Lab Investigation: Energy & Photosynthesis
PROBLEM
What factors affect the rate of photosynthesis in living leaves?
BACKGROUND
For this lab you should understand and summarize in your background:
a)Give the overall chemical reaction for photosynthesis. (What are the sources of the reactants and how do they enter the plant for use?)
b)Briefly discuss the visible light spectrum and how this relates to photosynthesis.
c)Describe how the rate of photosynthesis can be measured in an experiment and discuss specifically how you will indirectly measure the rate of photosynthesis in this lab. (What is ET50 and how will it be used? Include a brief description of the Floating Leaf Assay!)
d)Draw a cross section of the leaf structure and briefly describe the functions of the different parts. (How is understanding this information important to understanding the technique used in this lab?)
FLOATING LEAF ASSAY
Photosynthesis fuels ecosystems and replenishes the Earth’s atmosphere with oxygen. Like all enzyme-driven reactions, the rate of photosynthesis can be measured by either the disappearance of substrate or the accumulation of product (or by-products).
Write the general summary equation (balanced)for photosynthesis below:
______
To measure the rate of photosynthesis, we can measure either the production of O2 or
the consumption of CO2. In this investigation, you will use a system that measures the accumulation of oxygen.
Figure 1 – Cross Section of a Leaf
We will be using hole-punchers to make “leaf discs” in this investigation. Because the spongy mesophyll layer of leaves (shown in Figure 1) is normally infused with gases (O2and CO2), leaves (or disks cut from leaves), they would normally float in water.
What would you predict about the density of the leaf disk if the gases are drawn from the spongy mesophyll layer by using a vacuum and replaced with water? ______
How will that affect whether or not the leaf floats? ______
(These questions will help you develop a discussion in your background.)
If the leaf disk is placed in a solution with an alternate source of carbon dioxide in the form of bicarbonate ions (baking soda), then photosynthesis can occur in a sunken leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy mesophyll, and the leaf disk will once again become buoyant and rise in a beaker of water. Therefore,the rate of photosynthesis can be indirectly measured by the rate of rise of the leaf disks. However, there’s more going on in the leaf than that! You must also remember that cellular respiration is taking place at the same time as photosynthesis in plant leaves. (Remember that plant cells have mitochondria, too!) Aerobic respiration will consume someoxygen that has accumulated in spongy mesophyll. Consequently, the two processes counter each other with respect to the accumulation of oxygen in the air spaces of the spongy mesophyll. So now you have a more robust measurement tool — the buoyancy of the leaf disks is actually an indirect measurement of the net rate ofphotosynthesis occurring in the leaf tissue.
To make comparisons of the rate of photosynthesis between experiments, a standard point of reference is needed. Repeated testing of this procedure has shown that the point at which 50% of the leaf disks are floating(the median or ET50,the EffectiveTime it takes 50% of the disks to float)is a reliable and repeatable point of reference for the Floating Leaf Assay.
In this example, by extrapolating from the graph, the 50% floating point is about 11.5 minutes. Using the 50% point provides a greater degree of reliability and repeatability for this procedure.
What is the relationship between the rate of photosynthesis and the ET 50? It is an ______relationship!
As the rate of photosynthesis goes up, the ET50 will ______!
OBJECTIVE/PURPOSE
- PART A -To determine the relative rates of photosynthesis, using the floating disc method, on discs with and without a carbon source.
- PART B -To design and conduct an experiment to explore the effectof certain factors, (either a physical or environmental variable), on the rate of photosynthesis. (Your group will determine the factor you’d like to investigate!)
*Make an appropriate hypothesis for Part A of this lab.
MATERIALS
• 1%Baking soda(sodium bicarbonate) the carbon source!
• Liquidsoap (approximately 5 mL soap/ 250 mL of water)
- Beakers to hold the stock solutions
• 2plastic syringes (10 mL) * Light Source
• Living leaves (spinach) * Heat sink with H20 (deep petri dish)
- Hole punch * Pipettes
- 2 clear plastic cups * Stop Watch
- Ring Stand
PROCEDURE
GettingStarted
While going through this activity, record any questions in your laboratory notebook. Thesequestions and others that occur to you while working through the steps in the Procedure can serve as a basis for your own investigation in Designing and Conducting Your Investigation.
