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The Story of the CalvinCycle: Bringing Fixation to Life- H- FARDADFIROOZNIA 0-

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H A \ s teachersand professors,we aim to In my classroom, the details of the (for- prepare our students to think critically and analytically, mally the Calvin-Benson-Bashamcycle) are presented after and to provide a set of conceptual and practical skills the a general discussion of the big picture of in

0- students will need to continue their education in biology the global of the planet. By the time the students Downloaded from http://online.ucpress.edu/abt/article-pdf/69/6/364/54241/4452179.pdf by guest on 24 September 2021 during and after their college years. Another of our general are focusing on the chemical reactions in the Calvin cycle, goals in teaching a biology class is to convey to our students they have studied the overall goals of photosynthesis and its our interest and excitement about science, and biology in role in the . They have also reviewed, through particular. This is not an easy task, especially in required various activities, the photochemical reactions of photosyn- introductory courses or less popular plant biology courses, thesis leading to the production of ATP and NADPH and are as student motivation is usually lacking. Since most biology ready to find out the details of how these products are used. students think of plants as inherently less exciting than This way, we understand why the Calvin cycle is dependent animals, getting students excited to learn something about on the "light reactions" and why we do not refer to the physiological processes in plants is especially challenging. Calvin cycle as the "darkreactions." Add to that the difficulty in visualizing complex biochemical steps, and the introductory biology student, biology major or non-science major is ready to throw in the towel. Learning Objectives Many instructors of biology have noted that "students To understand: frequently have trouble visualizing what happens in a cell" * the three major steps of the biochemical reactions in (Chinnici et al., 2004). Techniques that require active partic- the Calvin cycle ipation to work out the details of a sub-cellular process help * the significance of Rubisco to the process students visualize and understand abstract concepts better. * Examples include hands-on exercises that require building the significance of to the plant and using a model (see Stavroulakis, 2005), or role-play- * the importance of regeneration to the cycle ing (Chinnici et al., 2004). In my introductory courses, I * the relationship between the Calvin cycle and the have resorted to role-playing and biological "plays"to help products of the photochemical reactions students visualize more abstract subjects in a manner that is easier for most students to understand. This is especially true for the inexperienced science students, especially first Pertinent Vocabulary/ year or non-major students. An example is The Story of Background the Calvin Cycle, described here, in which students trace (Mauseth, 2003; Taiz & Zeiger, 2002) the biochemical steps of the Calvin cycle through a musical play. Although introductory textbooks such as Campbell and Reece (2005) and Tobin and Dusheck (2005), and , an adenine plant biology texts such as Graham et al. (2006) and Rost et containing triphosphate that releases free when its al. (2006) provide beautiful diagrams showing the biochem- ATP bonds are hydrolyzed. ical steps of the Calvin cycle, a review of these diagrams This energy is used by the cell to is just one passive step used in my classroom. Through carry out endergonic reactions. the role-playing exercise described here, students will use reading, seeing, hearing, and physical participation to learn The first major step in the Calvin the subject and, as suggested by Chinnici et al. (2004), this cycle, referring to a combination enhances learning. ultimately Carboxylation through which a carbon-carbon bond is formed between CO2 and a carbon in an (RuBP in the Calvin cycle) FARDADFIROOZNIA (e-mail: [email protected])is a Two cells that flank the and former assistant professorof biology at Denison Universityin Guard Cells Granville,Ohio. He currentlyworks as an instructorat Lion regulate its opening and closing TechnologyInc. in Lafayette,New Jersey,a firm providing Glyceraldehyde-3-phosphate, the 3- compliancetrainingfor EPA, OSHA, and other environmental G-3-P (PGAL) carbon aldehyde produced directly regulatoryagencies. from the Calvin cycle

364 THEAMERICAN BIOLOGY TEACHER, VOLUME 69,NO. 6, AUGUST2007 The inside the mem- Lumen space The Cast of Characters branes Mesophyll Ground tissue, typically of a leaf, doors of the spe- Open guard cells with an open stoma Mesophyll______cialized for photosynthesis classroom Reduced form of nictoinamide Barrierssuch as chairs the porous cell wall adenine dinucleotide phosphate, Other barriers such as the plasma membrane and the NADPH a strong reducing agent, capable outer membrane of the of donating energized electrons to ropes reduce an acceptor CO2 entering through the open The bride stoma and diffusing to the site of The second major step in the Calvin carboxylation cycle, referring to the chemical The groom and his 4- Reduction reactions that reduce the 3-carbon person entourage, each organic acids to 3-carbon aldehydes the 5-carbon RuBP,floating in the with a C sign to desig- through addition of nate them as individual The third major step in the Calvin The minister the Rubisco cycle, referring to the chemical reac- Regeneration tions that use 3-carbon aldehydes to * ATPs, each with a triphosphate Downloaded from http://online.ucpress.edu/abt/article-pdf/69/6/364/54241/4452179.pdf by guest on 24 September 2021 produce more of the 5-carbon sugar tail RuBP * NADPHs, each with a remov-

