
thylakoid lumen and

Chemiosmosis
NADPH Formation
Carbon Fixation

N/A
bonded in CO2;

chloroplast stroma
bonded to NADP+
free ions in chloroplast

N/A
bonded in CO2;

enters through

stroma; also held by

enters through

stomata
bonded to RuBP

NADPH
free ions in chloroplast

stomata
bonded to RuBP

Intermediate

Phosphorylation

Intermediate Reduction

Formation of G3P

Reformation of RuBP

and a phosphate

stroma; also held by

and a phosphate

group; remains in

NADPH; bonded in

group; remains in

stroma

Calvin cycle

stroma

bonded to RuBP,

intermediates
free ions in chloroplast

bonded to RuBP,

phosphate group,

stroma; hydrolyzed

phosphate group,

and electrons;

from NADPH; bonded

and electrons;

remains in stroma

in Calvin cycle

remains in stroma

rearranged in the

intermediates
free ions in chloroplast

rearranged in the

formation of

stroma; hydrolyzed

formation of

sugars; remains in

from NADPH; bonded

sugars; remains in

stroma

in Calvin cycle

stroma

sugars released for

intermediates
reacts with G3P in the

sugars released for

plant use; some

stroma

plant use; some

remains in stroma

remains in stroma

during organic

during organic

reactions

reactions

Analysis/Conclusion:
- Possible Sources of Error:
Deviations from the expected results may arise from improper tracing of
radioisotopes. For example, the oxygen atom that is split from H2O may have
accidentally been followed instead of the oxygen atom from CO2. Although both the
oxygen atoms in H2O and in CO2 are to be labeled, it must be recognized that it is the
purpose of photosynthesis to produce organic sugars; thus, we must follow the oxygen
atom that will be incorporated into the sugars. The origin of hydrogen within the
produced sugars may have also been traced incorrectly. It must be remembered that when
NADPH oxidizes or when chemiosmosis occurs in the light reactions, the hydrogen

atoms and ions are still present in the chloroplast stroma. These are then incorporated into
the Calvin cycle to produce sugars when it is used to reform RuBP.
From this experiment, it was determined through a radioactive tracing model that carbon
and oxygen originate from carbon dioxide and hydrogen originates from water. Carbon
and oxygen are directly incorporated into photosynthetic sugars by means of the Calvin
cycle. Hydrogen is incorporated into photosynthetic sugars by the means of organic
reactions that generate RuBP from G3P, which is a necessary precursor for carbon
dioxide to bond to in order to create sugars in the Calvin cycle.