Watch this video by Bozeman Science to see how to remove air from the leaves.
In this part of the lab, you will learn how the floatingleaf disk technique can measure the rate of photosynthesis by testing a variable that you know affectsphotosynthesis. Later, you will apply this technique to test a variable that you choose. It is important for you to develop a few skills during this part of the investigation in order to carry out your own investigation. For the floatingdisk technique, the most challenging skill is getting the disks to sink. Don’t just watch someone do this; make sure you can get the disks to sink as well.
When immersed in water, oxygen bubbles are usually trapped in the air spaces of the spongy mesophyll in the plant leaf. By creating a vacuum in this experimental procedure, the air bubbles can be drawn out of the spongy mesophyll, and the space is refilledby the surrounding solution. Thisallows the leaf disks to sink in the experimental solution. If the solution has bicarbonate ions and enough light, the leaf disk will begin to produce sugars and oxygen through the process of photosynthesis. Oxygen collects in the leaf as photosynthesis progresses, causing the leaf disks to float again. Thelength of time it takes for leaf disks to floatagain is a measure of the net rate of photosynthesis.
Figure 3: Removing the Gasses from the Spongy Mesophyll
Step1(I will do this for you!)
STOCK SOLUTIONS FOR THE CLASS
- Prepare 500 mL of 1% bicarbonate solution (1 gNaHCO3/ 100 mL dH2O). Thebicarbonate will serve as a source of carbon dioxide for the leaf disks while they are in the solution. This will be prepared for you.
- The liquid soap will also be prepared for you 5 mL soap in 250 mL of dH2O
Step2
A.Pour the bicarbonate solution into a clear plastic cup to a depth of about 3 cm. Label this cup “With CO2.”
B.Fill a second cup with only distilled waterto be used as a control group. Label this cup “Without CO2/ Control”
C.Throughoutthe rest of the procedure you will be preparing material for both cups, so do everything for both cups simultaneously
Step3
Using a pipette, add one drop of a dilute liquid soap solution to the solution in each cup. It is critical to avoid suds. If either solution generates suds, then dilute it with more bicarbonate or water solution. Thesoap acts as a surfactant or “wetting agent” — it wets the hydrophobic surface of the leaf, allowing the solution to be drawn into the leaf and enabling the leaf disks to sink in the fluid.
Step4
Using a holepunch, cut 10 or more uniform leaf disks for each cup. Avoid major leaf veins. (Thechoice of plant material is perhaps the most critical aspect of this procedure. Theleaf surface should be smooth and not too thick.)
Step5
Draw the gases out of the spongy mesophyll tissue and infiltratethe leaves with the sodium bicarbonate solution by performing the following steps:
(One set of discs receives the NaHCO3, the other set receives distilled H2O)
*Watch the video above again, if necessary.
a.Remove the piston or plunger from both syringes. Place the 10 leaf disks into each syringe barrel.
b.Replace the plunger, but be careful not to crush the leaf disks. Push in the plunger until only a small volume of air and leaf disk remain in thebarrel (<10%). Figure 5 - Leaf Disks
c.Pull a small volume (5 mL) of sodium bicarbonate plus soap solution from your prepared cup into one syringe. Tap the syringe to suspend the leaf disks in the solution. Makesure that, with the plunger inverted, the disks are suspended in the solution. Make sure no air remains. Move the plunger to get rid of air from the plunger beforeyou attempt Step d. (Repeat for the distilled water and soap solution in a different syringe)
d.You now want to create a vacuum in the plunger to draw the air out of the leaf tissue. Thisis the most difficultstep to master. Once you learn to do this, you will be able to complete the entire exercise successfully. Create the vacuum by holding a fingerover the narrow syringe opening while drawing back the plunger (see Figure 6a). Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspend them in the solution. Now release the vacuumby letting the plunger spring back. Thesolution will infiltratethe air spaces in the leaf disk, causing the leaf disks to sink in the syringe. If the plunger doesnot spring back, you did not have a good vacuum, and you may need a different syringe. You may have to repeat this procedure two to three times in order toget the disks to sink. (If you have any difficultygetting your disks to sink after three tries, it is usually because there is not enough soap in the solution. Try adding a few more drops of soap to the cup and replacing the liquid in the syringe.)Placing the disks under vacuum more than three times can damage the disks.
