LABORATORY EXPLORATION

The Light-dependent Reactions of Photosynthesis

Plants are autotrophs, self-feeding organisms that capture light energy (photons)
and store it as chemical energy (carbohydrates). During the light reactions of
photosynthesis, the light energy of photons is converted briefly to electrical energy
(the flow of electrons through chlorophylls in the photosystems), and then into chemical
energy (in the bonds of ATP and NADPH). During the Calvin Cycle (sometimes
known as the light-independent reactions, since it can proceed either in dark or light),
the chemical energy briefly stored as ATP and NADPH is transferred for long-term
storage into the bonds of sugar.
I. Photosystems
The light-dependent reactions take place on the membranes of the thylakoids,
located inside the chloroplasts. Chlorophyll and carotenoid pigments are embedded in
the thylakoid membranes to form photosystems, designed to maximize the capture of
photons. A photosystem consists of a few hundred molecules of chlorophyll a,
chlorophyll b, and carotenoid pigments. As these pigments absorb photons, their
electrons are boosted to a higher energy level. In this excited state, electrons pass from
one pigment molecule to another until they reach a specific chlorophyll a molecule
positioned beside a Primary Electron Acceptor protein. At this Reaction Center,
chlorophyll a passes the excited electron to the Primary Electron Acceptor, starting an
electron transport chain that will result in the production of ATP (from ADP) and NADPH
(from NADP+). The energy that was once a photon is now stored as chemical energy in
the bonds of ATP and NADPH.
Note that the oxidation-reduction reaction that takes place between chlorophyll a and
the Primary Electron Acceptor cannot occur unless the two molecules are in their proper
positions in the thylakoid membrane. When you observed fluorescence in your
isolated chlorophyll in the last lab, you were observing the re-release of light energy that
would have been captured by the Primary Electron Acceptor if the chlorophyll was still
normally embedded in a living thylakoid membrane.
Two different photosystems--Photosystem I and Photosystem II--are found on
thylakoid membranes. The main difference between these two systems (named in order
of their discovery) is the absorbance maximum of their respective chlorophyll a
molecules. In Photosystem I, chlorophyll a (also known as P700) has a maximum
absorbance at 700nm, and in Photosystem II, a chlorophyll a (also known as P680)
associated with slightly different proteins absorbs maximally at 680nm. As perceived by
the human eye and brain, what "color" are these two wavelengths? .
II. Non-cyclic Photophosphorylation
In both photosystems, light drives the synthesis of ATP and NADPH by triggering a
flow of electrons through the photosystem pigments and associated proteins. Two
possible routes for electron flow--cyclic and non-cyclic--are known. Of these, plants
use primarily the non-cyclic route, and it is this pathway we will study today. Review the
figure in your text illustrating non-cyclic electron flow, and be sure you understand the
process before you continue.

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A. Overview of Non-cyclic Photophosphorylation in a Live Plant Cell
The essential steps in the light reactions are...
• water molecules are split, producing electrons, protons, and oxygen gas (O2).
• electrons from the water molecules are passed along the electron transport
system on the thylakoid membranes
• as electrons are passed along the chain of proteins, energy is lost at each
transfer, and this is packaged in the high-energy phosphate bonds of ATP.
• some of the protons from the split water molecules reacts with another energy
courier molecule, NADP (nicotinamide adenine dinucleotide phosphate) to form
NADP-H, which stores the energy of the proton.
• both ATP and NADP-H shuttle the energy of captured photons, now stored as
chemical energy, to the stroma (the thick gel matrix of the chloroplasts) where it
will be transduced yet again and stored in the covalent bonds of sugars.
•
Heart of the Light Reactions: The Photosystems
A photosystem is a light-gathering complex composed of a proteinaceous reaction
center complex surrounded by several light-harvesting complexes. These are
embedded in the thylakoid membranes inside the chloroplast.
• Each light-harvesting complex consists of various pigment molecules (chlorophyll
a, chlorophyll b, carotenoids) bound to proteins in the thylakoid membrane.
• The systems are spread out over the surface of the thylakoid, providing a large
surface area for light harvesting as well as a variety of pigments with different
absorbance spectra.
• Antenna pigments, such as carotenoids and chlorophyll b, pass their excited
electrons to one another until the e- reaches the reaction center complex.
• The reaction center complex contains a pair of chlorophyll a molecules
associated with a large protein, the primary electron acceptor.
• As the chlorophyll a molecules accept excited electrons from other pigments in
the photosystem, they pass them along to the primary electron acceptor (a redox
reaction).
• The primary electron acceptor passes the excited electron to a small "shuttle"
protein that carries it to a cytochrome complex of proteins that form an
electron transport chain.
• And you know what happens when an excited electron passes along such a
chain: at each transfer, energy is lost. But in a live photosystem, it isn't puffed off
into space. There are small, energy-courier molecules just waiting to package
that energy: ADP and NADP. These are converted to energy-storing ATP and
NADP-H, respectively.
• The ATP and NADP-H travel to the stroma, where their energy will be packaged
in the covalent bonds of sugar.

