Topic 8. Exercises on PhotosynthesisThe pathways of photosynthesis and respiration are quite different. However, at theglobal level, one is perfectly complementary to the other as the end products ofrespiration are the reagents for photosynthesis. The oxygenated air that sustains allanimal life is due to photosynthesis. This fact was only discovered in the lateeighteenth century when Joseph Priestly found that plants could “fix” air exhaustedby fire and/or respiration to allow a mouse to survive in a closed jar.It is worth considering the overall reactions of photosynthesis and respiration.

Respiration C6H12O6 + 6 O2 —–—> 6CO2 + 6 H2OPhotosynthesis 6CO2 + 6 H2O —–—> C6H12O6 + 6 O2

Web Lesson@ http://botit.botany.wisc.edu/botany_130/photosynthesis

I. Where Photosynthesis Occurs in Plant Cells

While some parts of respiration occur outside the mitochondrion, all parts ofphotosynthesis occur in the chloroplast. Inside the chloroplast is an internalsystem of membranes called thylakoids, some of which are clustered into stackscalled grana. The space not occupied by these membranes is called the stroma.

The Light Dependent ReactionsThe light reactions require the spacial structure generated by the intactthylakoids. The pigment systems of light reaction I and II are both bound tothese membranes and are each coupled with an electron transport chain. As inthe mitochondrion, these electron transport chains generate a hydrogen ionconcentration across a boundary which is harnessed to generate ATP. Theultimate electron receptor in the process is not oxygen, as in the mitochondrion,but NADP+ which generates NADPH.

The Light Independent ReactionsThe Calvin Cycle, which fixes carbon, occurs in the stoma. It requires thereducing potential (NADPH) and the energy (ATP) provided by the lightreaction, but is not, itself, dependent upon the structure of the internalmembranes of the chloroplast (other than to keep the whole mix together).To produce sugars and to evolve oxygen, the light reaction and the CalvinCycle must work in tandem. While the light reaction can produce ATP withoutthe Calvin Cycle, it cannot split water without the regeneration of NADP+ fromNADPH accomplished by the Calvin Cycle. The Calvin Cycle, in turn, cannotfix CO2 without a constant supply of ATP and NADPH from the light reaction.

Consider the figure on the next page

1. Where do the light reactions occur? __________

2. Where does the Calvin Cycle occur? __________

II. The Necessity of Chlorophyll for Photosynthesis.To ‘eat’ light as plants do, it must first be absorbed. As plants are universallygreen due to chlorophyll, it seems obvious that a green pigment is necessary forphotosynthesis. However, correspondence doesn’t constitute proof of causality.The following is a simple exercise to provide evidence of that relationship.

In this exercise we will consider the hypothesis

Chlorophyll is necessary for photosynthesis.At the side bench is a variety of Coleus with variegated leaves. There are twoobvious pigments to be found in various regions of its leaves. Observe leaves on theplant, and note that some leaves have areas with...

- just chlorophyll colored green;- just anthocyanin colored red;- regions of overlap, and- regions with neither which are white.

Because chlorophyll is not found everywhere in the leaf, we can use this plant toevaluate whether chlorophyll is necessary for photosynthesis. To do so, however, werequire a method for determining photosynthetic activity.One approach is to consider where in the leaf starch forms. Starch is derived fromsugars produced by photosynthesis and we can detect starch using iodine in I2KI.

Procedure: Work in pairs.

1. Take a leaf and draw it. Clearly indicate where chlorophyll and anthocyaninare located and where they overlap.

2. Boil the leaf in water to remove the water soluble anthocyanin pigment.Make a second drawing clearly showing where the chlorophyll is located.

3. Boil the leaf in alcohol to remove the chlorophyll pigmentation.

4. Carefully place the bleached and brittle leaf on a watch glass and flood itwith I2KI.

5. Again draw the leaf clearly indicating where the purple stained starch islocated.

Drawing 1 Drawing 2 Drawing 3Untreated leaf Anthocyanin removed Stained with I2K

Discussion:

Are your results consistent with the hypothesis?

____________________________________________________

Consider a second hypothesis:

Anthocyanin is necessary for photosynthesis.

Do your results support that statement?

