Lesson 2: Photosynthesis and RespirationThis exercise was written by V. Muilenburg and K. Winnett-Murray (2014). Adapted from a laboratory entitled "Respiration & Photosynthesis" written for by T. Bultman, J. Li, and J. Hielkema (2013).

Questions What are photosynthesis and respiration and why are they important to plants? How can we find rates of photosynthesis and respiration in plants? Why might plants grown under high vs. low concentrations of CO2 have different rates of

photosynthesis and respiration? Why might plants grown under high concentrations of CO2 have different rates of

photosynthesis and respiration when later exposed to ambient CO2 concentrations?

Learning ObjectivesUpon completion of this exercise, you should be able to:

1. Apply previous knowledge to make predictions and informed hypotheses about new phenomena.

2. Use a CO2 Gas Sensor to measure the amount of CO2 consumed or produced by a plant during respiration and photosynthesis.

3. Use regression analysis to find rates of photosynthesis and respiration.4. Determine the rate of photosynthesis and respiration of a plant.5. Articulate the relationship between atmospheric CO2 concentrations and rates of

photosynthesis and respiration.6. Describe what it means for plants to acclimate to environmental resources.7. Calculate means and graphically display means using statistical software.8. Perform t-tests using statistical software.9. Write reports in the style of scientific manuscripts.10. Write or record the following in a laboratory notebook: a working hypothesis with a

biological rationale, data, observations, results with supporting statistical evidence, and conclusions.

Introduction

Photosynthesis and respiration: the basics

Carbon fixation is the conversion of inorganic CO2 from the atmosphere to organic compounds during photosynthesis. This process is essential for growth and survival of plants. As we learned in the previous lesson, CO2 enters leaves through the stomata, diffuses through the intracellular

1

spaces, and diffuses into cells and the chloroplasts inside the cells. The chloroplasts contain specialized green pigment molecules called chlorophyll. Chlorophyll absorbs light energy, which drives the synthesis of organic molecules (primarily sugars) from inorganic CO2. The chemical changes during photosynthesis can be summarized by the following reaction:

light energy + CO2 + H2O O2 + organic molecules

Environmental factors can drastically impact rates of photosynthesis. The intensity of light energy and concentration of atmospheric CO2 are two major factors that can limit the rate of photosynthesis. However, plants can acclimate to environmental conditions, including light intensity, to maximize their photosynthetic rates. For example, plants growing in low light conditions have leaves that are thinner and contain more chlorophyll that plants of the same species growing in high light conditions (Anderson 1986, Taiz and Zeiger 2010).

During respiration in plants and all other organisms, organic molecules are broken down by chemical (metabolic) reactions, producing energy in the form of ATP and carbon precursors of molecules needed for growth. In plants, the organic molecules used in respiration are produced by photosynthesis. The chemical changes during respiration can be summarized by the following equation:

organic molecules + O2 CO2 + H2O + ATP (chemical energy)

Considering all reactions in a plant over a 24-hr period, approximately half of the carbon in the organic molecules produced by photosynthesis is used in respiration (Lambers et al. 2008). Thus, the plant breaks down about half of the organic molecules produced by photosynthesis to produce chemical energy for the plant, with CO2 as a byproduct. The rate of plant respiration may be altered by many factors including light, CO2 concentrations, temperature, oxygen availability, energy demand by the plant, and availability of organic molecules produced by photosynthesis (Covey-Crump et al. 2007, Lambers et al. 2008).

Effects of elevated CO2 on photosynthesis and respiration

A plant grown in elevated concentrations of CO2 typically has a higher rate of photosynthesis than a plant exposed to low concentrations of CO2 because more CO2 is available for carbon fixation (conversion of CO2 to organic molecules) (Curtis and Wang 1998). Rates of respiration in plants, however, can change in plants growing under altered CO2 conditions (Curtis and Wang 1998, Tjoelker et al. 2001, Gonzalez-Meler et al. 2004). If we move a plant from a high CO2 environment to lower CO2 conditions, will it have the same rates of photosynthesis and respiration as before? Or, will it acclimate to the new environment by adjusting its rates of photosynthesis and respiration in response to lower CO2 levels? (Remember, acclimation means that an individual plant adjusts its morphology and/or physiology to better perform under its present environmental conditions).

In this lesson, we will investigate how growing B. rapa in high (800 ppm) CO2 concentrations affects rates of photosynthesis and respiration when those plants are later exposed to lower (ambient) levels of CO2. Ambient levels are the conditions in the immediate surroundings, which in this case is ~400 ppm of CO2. In other words, we will analyze whether or not plants grown in high concentrations of CO2 can acclimate their physiology (i.e., photosynthetic and respiratory rates) if transferred to lower (ambient) CO2 concentrations by comparing photosynthetic and respiratory rates to control plants. In this case, control plants are B. rapa grown and assessed in ambient (400 ppm) CO2 concentrations. Optimizing photosynthetic rates (which is directly linked

2

to respiratory rates) to environmental conditions is important because it determines a plant’s ability to grow, survive, and reproduce.