There are two types of photosystems (named in order of their discovery, not in order of
their function) in the thylakoid membranes:
• Photosystem II (PS II) -
o reaction center contains chlorophyll a molecules (P680) that have peak
absorbance (λmax) of 680nm.

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 • Photosystem I (PS I) -
o reaction center contains chlorophyll a molecules (P700) that have λmax of
700nm.
The chlorophyll a molecules of the two photosystems are nearly identical, but
each is associated with different proteins in their respective reaction centers.
Here's how it works...
A photon is absorbed by a chlorophyll or carotenoid molecule in the thylakoid
membrane. As it falls back to its ground state, its energy is transferred to the electron of
an adjacent pigment, raising it to an excited state. This continues until the excited
electron belongs to the famous P680 chlorophyll of PS II.
The excited P680 electron is transferred to a primary electron acceptor protein. The
oxidized P680 is now P680.
Nearby, an enzyme splits water to yield
• two electrons - these replace those lost by the two P680 molecules in the
reaction center
• one oxygen atom - this combines with another oxygen atom from a different
split water molecule to form oxygen gas (O2)
• two protons (hydrogen ions) - some will combine with NADP (to form NADP-H)
to store energy to be shuttled to the stroma for the light-independent reactions.
The excited electrons from PS II travel to PS I via an electron transport chain (similar
components as those found in the mitochondrial electron transport chain) consisting of a
cytochrome complex known as plastoqinone (Pq) and another protein, plastocyanin
(Pc).
As electrons "fall" exergonically from one component of the electron-transport chain
to the next, our old pal the Second Law of Thermodynamics rears its head: energy is
released at each transfer, but quickly captured and packaged in the high-energy
phosphate bonds of ATP. (Electrons passign through teh cyctochrome comlex results in
the pumping of protons out of the membrane, and the resulting potential difference is
used in chemiosmosis.
Meanwhile, back at PSI, chlorophylls and carotenoids are behaving in a similar
manner, doing the wave, and transferring photon energy (not converted to electrical
energy--the flow of electrons) to the pair of P700 chlorophylls at the reaction center.
P700 transfers its excited electron to its own primary electron acceptor, and becomes
P700+ (redox again!).
The electron reaching the "bottom" of the electron transport chain in PSII is shuttled
to PSI, where it replaces the lost electron of P700+, restoring it to its original P700
configuration.
PSI excited electrons travel along a different electron transport chain via the protein
ferredoxin ((Fd). There is no proton pump at the PSI electron transport chain, so no
ATP is produced there.
A special enzyme, NADP+ reductase, catalyzes the transfer of two electrons from
Fd to one NADP+, reducing it to energy-storing NADP-H. An overview of the entire non-
cyclic photophosphorylation process can be found in Figure 2 (reproduced here from
your textbook for your convenience).

