Module 3 - Photosynthesis
Week 1
Introduction: Photosynthesis
• Energy inherent in photons of light perform
useful work:
– Cleaves covalent bond in water to form O2
– High-energy electrons transferred to NADPH and
ATP
• Drives synthesis of glucose through anabolic pathway
Three stages of photosynthesis
• All occur in organelles
called chloroplasts
• Two stages depend on
light absorption by
chlorophyll
– Photosystem I (P700)
– Photosystem II (P680)
• Third stage is Calvin
cycle, and is
independent of light
Redox reactions in thylakoid membrane
• PS-II e- transfer: chlorophyll  PQ  cyt b6f  PC
– PS-II also generates protons for electrochemical gradient
• PS-I e- transfer: chlorophyll  multi-subunit complex
 reduction of NADP+ to NADPH
• Proton gradient generates ATP via ATP synthase
Measuring photosynthetic rate
• Multiple ways to assess the rate at which light
energy converted into useful chemical energy
– Electron transfer (what we will study)
– Formation of ATP
– Change in pH in lumen
Hill reaction
General equation
Use of colorimetric
reagent as electron
acceptor
DCPIP
DCPIP
• Electron transfer during photosynthesis can be interrupted by adding an
artificial electron acceptor (e.g., DCPIP)
– Acceptor must have lower energy than donor
– Colorimetric compounds change colors upon accepting/donating an electron
Quantifying photo-reduction activity of
chloroplasts with colorimetric DCPIP assay
• Spinach chloroplasts prepared for you
• DCPIP used to assay rate of photo-reduction
as determined in spectrophotometer
• Use of a “blank” to calibrate spec
• Positive and negative controls included
• Test effects of a herbicide on rate of photoreduction
Use of spectrophotometer
• Monochromator tunes the wavelength of light passing through sample
• Amount of light transmitted through sample measured by photodetector
– 100% transmission = no absorption of light by sample
– 0% transmission = sample absorbs all light
• DCPIP is initially blue color reagent and absorbs light at 600 nm
• As DCPIP accepts electrons it is converted to a colorless compound (no
absorption of light)
• Increase in light transmission indicates electron transfer from
photosystem components to DCPIP
Set up Spectronics 20+
• Set up for Spectronics 20+
• Turn on spec (knob on front face on left). Allow to warm up
for 15-20 minutes.
• Set wavelength to 600 nm (knob on top)
• Adjust filter to 600-950 nm (bottom left)
• Set spec to 0% T with nothing in the compartment
– Close lid of empty compartment (no cuvette, no light)
– Adjust left knob to 0% T
• Set spec to 100% T with blank
– Fill cuvette (glass 0.5” diameter tube) with water. Make sure to
use at least 4 mL (about half-way filled).
– Insert cuvette in compartment, close lid.
– Using the front right knob, adjust to 100%T
• Remove blank cuvette.
• Insert cuvette containing sample and read %T
Set up your different samples
Table 1
Tube 1
Tube 2
Tube 3
No C'plasts
3
1
1
0
Dark C'plasts
3
1
1
0
Light C'plasts
3
1
1
0
Tube 4
Light C'plasts +
Herbicide
2.5
1
1
0.5
water
Rxn buffer
65 µM DCPIP
Herbicide
Chloroplasts?
0
0.3
0.3
Numbers are volume of each solution to add. Units=millimeters (mL)
0.3
• Number the glass cuvettes 1 through 4 and place them in a tube rack.
• Fill each of the tubes with the indicated amount of water:
– P1000 pipette (dial set to 1.00) with a blue tip attached
•
•
•
•
Add 1 ml Reaction Buffer to each of the four tubes; P1000 set to 1000 μl
Add 1 ml of DCPIP to each tube.
To Tube #4, add 0.50 ml of your assigned herbicide.
Add a small piece of Parafilm to seal the top of each tube, and invert several
times to mix.
• Visually inspect the height of the liquid in each of the four tubes; they should all
be the same if you pipette your ingredients correctly; they should also all have the
same bluish color.
• Wrap the tube to be kept in the dark (Tube #2) in foil to block out incident light.
Start your experiment
• At Time = 0 (check the clock time or start a timer), add 300 µL of
the chloroplast preparation to your Tube. Immediately cover with
Parafilm and invert/mix the contents.
• As soon as possible after adding chloroplasts to your tube and
mixing, measure the % Transmission of the Tube in the spec.
Record the Time and the %T in Table 6.3.
• For Tubes #1, #3, and #4, place the Tubes under the light provided
on your lab bench. Keep Tube #2 in darkness (wrapped in foil).
• Each person in your group should do one Tube (stagger 5 min):
– Note exact time chloroplasts were added,
– Note time at which each measurement was made
– Record Time and %T.
• Repeat your measurement of the %T at various times from 1 to 16
minutes.
• Be sure the dark Tube #2 is returned into foil after each
measurement.
Data collection
Table 2
% Transmission measurements
Tube 1
Time (min)
1
2
4
8
16
Tube 2
Tube 3
Tube 4
Light
No C'plasts Dark C'plasts Light C'plasts C'plasts +
Herbicide
Data analysis with Excel
Write up answers to questions
1.
2.
3.
4.
5.
Compare the time course you obtained for samples with no chloroplasts
with that of chloroplasts in the light. What happened to the absorbance
at 600 nm in the tubes with chloroplasts? What specifically is occurring
at the level of individual molecules to produce the effects you have
observed?
Compare the time course for samples with chloroplasts kept in light
versus those kept in darkness. Explain what is happening at the
molecular level and how it produced the observed results.
Each lab group tested a different herbicide to determine if it inhibited
photosynthetic redox reactions. Consult with the other groups in the lab:
Which compound(s) appear to inhibit electron transport? Which do not?
If more than one altered electron transport, rank them in relative order
of inhibition.
Some of the herbicides you tested might not have affected electron
transport. What are some other potential targets for herbicides? (Hint:
Most commercially available herbicides are not particularly toxic to
animals because they interfere with processes that are unique to plants.)
What other environmental factors or conditions do you think could
increase or decrease the rate of redox reactions by chloroplasts? What is
your rationale?
Create your own experiment…
• Discuss potential experiments
• Consult with faculty/TA about your proposed
experiment
• Fill out “My proposed experiment” form and
turn it in before leaving