Ribulose bisphosphate carboxylase/ Other participants able proton oxygenase, the enzyme that carries * four G-3-P (3 stu- out the carboxylation step in the dents per molecule represent- Rubisco Calvin cycle by attaching a CO2 to ing the 3 carbon atoms, each the 5-carbon sugar RuBP to form two with a C sign) molecules of 3-phosphoglycerate, a 3-carbon organic acid The Musical Ribulose-1,5-bisphosphate, the 5- The lyrics and directions for the musical are presented RuBP carbon sugar that is carboxylated by below. I wrote the lyrics to the theme from the TV show The Rubisco in the first step of the Calvin BradyBunch, written by Sherwood Schwartz and Frank De Vol. cycle As one who only knows The BradyBunch from TV reruns him- self, I am happy to say that all of my teenage college students The pore, surrounded by guard cells, recognized and knew the theme song and the story line. Others Stoma in the epidermis of leaves and stems through which gas exchange occurs may choose to adopt this approach and write their own lyrics to a different tune; however, this exercise, as written, works with the theme song from The BradyBunch. Stroma The solution surrounding the thyla- koid membranes As we perform this musical in the class, the characters sing Thylakoid The photosynthetic membranes the lyrics (in italics below) and the following events, written inside the chloroplast under Directions for each segment, are acted out: The Story of the Calvin Cycle Materials Required Here'sthe story of RuBP This exercise requires the willingness of the students to Who wasfloating in the stromaon its own. in participate the musical. Not much else is required. Due to my Therewere lots of other RuBPsaround general enthusiasm for biology, and all of the other biological But each was all alone. plays and activities I employ in teaching, my students are used to and expect active participation in learning a complicated pro- Directions: We meet RuBPwith its five carbons (the groom cess such as the of the Calvin cycle. Thus, I have and his 4-person entourage), floating in the stroma. Other not found student willingness to be a problem for any of the compounds in the stroma may wander in the background. activities I employ in my class, including this activity. Here'sthe story of * I make simple printout labels with large print that the That had come in throughan open stoma. students pin to their shirts to designate them as carbon atoms or NADPH (with H+ labels to pass on) or ATP It diffusedthrough mesophyll cytoplasm (with a tri-phosphate tail pinned on them) or the C02- Ending in the stroma. fixing enzyme Rubisco (Ribulose bisphosphate carbox- Directions: One CO2 (the bride) enters the leaf (the class- ylase/oxygenase). room) through an open stoma (the open doors) and diffus- * To make the play more interesting, I borrowed a couple es through the leaf (barriers set up using chairs and ropes of simple costumes, such as a wedding veil and a minis- in the classroom represent the diffusion path through the ter's collar, from the theatre department; these costumes mesophyll cell wall, plasma membrane, and outer envelope are used by the characters involved in the carboxylation of the chloroplast) till it reaches the stroma inside a chloro- step, as described below. plast where the RuBP is floating.

CAlVINCYClE 365 'Till Rubiscobrought these two togetherat last Directions: Regeneration step = The other five remaining And in one magical momentof 3-carbon aldehydes (PGALs or G-3-Ps) go through the complicated regeneration steps to yield three 5-carbon They werejoined as one and seemed to have a blast. RuBPs with the help of more hyper-energetic ATPs. This is But their union was not really meant to last ... done during the singing of the above two stanzas, possibly Not meant to last, not meant to last, their union was not really extending to the final stanza below, to show the general meant to last. steps involved in regeneration. The regeneration steps dem- Directions: = Carboxylation step; Rubisco onstrate how the 3-carbon aldehydes join and split through (the minister) joins the two together to form a 6-carbon a series of reactions that involve 3, 4, 5, 6, and 7-carbon compound. The bride and groom put up a good fight after compounds to yield the regenerated RuBPs in the final step they are joined to show the instability of the 6-carbon com- of the Calvin cycle. We do not dwell on all of the pound and get ready for the split. involved in the regeneration steps. However, to show the general picture, the molecules go through the joining and When their blissfulunion so ended splitting motions: The 6-carboncompound had split into two a) Two 3-carbon compounds join to make a 6-carbon Formingtwo moleculesof Phosphoglycerate. sugar, So what's a plant to do? b) which then joins with another 3-carbon compound only