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Figure 1. Non-cyclic photophosphorylation. (c) 2008 Pearson Education

B. The Hill Reaction

As described above, the first event in photosynthesis is the light-activated transfer of
an electron from one molecule to another against an electro-chemical potential. This
reduction results in the conversion of light energy into chemical energy.
2H2O + 2NADP+ ----------> O2 + 2NADPH + 2H+
In 1939, Dr. Robin Hill discovered that isolated, illuminated chloroplasts will produce
oxygen when in the presence of a suitable electron acceptor. Hill used iron salts in the
place of NADP, generating the following chemical reduction:
2H20 + 4Fe+++-----------> O2 + 4H + + 4Fe++
Hill's experiment was a landmark in the elucidation of photosynthetic processes, as it
was the first to demonstrate that
(1) Oxygen evolution during photosynthesis occurs without carbon dioxide
reduction.
(2) Oxygen evolved during photosynthesis comes from water, not carbon dioxide
(as previously believed), since no CO2 was used in Hill's experiment. (In the
1940's, another experiment using water labeled with heavy oxygen (18O)
confirmed this result.)
(3) Isolated (and in Hill's reaction, fragmented) chloroplasts could perform a
significant partial reaction of photosynthesis.

In our last laboratory, you disrupted the thylakoid membranes of spinach plants to
extract chlorophyll and carotenoid pigments and study their physical properties. This
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week, you will extract living chloroplasts from spinach cells without disrupting their
membranes or their thylakoids, and perform a variation of Hill's reaction. Your isolated
"photosynthesis factories" may be experimentally manipulated to study the effects of
environmental variables on this first stage of photosynthesis, the photolysis of water.
You will use an artificial electron acceptor--a blue dye known as indophenol--to
mimic the NADP found in a live cell. (In Hill's reaction, what is the chemical equivalent
of indophenol? ). As shown below, when indophenol
(the R in the chemical formula indicates a variable functional group) accepts electrons
and is reduced, it changes from blue to colorless.

This property of our artificial electron acceptor makes it a simple matter to monitor
the rate of the light reactions via colorimetry. You will use a spectrophotometer (Figure
9-1) to monitor the rate at which your indophenol changes from blue to colorless as it is
reduced, mimicking the role of NADP in a live plant cell.
You will follow a procedure that will enable you to extract living chloroplasts from
plant cells, alloquating your sample into a series of small colorimetry tubes (test tubes
made from optical quality glass, also known as cuvettes) which you should separate
into equal numbers of treatment and control tubes. These must be kept on ice to
prevent degeneration of the chloroplasts, but this should not interfere with your
manipulating your choice of environmental variable that you suspect might have an
effect on the light reactions.

III. The Light Reactions: Identifying Problems

Work in groups of four. Before you begin your chloroplast extraction and
manipulation, you must decide what scientific problem you hope to address. Consider
what you know about chlorophyll absorption spectra, the activity of proteins and
enzymes in different environmental conditions. Consider which wavelengths of light
drive photosynthesis. Your laboratory instructor will tell you what supplies are available
in the lab for your experiments. Consider one of your observations about
photosynthesis, ask questions, formulate hypotheses. By now, you should be familiar
with the scientific process and be able to formulate interesting, relevant hypotheses
about some aspect of the light reactions.
Restrict your question to the environmental variable of LIGHT: presence vs.
absence; source, illumination period, intensity, or wavelength. Your hypotheses should
be informed by your knowledge of photosynthesis. We recommend that you compare
no more than 2 variables, since more than that will complicate any statistical analysis
you might do. Have your TA approve your design and give helpful suggestions.

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A. Experimental Design
What is your question/problem?

List as many hypotheses as possible that might explain the above.

If you plan to use statistical methods (e.g., comparing rates of fermentation of two
different sugars), then what are your null and alternative hypotheses for the particular
experimental protocol you will be using?

Ho: __________________________________________________________________

_____________________________________________________________________

Ha: __________________________________________________________________

______________________________________________________________________

rationale for Ha: _________________________________________________________

______________________________________________________________________

1. List the materials your group will use

2. Briefly describe your experimental set-up and procedure. (Remember: you need not
give a detailed explanation of equipment here, unless it is unique to your experiment
and really requires an explanation for the educated reader to understand what you will
be doing. Simply refer to the lab manual as a literature cited for equipment specifics.)

3. Do you think it is sufficient to run only one experimental run with each variable? Or
should you run multiple trials? Explain your answer:

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4. How many times will you replicate your experiment for each variable?

If you plan to run multiple trials at each value of your variable—a wise choice—then
you will need to calculate the mean rate of reaction of the runs. (For example, if you
ran your experiment three times with a particular variable, then you must calculate the
mean reaction rate for each variable from your three runs with that variable.
Create an appropriate table to record your raw data. You should be able to do this
on your own by now.