__________________________

Can you reject the second hypothesis?

__________________________

Have you “proved” the first hypothesis?

__________________________

III. The Necessity of Carbon Dioxide and Light forPhotosynthesis.

This activity introduces a simple way of determining photosynthetic activity, toevaluate the two hypotheses:

Carbon dioxide is necessary for photosynthesis.and

Light is necessary for photosynthesis.

Experimental Design All plant tissues contain intercellular spaces. Those in the leaf are filled with air,which allows for rapid diffusion of gases. They also make leaf tissue buoyant. If theair is removed, leaf tissue will sink. If the tissue is living and is then exposed to light,photosynthesis will generate O2 which will refill the spaces with gas and cause thetissue to again float.

In this exercise photosynthetic activity will be determined for three differentexperimental conditions as outlined below. 1. Light - discs in bicarbonate solution.

2. Light - discs in boiled distilled water. 3. Dark - discs in bicarbonate solution.

For each condition a beaker with 10 non-floating leaf disks will be tested. Thebeakers will all sit on a piece of white paper.

Procedure (To be in teams of six students).

Each team to work in three groups. One group to start part a, while another startspart c, while the third starts part e.

a. Punch out forty leaf disks from green Coleus leaves and place them into asyringe. Try to avoid the midrib and the major veins.

b. Add 7 ml (7cc) of dilute detergent solution to the syringe. Insert the plunger,invert the syringe, and push out all the air. While covering the opening at thefront with your thumb, pull the plunger back to create a vacuum. Maintain thevacuum for 10 seconds while agitating the syringe and then slowly release theplunger. Repeat this procedure for two to three minutes until most of the diskshave sunk. Remove the plug and immerse the syringe in a large finger bowlof tap water. Slowly pull the plunger out, sucking the water into the syringe.After removing the plunger carefully pour the disks into the water.

c. Two other people should heat 500 ml of bicarbonate solution to 35º C.

d. Fill two 250 ml beakers with the NaHCO3 solution heated in Part c. Fill anotherwith 200 ml of boiled and stoppered distilled water and label it. Then, usingforceps, add 10 disks to each of the three 250 ml beakers. Only use disks that arenot floating and introduce them into the water edge on to prevent bubbles frombeing captured on their surfaces.

e. During the preceding steps two members of the group should position two lamps50 cm above the bench surface.

f. Place one beaker with bicarbonate and the one with distilled water below thelights, Place the other beaker of bicarbonate on the bench and cover with foil.Turn on both lights.

g Run the experiment for 40 minutes or until you observe eight or more discsfloating in the illuminated beaker of bicarbonate.

h. Record the number of floating discs for each condition in the table.

Light + bicarbonate Dark + bicarbonate Light + Distilled water

7. In the balanced equation for photosynthesis, for every mole of CO2consumed, one mole of oxygen is produced. Why do we observe an increase inthe volume of total gas for photosynthesis in water?

___________________________________________________________

8. In the beaker of distilled water, was any form of photosynthesis occurring? Ifso what were its products?

___________________________________________________________

9. What is the source of the oxygen generated?

___________________________________________________________

10. What prevents the discs in the distilled water from producing oxygen?

___________________________________________________________

___________________________________________________________

IV. The Light Harvesting Pigments of PhotosynthesisIn the following activities you will observe specifically which colors of light areabsorbed by a pigment extract from banana leaf, and, hence, which colors areused by photosynthesis.

IVa. The Spectrum ViewerWork with a partner

See the illustration of the spectrum viewer on the next page. These are locatedon the side bench. Look through the front opening. If the light behind theviewer is on and is properly aligned with the opening at the back, you shouldview a spectrum of colors projected to the right. Note that this spectrum isprojected onto a scale. The scale is to be read in nanometers. Note that eachcolor is projected at the point on the scale corresponding to that color’swavelength in nanometers.

Spectrum Viewer Illustrations

Spectrum ViewerLook to the right to view the scale

The spectrum from the light source will beprojected against a scale in nanometers.

Only push the filters andpigment extract half waydown the opening at theback

Procedure:1. Take color filters and push them half way down the opening at the back as

you view the projected spectrum (see illustration). Note, if properlypositioned you will still view the total spectrum of the light source on thebottom of the scale while also viewing the light transmitted through the filter at the top.