Create a working hypothesis about how B. rapa grown in high atmospheric CO2 concentrations might have different rates of photosynthesis and respiration when they are exposed to ambient CO2 concentrations compared to control plants (B. rapa grown and assessed at ambient CO2 concentrations). Consider the potential role of acclimation and stomatal density (that you quantified last week) when developing the biological rationale for your hypothesis. Record this hypothesis in your lab notebook. Be aware that when we measure “photosynthetic rate” in a plant in a closed system, the plant is doing both photosynthesis and respiration at the same time. Respiration produces CO2, while photosynthesis removes it.

Therefore, what you will measure as the change of CO2 inside the chamber while measuring “photosynthesis” is actually the net photosynthetic rate (since you can’t stop the plant from respiring without killing it). How would you then determine the gross photosynthetic rate?

Gross photosynthetic rate = net photosynthetic rate + respiration

References and ResourcesAnderson, J. M. 1986. Photoregulation of the composition, function, and structure of thylakoid

membranes. Annual Review of Plant Physiology 37: 93-136.

Covey-Crump, E. M., N. V. Bykova, C. Affourtit, M. H. N. Hoefnagel, P. Gardestrom, and O. K. Atkin. 2007. Temperature-dependent changes in respiration rates and redox poise of theubiquinone pool in protoplasts and isolated mitochondria of potato leaves. Physiologia Plantarum 129:175-184.

Curtis, P. S., and X. Z. Wang. 1998. A meta-analysis of elevated CO2 effects on woody plant mass, form, and physiology. Oecologia 113:299-313.

Gonzalex-Meler, M., L. Taneva, and R.J. Trueman. 2004. Plant respiration and elevated atmospheric CO2 concentration: cellular responses and global significance. Annals of Botany 94:647-656.

Lambers, H., F. S. Chapin III, and T. L. Pons. 2008. Plant Physiological Ecology. Springer, New York, U.S.

Taiz, L., and E. Zeiger. 2010. Plant Physiology, Fifth Edition. Sinauer Associates, Inc.: Saunderland, United States.

Tjoelker, M. G., J. Oleksyn, T. D. Lee, and P. B. Reich. 2001. Direct inhibition of leaf dark respiration by elevated CO2 is minor in 12 grassland species. New Phytologist 149:419-424.

3

Procedures: Rate of Photosynthesis

1. If your Vernier sensor has a switch, set it to the low setting. Connect the CO2 Gas sensor to the Vernier interface.

2. Open Logger Lite 1.6.1 on your computer.

3. To equilibrate the CO2 concentration inside the chamber, wave the bottle around vigorously, away from other students who are respiring. Then cap the bottle.

4. Once Logger Lite is open, click “Experiment” and then “Data Collection.” Change the duration to 15 min. Change sampling rate to 2 samples/minute and 0.5 min/sample. Then click “Done.”

5. Obtain a B. rapa that has been grown in high CO2 and one that has been grown in low (ambient) CO2. Chose ONE plant, cut the stem at the soil surface level, and weigh your plant in mg. Record the mass in your lab notebook as in Table 1.

6. Place the plant into the CO2 chamber, using forceps if necessary (Fig. 1).

7. Insert the CO2 Gas Sensor into the screw top of the chamber (Fig. 1) and wrap parafilm around the joint where they come together to ensure an airtight seal.

8. Turn on the fluorescent lamp (1 globe electric utility clamp work lamp with a 100W equivalent fluorescent bulb). Place the lamp six inches from the leaves (MEASURE THIS DISTANCE). Do not let the lamp touch the respiration chamber (Fig. 2). Note the time. The lamp should be on for three minutes prior to beginning data collection.

9. As you wait, answer this question in your lab notebook: Do you expect the slope of the line representing photosynthesis to have a positive or negative value? Why?

Figure 1. Chamber set-up for photosynthesis and respiration (drawing by Jennifer Fuller, Hope College class of 2017).

Figure 2. Configuration for measuring (A) photosynthesis and (B) respiration using Vernier equipment and Logger Lite software.Image Source: V. Muilenburg 2017

B

A

4

10. After the three-minute time period is up, click the green Collect button on the computer to begin data collection. Collect data for 15 minutes.