B. Parameters and Data Analysis

What type of data are you collecting? Is it attribute, discrete numerical, or continuous
numerical data, as described in Appendix 2.
1. What parameter will you measure?

2. How will you analyze your data?

C. Predictions and Interpretations

In the spaces below, list all possible outcomes of your experimental trials. (As
before, we have given you more spaces than you might need for this.) Each outcome
should be accompanied by an explanation of how it supports or refutes each hypothesis
and its companion predictions.
Remember that the result is not the same as the interpretation. The outcome is the
summary of the results themselves, whereas the interpretation is the logical, reasoned
explanation for the observed results. The explanation of your outcome can serve as the
basis for the next level of posing hypotheses, so be prepared to go to that next level.
Outcome 1:

Explanation:

Outcome 2:

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Explanation:

Outcome 3:

Explanation:

Outcome 4:

Explanation:

IV. Laboratory Techniques for Chloroplast Extraction

To obtain a usable isolate of live chloroplasts, follow these directions carefully.
A. Preparation of Chloroplast suspension
THIS EXPERIMENT WILL WORK ONLY IF YOUR EXTRACTED CHLOROPLASTS
ARE KEPT COLD AND ARE PREPARED QUICKLY. READ AHEAD, AND BE SURE
TO ICE DOWN OR REFRIGERATE ALL GLASSWARE YOU WILL USE. KEEP YOUR
CHLOROPLASTS ON ICE (AND AWAY FROM HOT HANDS) AS MUCH AS
POSSIBLE.
1. In a blender, macerate about 25g of spinach leaves in 100ml of ice cold 0.35M
NaCl for about 30 seconds (or until obvious leaf fragments have disappeared,
giving way to a delicious-looking spinach smoothie).
2. Filter the homogenate through two layers of cheesecloth into a cooled 250ml
beaker. You will get plenty of extract by pouring your suspension through the
cheesecloth; don’t squeeze for more. (Don't waste cheesecloth! A section about 6"
square should be more than sufficient for your needs.)
3. Divide the extract between two chilled centrifuge tubes. Fill each tube to 1cm from
the top—no more. You will probably not need the rest of the extract, but save the
excess extract in case of any errors that might require you to do a procedure over
again. (This is simply good lab procedure: never throw anything away until you are
absolutely sure you don’t need it any more. Always label well to reduce the chance
of human error. )

4. Insert each tube into a metal centrifuge sleeve. Balance the tubes with their metal
sleeves carefully, and then insert them on opposite sides of the centrifuge rotor.

4a. To balance your tubes, use the double-pan balance available on the back table:
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 a. Place each plastic centrifuge tube into a stainless steel centrifuge sleeve.
b. Make sure there is a 250ml beacker on each balance pan, and that the balance
is zeroed. To zero the balance, use the sliding weight on the balance to
effectively make the two beakers weigh the same. When the balance is zeroed,
the pointer on the small scale in the middle of the balance will rest exactly in the
center of the scale. You are now ready to add your samples and balance them.
c. place one sleeved centrifuge tube in each beaker.
d. If the two tubes do not weigh the same, use the water droppers at the balance
at the station to carefully add water BETWEEN THE PLASTIC CENTRIFUGE
TUBE AND THE STAINLESS STEEL SLEEVE. DO NOT add water to your
chloroplast suspension. Add it only to the space between the tube and sleeve.
e. Once your two tubes are balanced, insert them into the centrifuge exactly
opposite each other in the centrifuge. (Don't run the centrifuge with only two
tubes. Wait for another team to load additional tubes, and always try to run a
full load. This will save time for everyone.)
5. Set the centrifuge at 1/2 speed (dial setting 6), and spin your filtrate for two minutes.
Time the spin with a watch or clock, as the centrifuge timers may be less accurate.
6. Pour the supernatant (liquid) from both the tubes into a chilled 250ml beaker.
Discard the precipitate (solid pellet at the bottom of the tube) in the trashcan
(NOT THE SINKS!). Rinse your centrifuge tube well with distilled water. (Distilled
water is available at the brushed aluminum tap at the back of the lab. It’s the tap you
must manually hold in the “on” position to keep the water flowing.)
7. Pour the supernatant from the beaker back into your rinsed centrifuge tubes. Re-
balance the tubes, and centrifuge at full speed for 8 minutes. This time, discard the
supernatant and SAVE THE PRECIPITATE/PELLET. The pellet is composed of
isolated chloroplasts.
8. To each of your centrifuge tubes, add 0.4mL of ice-cold NaCl solution. Mix the
contents with a clean, glass stirring rod to re-suspend the pellet. Then gently draw
the pellet into a Pasteur pipet and expel against the wall of the tube. Repeat 5 times,
or until the chloroplass are very evenbly suspended (i.e., no clumps are visible).
9. Combine the chloroplast suspension from your tubes into a chilled 30mL beaker.
Keep on ice.
10. Place 1mL of this chloroplast suspension into a new chilled 30mL beaker. Make a
1/10 dilution by adding 9mL of chilled 0.35M NaCL. Keep on ice.
11. Obtain the number of colorimetry tubes your team has decided to use. Label each
tube carefully, placing tape labels near the top of the tube, where they will not
obstruct the spectrophotometer’s light path. Chill the tubes.
12. In a chilled 50 or 100mL beaker, make sufficient phosphate buffer for your
experiment. To maek 20mL of 0.05M sodium phosphate buffer (pH 6.9), mix 9mL of
0.05M sodium phosphate monobasic (acid) with 11mL of 0.05M sodium phosphate
dibasic (base). Check the pH and adjust (titrate) as necessary. Make 10mL of buffer
for each tube you plan to use. Multiply the recipe proportionally if you have more
than two tubes. Do not make more buffer than you need!