Are the filters totally effective?- With the red filter do you see colors other than red? ______- With the green filter do you see colors other than green? ______- With the blue filter do you see colors other than blue? ______

2. Take a test tube of pigment extract, and, while viewing the projectedspectrum, push the tube half way down the opening in the back. Carefullynote that there are two regions where the pigment absorbs the light mostcompletely. If you cannot discern these regions, the pigment may be wornout and will need to be replaced. If you have trouble here ask your TA forhelp!

- The two regions of maximum absorption corresponds to what twocolors?

- The two regions of maximum absorption corresponds to what tworanges of wavelengths?

IVb. The Spectrophotometer:Work in pairs. The spectrophotometer is on the side bench. Instructions for itsuse are provided by the instrument, and your TA is available to assist.

With a spectrophotometer, we can quantify what you have just observed withthe spectrum viewer. Each pair of students will measure the transmittance oflight through a solution of pigment extract at each of the wavelengths given inthe table. Transmittance is simply a measure of the portion of light notabsorbed as passes through a sample. From your observations with thespectrum viewer it should be clear that this value will vary with color. The datatable below will dictate your choice of wavelengths. Measure transmittance ateach of these wavelengths. Record your readings below, and graph the data onthe following page. In class, check with other students to verify that you didn’tmake mistakes. Turn in this table and graph in discussion.

Wave Length (nanometers) Transmittance

400

425

450

475

500

525

550

575

600

625

650

675

700

725

1. Do the results in Part VIb. Correspond to you observations in VIa?

2. Are they consistent with the information given in your book about the actionspectrum of photosynthesis?

Name ________________________ Partner_________________________

500

700

600

350

400

90% 70% 50% 30% 10%

IVc. Fluorescence of ChlorophyllIf the light energy absorbed by chlorophyll is not transferred to a reactioncenter, then the energy is lost either as heat, or as fluoresced light, or somecombination of the two. View the demonstration of fluorescence at the frontbench. Do not confuse this fluoresced light with the transmitted lightviewed by the spectrum viewer.

What emits the green light viewed through the spectrum viewer?

__________________

What emits the red, fluoresced, light?

__________________

IVd. Isolating Pigments Using Paper Chromatography

Work in groups of two

The glassware required for this activity is at the front of your bench. Thechromatography paper and solvent, and the pigment extract for use in thisactivity are at the front bench.

Procedure:

1. Take a piece of chromatography paper, roll it into a cylinder, and staple itin three places (see illustration on the next page).

2. Remove the 500 ml lipless beaker from the larger of the two petri dishes(see illustration). Take the smaller petri dish and add enough pigmentextract to cover the bottom.

3. Stand the paper cylinder in the smaller dish until the pigment has movedabout 1.5 cm up the cylinder.

4. Set the paper aside for 5 minutes to dry.

5. Load the paper two additional times (repeat “c” and “d” twice).6. After loading the paper with pigment the third time, allow to dry at least

10 minutes. Then return the pigment extract to the stock bottle; wipethe small petri dish with a tissue, half fill that dish with chomotographysolvent and place it in the center of the larger petri dish; sit the papercylinder in the pigment extract, and cover with the 500 ml lipless beaker.

7. If you properly dried your paper, you should quickly see thechromatography solvent move through the previously loaded pigment bandwith the immediate result that bands of pigment separate from each other.

Chromatography Glassware Chromatography Paper withsmaller and larger petri dishes

Explanation: These pigments are separating by their physical affinity for water(determined by the extent of their molecular polarity). Water is bound to the fibersin the paper, and as the chromatography solvent moves up the cylinder of paper, itacts as a drag on the pigment molecules. The extent of this drag is directly related toeach molecule’s attraction to water. At the top of the cylinder you should see a deepyellow band made up of the carotinoid pigment, carotene. This layer moves freelywith the moving solvent front. It is non polar with no affinity for water. The nextpigment down, represented by one or two yellow bands, is the carotinoid pigment,xanthophyll. Next going down the cylinder, is a green band made up of Chlorophylla, and the lowest band is made up of the pigment with the greatest affinity forwater, Chlorophyll b.