11. When data collection has finished, determine the rate of photosynthesis:

a) Move the mouse pointer to the position where the data values begin to decrease (which indicates that the plant is photosynthesizing more than it is respiring). Hold down the left mouse button. Drag the pointer to the point where the data ceases to decline and release the mouse button (the selected part of the graph will now appear gray).

b) Click on “Analyze” and then “Linear Fit” from the pull-down menu to perform linear regression. A floating box will appear with the formula for a best-fit line.

c) Record the slope of the line, m , as the net rate of photosynthesis in your lab notebook as in Table 1 (below).

d) In your lab notebook, answer this question: Occasionally, a positive value for photosynthetic rate will be measured. Is this measurement possible? What does it mean?

12. Review your data. Do they make sense? Were any mistakes made that would warrant you to repeat your measurements?

13. Close the linear regression floating box.

14. Divide the photosynthetic rate by the plant mass to express the rate by mg of plant tissue. Record this in your lab notebook as in Table 1.

15. Remove the plant from the CO2 chamber, using forceps if necessary. Clean and dry the chamber.

Procedures: Rate of Respiration

1. Wrap the respiration chamber in aluminum foil so that no light reaches the leaves and place the chamber such that the surface of the lamp is six inches from the leaves. (Note: keep the lamp on to maintain constant environmental conditions throughout the photosynthetic and respiratory measurements).

2. Wait five minutes for the sensor readings to stabilize before proceeding to Step 5.

3. As you wait, answer this question in your lab notebook: Do you expect the slope of the line representing respiration to have a positive or negative value? Why?

4. Once Logger Lite is open, click “Experiment” and then “Data Collection.” Change the duration to 15 min. Change sampling rate to 2 samples/minute and 0.5 min/sample. Then click “Done”

5. Click the green Collect button on the computer to begin data collection.

6. When data collection has finished, determine the rate of respiration:

5

a. Move the mouse pointer to the point where the data values begin to increase. Hold down the left mouse button. Drag the pointer to the position where the data ceases to rise and release the mouse button (the selected part of the graph will now appear gray).

b. Click on “Analyze” and then “Linear Fit” from the pull-down menu to perform linear regression. A floating box will appear with the formula for a best-fit line.

c. Record the slope of the line, m , as the rate of respiration in your lab notebook as in Table 1 (below).

7. Review your data. Do they make sense? Were any mistakes made that would warrant you to repeat your measurements or to exclude the data if you are short on time?

8. Close the linear regression floating box.

9. Divide the respiration rate by the plant mass to express the rate by mg of plant tissue. Record this in your lab notebook as in Table 1.

10. Why do we measure changes in CO2 in the dark to quantify respiratory rates of our plants? Record the answer in your lab notebook.

11. Do you have evidence that photosynthesis (measured previously) and respiration occurred in your plant? Explain. Record this explanation in your lab notebook.

Graphing, Data Analysis & Conclusions – Lab Notebook

1. Once you have reviewed your group’s data, enter the data into the master file. Analysis will be completed on course data representing all lab sections.

2. Import the Excel file with the entire class data into SPSS and determine the mean and standard error (± 1 SE) of the mean for the photosynthetic and respiratory rates for plants growing in high vs. ambient concentrations of CO2 but later transferred and assessed at ambient CO2 conditions. Record these in your lab notebook.

3. Prepare a bar graph to compare the mean net photosynthetic rates and a bar graph comparing the respiratory rates of plant growing in high vs. low (ambient) concentrations of CO2 but then transferred and assessed at ambient CO2 conditions. Import or copy the graph into Word and include a figure caption below the graph. Print and place the completed figures and captions in your lab notebook.

4. Statistically compare the net photosynthetic rates and the respiration rates of plants grown in high vs. low (ambient) CO2 concentrations but then transferred and assessed at ambient CO2 conditions using independent samples t-test. Do we have evidence that growing plants in high or low CO2 concentrations affects rates of photosynthesis and/or respiration when the plants are later exposed to ambient CO2 concentrations? Print and include the t-test tables with written results and conclusions about your working hypotheses in your lab notebook.

6

5. Compare your results with the prediction that you made about rates of photosynthesis in the stomatal density lab. Do your results agree or disagree with your predictions? Explain why or why not by giving a potential biological explanation for your results.

7

Table 1. Rates of respiration and net photosynthesis in Brassica rapa plants grown in different CO2 concentrations

CO2 Level

Mass

(mg fresh weight)

Rate of respiration*

(ppm/min)

Rate of respiration per mg

((ppm/min)/mg)

Net Rate of photosynthesis*

(ppm/min)

Net Rate of photosynthesis per mg

((ppm/min)/mg)

Lowa CO2

Highb CO2

* As given by the slope (m)aLow (ambient) corresponds to 400 ppm CO2 and Highb corresponds to 800 ppm CO2

Note: this table is for your group’s data (not course data). Include this table in your lab notebook.

8