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13. Into your tubes, aliquot (to aliquot is to divide a total amount of a solution or
suspension into multiple portions of the same size) 8.0mL of pH 6.9 phosphate buffer
solution and 0.2ml of the 1/10 dilution of the chloroplast suspension. Show the first
tube to your TA before preparing the others.
14. Make a mini ice water bath for each treatment by filling 100mL beakers 2/3 with ice
and water. Place each colorimetry tube into its own mini ice water bath.
15. Your samples are now ready for the experiment you designed in Part II. Set up your
treatments so that you can keep the tubes in their mini ice-water baths during the
experiment, and remove them only to take spectrophotometer readings.

B. Measuring Rate of Reaction via Colorimetry

1. Spectrophotometer (Figure 2) instructions are available at each machine. Read them
carefully before you begin.
2. Be sure the spectrophotometer is set at 610nm, as transmission of this wavelength
(red) will increase as indophenol blue fades to colorless. (Why does this make
sense? Why do we not use blue or green light?)
3. When you are ready to begin taking readings, bring your iced sample tubes and
bottle of indophenol to the spectrophotometer. Add 0.15ml of the indophenol dye
solution to each test tube in turn, mix very well, and immediately take a colorimetry
reading. This will be your "zero" data point.
(IMPORTANT: Be sure to mix the contents of the tubes thoroughly, and wipe dry
before each reading. Mixing procedure should be standardized.)
4. After taking readings, immediately put the tubes back in their treatment conditions
(and mini ice water baths).
5. Measure the color change in your tubes at set intervals (two minutes works well).
Record your data in the grid provided. Label the grid appropriately.

6. For your presentation, make two graphs using Excel or similar software:
a. a raw data graph showing rate curves for each treatment. Obtain the rate of
reaction for each treatment as the slope of the best-fit line for the linear portion of
the rate curve only.
b. a results graph comparing the rates of reaction for all treatments.

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Grid for data collection:

2.
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Figure 2. The Spectronic 20D+ spectrophotometer. 1-sample compartment.
2-digital readout. 3-mode indicators. 4-mode selector button. 5-decrease. 6-
increase. 7-print. 8-wavelength control. 9-transmittance/absorbance control
(100%T/OA). 10-power switch/zero control. 11-filter lever.

V. Presenting Your Data

You should now be able to analyze your data and present your findings in such a way
that further hypotheses about the mechanism of the light reactions can readily be
proposed. Remember to present this next step of your research in next week’s
symposium, and to propose further experiments that would allow you to address the
multiple, competing hypotheses that might be supported by any given outcome of those
experiments.

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