Which of these pigments are integrated into the reaction centers of bothphotosystems I and II?

Which act as accessory pigments in the antenna complexes of photosystems I andII?

_________________________________________________________

Which serve to protect the photosystems from photo oxidative damage?

Which is the plant source for vitamin A in our diet?

V. C-4 Photosynthesis

As discussed in your text, oxygen disrupts the Calvin cycle at the point of CO2fixation: RuBP carboxylase (Rubisco) catalyzes CO2 fixation through a reactionbetween CO2 and ribulose 1,5,-bisphosphate. However, this same enzyme will alsocatalyze a reaction between oxygen and ribulose 1,5,-bisphosphate. The reactionwith oxygen is not productive and the biochemical pathway required to salvage theresulting product consumes oxygen and releases CO2. This process is termedphotorespiration. The name is deceiving as no ATP is produced. Photorespirationis particularly maladaptive in hot dry conditions where a plant’s stomata are morelikely to be closed. C-4 plants have evolved in response to this problem, and C-4photosynthesis has evolved independently in many different lineages.

Details of C-4 physiology are outlined in your text. Simply stated C-4 plantschange the ratio of carbon dioxide to oxygen in the cells where the Calvin Cycleoccurs. By increasing the level of CO2 relative to O2, the rate of photorespirationcan be reduced. This is accomplished by first capturing CO2 in a part of the leafwhere the Calvin Cycle does not occur using the enzyme PEP carboxylase. Thisresults in the formation of a four-carbon compound (hence the name C-4Photosynthesis). This four-carbon compound is moved into another part of the leafwhere it is broken down into a 3-carbon compound and CO2. This CO2 releasingreaction also produces NADPH from NADP+. The CO2 and NADPH then entersinto the Calvin Cycle.

Procedure: On the side bench are corn plants. Corn is a C-4 plant. These plantswere subject to 48 hours of continuous light. Under these the conditions, a leaf cellundergoing the Calvin cycle cannot export photosynthate as rapidly as it isproduced resulting in the accumulation of starch in the cell. Hence, the presence ofstarch corresponds with where the Calvin Cycle occurred in the leaf.

1. Cut several leaf squares from the plant. Place these in a syringe with detergentwater and subject the tissue to a vacuum to remove the air (your TA willdemonstrate). These are ready for the next step when they loose buoyancy.

2. Pour your sunken squares into a communal bowl by the plants. Take a leafsquare, place it on a microscope slide in a drop of water. Then, using a stainless

steel razor blades from the front of your bench, cut thin cross sections from theleaf square. This will be difficult. One approach for cutting thin sections is tomake numerous choppy cuts and save only those cross sections narrow enoughto be seen in profile. Another is to use two blades where one holds the leaftissue down while the other makes the slice. One other technique is to place theleaf tissue between two sections of carrot and to make thin sections through thecarrot tissue. Give yourself time as it may take several minutes before yougenerate a useful section. Ask you TA for help if you don’t succeed after tenminutes or so of effort.

3. Make a wet mount and observe the leaf tissues in cross section. Pay closeattention to the enlarged bundle sheath cells.

Cross section of Zea leaf showing enlarged bundle-sheath cells

3. After making these observations treat your slide with I2KI by adding the stainto one side of the cover slip while soaking up water on the opposite side usingtissue.

4. Again observe your leaf section. Make a quick sketch below showing where thestarch is located.

5. In which part of the leaf is the starch located? This is also where the Calvincycle occurs.

6. Observe the electron micrograph of corn leaf on the side bench and correlateyour observations with it. Compare it with the ER of a C-3 plant. Why does theC-3 plant have more microbodies?

Discussion Topics for Your Consideration1. Consider the argument that the first instance of organisms polluting their

environment were the Cyanobacteria releasing noxious diatomic oxygen.

2. How has the presence of oxygen gas made life on land possible?

3. As oxygen is cycled between respiration and photosynthesis, in what molecularforms is it found?

4. What came first, aerobic respiration or photosynthesis?

4. What came first, glycolysis or photosynthesis? On what basis can you evaluatethe question?

Web Lesson @ http://botit.botany.wisc.edu/botany_130/photosynthesis/