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Option 9.9 Biochemistry: 3. The
discovery of chloroplasts as the site of
photosynthesis
Extract from Biology Stage 6 Syllabus (Amended October
2002) © Board of Studies, NSW.
[Edit: 18 June 09]
Prior learning: Stage 4, Structures and Systems 4.8.2 (c), 4.8.4 (d) Models,Theories and Laws 4.7.5 (d).
Recall statements in Preliminary course: Preliminary module
8.3 (subsection 4 and 6)
Background: By the late 19th century, the
main features of photosynthesis were known. At this point it
was discovered that chloroplasts were the site of
photosynthesis.
identify data sources, plan and choose equipment or resources to perform a first-hand investigation to gather data to determine the effect of light intensity and temperature on gas production in a suitable pond weed
- Identify your data sources by determining the type of data that needs to be collected and explain the qualitative and quantitative analysis that will be required for this data to be useful. Will you measure the light intensity quantitatively or just move the specimen away from the light and describe the light as greater or lesser?
- Plan your experiment to recognise the variables that need to be kept constant and demonstrate the use of a control. Variables to consider include temperature, volume of the water and quantity of pondweed.
- Choose equipment by identifying technology that could be used during the investigation and determine its suitability.
- Perform the investigation by carrying out the planned procedure recognising any modification that need to be made.
- Gather the first hand information by measuring, observing and recording results and carry out repeat trials if necessary.
Sample method
For light intensity, set up several test tubes with fresh
water and a suitable pondweed. Adding the same amount of
NaHCO3 to each tube can ensure a good supply of
CO2. Different light intensities can be produced
by placing the test tubes at different distances from the
same light source (a fluorescent desk lamp for example).
Oxygen evolution can be determined using an oxygen sensitive
probe and a data logger over time or bubbles of gas evolved
per minute can be counted over time.
explain that Sachs proved that chlorophyll is located in special bodies within plant cells and relate his finding to the site where glucose is made
- In 1864, Sachs observed grains of starch were being formed in leaves exposed to light. He showed that chlorophyll is not distributed randomly throughout the plant but is located in special bodies (later called chloroplasts) within plant cells. He found that the site where glucose is made is in these bodies and glucose is usually stored as starch.
describe homogenisation as a process that breaks up cells and allows study of cell fractions, suspensions and solutions
- Although electron microscopes have allowed detailed pictures of cell organelles, in order to study reactions it is necessary to isolate components of cells. The first step in isolating cell components is to break the cell membrane without destroying the internal parts. This is done by sonication (exposure to high frequency sound) or grinding in a blender (homogenisation).
outline the role of centrifugation in removing cell debris and sedimenting cell organelles, such as chloroplasts
- Spinning in an ultra-centrifuge (100 000 rpm or 500 000
times gravity) separates the fractions or parts of the cell.
Cell components move to the bottom of the centrifuge at a
rate determined by their size and density, the largest and
heaviest falling to the bottom first. If a suspension of
broken cells (called an homogenate or lysate) is spun at
different rates, the following fractions can be separated:
- low speed – unbroken cells and nuclei can be removed in a pellet (sediment) at the bottom of the tube leaving the rest of the cell components in solution above (the supernatant).
- centrifuging the supernatant at higher speed sediments the chloroplasts, mitochondria and lysosomes.
- re-centrifuging the supernatant at an even higher speed sediments the plasma membrane and the endoplasmic reticulum.
- a fourth centrifugation at a still higher speed sediments the ribosomes, leaving only the cytosol (cytoplasm) in the supernatant.
- To produce a pure chloroplast preparation density gradient centrifugation is preferred. To make a density gradient, different concentrations of sucrose solutions are used in turn, layered on top of one another in a centrifuge tube. The most concentrated solution is at the bottom and the least concentrated is on the top. The homogenate is placed carefully on top of the layers and spun in a centrifuge. Organelles of different sizes settle out as discrete bands through the gradient at the interface between sucrose solutions, depending on their size and density.
outline the discoveries of Englemann and explain why Englemann’s work led to the description of the action spectrum of photosynthesis
- Englemann performed two experiments. In 1894 he used a modified microscope condenser to shine thin rays of light on the green alga Spirogyra, which has chloroplasts arranged in spirals (hence the name) with clear cytosol between them. Englemann dispersed the Spirogyra in a bacteria suspension and found that the bacteria migrated to the areas where the Spirogyra was green or where there were chloroplasts. The bacteria were attracted to the oxygen produced by photosynthesis.