Figure6a -Creatinga Vacuum in the Plunger
Figure6b–Sinking Leaf Disks
Step6
Pour the disks and the solution from the syringe into the appropriate clear plastic cup. Disks infiltratedwith the bicarbonate solution go in the “With CO2” cup, and disks infiltratedwith the water go in the “Without CO2” cup.
*Remove any floating discs! (Be sure there are at least 8 discs before starting.)
*PLACE THE HEAT SINK (use regular tap water for this) ON TOP OF EACH CUP TO ABSORB EXCESS HEAT. THIS WILL REDUCE THE CHANCE OF DENATURING CRUCIAL ENZYMES DUE TO THE HEAT FROM THE LIGHT SOURCE.*
Cover the cups with tinfoil until you are ready to start both trials at the same time.
Step7
Place both cups under the light source and start the timer. At the end of each minute, record the number of floatingdisks for each cup. Every 30 seconds,gently swirl the cup to dislodge any disks that stuck against the side of the cups. Continue until all of the disksare floatingin the cup with the bicarbonate solution.
Be sure to enter all of your data in an appropriate table. (This should be prepared BEFORE the lab starts.)
Figure7a - Cupwithout BicarbonateFigure7b –Cupwith Bicarbonate
To make comparisons between experiments, a standard point of reference is needed. Repeated testing of this procedure has shown that the point at which 50% of the leaf disks are floating(the median or ET50,the EffectiveTime it takes 50% of the disks to float)is a reliable and repeatable point of reference for this procedure.
Step8
Repeat this procedure 3 times (if time allows do 3…but do 2 times minimum) to ensure that the ET50is comparable.
Record and report findings in your data table.
You will make another column in your data table for “Average time” of all trials.
Determine the ET50 for the control and experimental group.
Data and Observations
- Make an appropriate table for the data you collect in this experiment.
- Graph the data, placing the dependent and independent variables on the appropriate axis. (You should only plot one line (the average) for each set of discs.)
- Indicate the average ET50 for the control and experimental group on the graph both on the graph and below the graph.
Conclusion Section.
Write a thorough conclusion, referencing your data and data analysis!
Error Statement (separate from the conclusion)
Don’tforget to indicate the variability in your data (from the different trials). Were any of the times very different? Why do you think? What others factors may have skewed the data?
How would you improve the design for next time?
PART B: DESIGNING AND CONDUCTION YOUR OWN INVESTIGATION
- Be sure to include another background referencing your new variable, and how that factor affects the rate of photosynthesis. Be sure to define your dependent and independent variable in your background!
- methods you’ll use to measure the rate of photosynthesis.
- Include a new Purpose, hypothesis, materials, & procedure., etc.
To help get you started….
What factors affectthe rate of photosynthesis in living plants?
1.Once you have mastered the floatingdisk technique, you will design an experiment to test another variable that might affectthe rate of photosynthesis. Some ideas include the following, but don’t limit yourselfto just these:
• Whatenvironmental variables might affectthe net rate of photosynthesis? Why do you think they would affectit? How do you predict they would affectit?
• Whatphysical features or variablesof the plant leaves might affectthe net rate of photosynthesis? How and why?
• Couldthe way you perform the procedure affectthe outcome? If the outcome changes, does it mean the net rate of photosynthesis has changed? Why do you think that?
Note: If you are truly stumped, your instructor can give you some guidance. Keep in mind that leaves with hairy surfaces should be avoided. Ivy and spinach work well, but many others do as well. Differencesbetween plants may be one of the ideas that you want to investigate.
Brainstorm some ideas below with your lab partners.