- In an earlier experiment in 1882, Englemann split white light into its spectral components using a prism made for him by Carl Zeiss. He shone the spectrum onto the green alga, Chladophora. In Chladophora, unlike Spirogyra, the cells are completely and evenly filled with chloroplasts. The bacteria accumulated in the violet and red wavelengths of the spectrum, attracted by the regions of high oxygen concentration. Englemann had produced an action spectrum, showing the regions in which chlorophyll absorbs light.
explain how the role of pigments, other than chlorophyll, in photosynthesis was inferred
- The action spectrum of photosynthesis does not match that determined by Englemann for chlorophyll. It shows more activity in the areas where chlorophyll reflects light, the wavelengths corresponding to green , yellow and orange light. Other pigments, such as the yellow and orange carotenoids and the phycobilins, absorb light in these other wavelengths and pass the energy on to chlorophyll. These accessory pigments allow the plant to absorb a broader range of wavelengths than chlorophyll alone and explain why the action spectrum of chlorophyll and photosynthesis do not match exactly.
gather information from secondary sources to produce a time-line indicating improvements in microscopy that would have assisted Englemann in his work with Spirogyra
- Gather information by summarising and collating information from a range of resources.
- Look for trends and patterns in the information you have gathered.
- Illustrate these patterns by drawing a time-line of the important events.
The web sites below are a starting point.
History of the Light Microscope 
About.com, Inventors
Microscopy
Microscopy-UK and Onview.net Ltd, UK
| Sample timeline | |
|---|---|
| Year | Improvement |
|
1886
|
Zeiss made a series of lenses that allowed structures
to be resolved at the theoretical limits of visible
light, which would have improved the detail (resolution)
of what Englemann saw
|
|
1924
|
Lacassagne developed the first autoradiographic method
to see where radioactive polonium accumulated in
biological specimens. If this technique had been
available to Englemann, he could have used radioactively
labelled water and autoradiographic techniques to
visualise the production of oxygen in the violet and red
wavelengths under the microscope.
|
|
1930
|
Lebedeff designed and built the first interference
microscope
|
|
1931
|
Ruska built the first transmission electron microscope. The development of electron microscopes, scanning and transmission electron microscopes occurred in the 1940s and continued through the rest of the century, concentrating mainly on methods of fixing, staining and computer enhancement of images. These developments would not have helped Englemann. He required the plants to be actively photosynthesising and so fixing, staining or freeze fracture techniques developed recently would not have assisted him. |
|
1934
|
Zernike invented the phase contrast microscope. These
developments allowed unstained living cells to be seen in
detail for the first time. It was important for Englemann
to view unstained living cells and he would have been
able to watch the bacteria migrating to the oxygen rich
areas. (After 1981 he would have been able to video tape
it!)
|
|
1952
|
Nomarski patented the system of differential
interference contrast for light microscopes. This
development would have assisted Englemann in a similar
way to a phase contrast microscope.
|
perform a first-hand investigation to:
- extract the mixture of pigments from leaves
- examine the absorption spectrum of these pigments
- separate the pigments using chromatography
- Choose equipment by identifying technology that could be used during the investigation. For example, use a hand held spectrophotometer to view the spectrum of your extracted pigments. Choose a suitable adsorption medium and solvent for the chromatography. Chromatography paper and methylated spirits is adequate.
- Perform the investigation by carrying out the planned procedure recognising any modification that need to be made.
How you can measure the amount of pigment in plants Mad
Science Network, Washington University, USA
Be aware that more sophisticated chromatography techniques
than using solvents are now available to separate pigments in
plants. Advanced spectrophotometers can precisely determine the
absorption spectrum of plant pigments.
Extraction of pigments from spinach Classroom bats, Bermuda
Biological Station for Research, Inc, Florida, USA
process information from secondary sources to outline the importance of Tswett’s invention of chromatography for the separation of leaf pigments
- Gather information from a range of resources including popular scientific journals, CD ROMS and the Internet. Use the websites below as a starting point.
Mikhail Tswett original paper
History of chromatography
- Process the information to assess the reliability of first hand and secondary information in relation to the area of investigation.
Sample information
Tswett invented chromatography which is the separation of parts of a mixture by selective adsorption (not absorption). He isolated plant pigments using CaCO3 or chalk as the column of adsorbent and CS2 (carbon disulfide) as the solvent. He recognised two types of chlorophyll and four types of xanthophylls (or carotenoids as they are now known), proving that the green colour in plants is a mixture of pigments.
Students may be interested to read the following from
Tswett’s original paper.
“The green pigment of the leaves, the chlorophyll, is known to be a mixture of pigments, the complexity of which was differently estimated by different investigators. Chromatographic analysis is called upon to settle finally this degree of complexity... The chromatograms obtained from a CS2 solution have the following form:
- (Top) Zone. Colourless
- Zone, especially less sharply separated from the next. Yellow due to xanthophyll β (identical with Sorby’s yellow xanthophyll – original note)
- Zone. Dark olive green. Chlorophyllin β.
- Zone. Dark blue green. Due to chlorophyllin α (Sorby’s blue chlorophyll).
- Zone. Yellow (xanthophylls α’ and α”).
- Zone. Colourless
- Zone. Orange yellow xanthophyll (α).”
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