Directions: Following the fight between the bride and to split into a 4-carbon and a 5-carbon compound. Downloaded from http://online.ucpress.edu/abt/article-pdf/69/6/364/54241/4452179.pdf by guest on 24 September 2021 groom, the 6-carbon compound splits in two to form c) The 4-carbon compound joins with another 3-carbon two phosphoglycerates (phosphoglyceric acids, PGAs). compound to make a 7-carbon compound, Depending on how many students there are in the class, there could be three sets of such reactions, producing 6 d) which joins with another 3-carbon compound only to molecules of phosphoglycerate. This will help balance the split into two 5-carbon compounds. reactions. e) The three 5-carbon compounds formed above in Steps b and d then receive phosphate groups from 3 ATP mol- NADPH, and ATP too ecules to become RuBPs, thus completing the regenera- Bringenergy and electronsforthe ride. tion process. They reducethe acid on phosphoglycerate Alternatively, in a course less concerned with details of Formingan aldehyde. these steps, the 15 carbon atoms from the five 3-carbon alde- Directions: Reduction step = The NADPH and the hyper- hydes can gradually separate and rearrange into three 5-carbon energetic ATP that are present in the stroma act their parts RuBPswith help from the ATPs without necessarily demonstrat- (showing their energy and enthusiasm) to change the acids ing all of the steps discussed above. to aldehydes. First the ATP removes one phosphate group The Regenerationsteps are complicated from its tail and attaches it to the phosphoglycerate to make But it's absolutelyessential that they'redone, 1,3-bisphosphoglycerate. Then NADPH donates its proton and removes the phosphate group from 1,3-bisphospho- Since Rubiscocan act its part once again. glycerate to create glyceraldehyde 3-phosphate (G-3-P or The Calvin-Benson-BashamCycle must go on ... PGAL). It mustgo on, it mustgo on, the Calvin-BensonCycle must go on. Now some aldehydesare used to make the sugars Directions: The regeneration steps may extend into the ,or other carbon need a plant may see. singing of this stanza. Otherwise, after the regeneration step is complete, the final stanza can be sung by the whole But no more C02 can be taken up now cast before it takes its final bow. Unless something'sdone about RuBP... RuBP,RuBP, something is done about RuBP. Review Directions: One of the 3-carbon aldehydes (PGAL or G-3- Following the musical, there is a quick review of the major P) is used to make starch in the chloroplast or exported to steps of the Calvin cycle and their relation to internal be used in other biochemical steps to make sugars in the of the leaf and the photochemical reactions in the . . In the play, one 3-carbon aldehyde floats away to We perform and review the Calvin cycle during one 50-minute signify its separation from the rest that will go through the class period. The musical itself only takes about 10-15 minutes, next few steps: the regeneration process. including assigning roles and costumes. Sample review ques- tions on the Calvin Cycle may include: The aldehydeseach have 3 carbons; 1. What is accomplished in each of the three major steps in 5 in each RuBP: Thereare carbons the Calvin cycle? Making a 5 carboncompound from one with 3 2. What do NADPH and ATP do in chemical reactions in A problem,as you see. the Calvin cycle? Nowtak-e 5 of the 3-carboncompounds, 3. Why is it important for the plant to use some of the A total of 15 carbonsfor your use. 3-carbon aldehyde (glyceraldehyde-3-phosphate) that Youcan then make 3 of the 5-carbonsugars. is produced in the Calvin cycle to make more 5-carbon Problemsolved! What good news! RuBP (Ribulose-1,5-bisphosphate)?

366 THEAMERICAN BIOLOGY TEACHER, VOLUME 69, NO.6, AUGUST2007 4. Why is it important for the plant to export some of the I usually precede this musical with other in-class activi- 3-carbon aldehyde (glyceraldehyde-3-phosphate) that is ties that demonstrate the photochemical reactions leading to produced in the Calvin cycle? the synthesis of NADPH and ATP as the final products. Thus, 5. If we turn off the lights in the classroom to simulate the connection between the photochemical and biochemical night time, how will that affect the steps of the Calvin reactions through ATP and NADPH becomes an easy point cycle, as demonstrated by you, the students? This ques- of discussion and a fun activity. In a larger class with enough tion requires students to think of the overall picture of participants for all of the roles, such activities can be easily photosynthesis, and their knowledge of both the Calvin combined. cycle and the photochemical reactions. Analysis of the performance of the students, as described above, shows that this activity has educational merit in addition to its entertainment value. Anecdotal responses by students Assessment and feedback from colleagues I compared the performance Table1. Statisticalanalysis comparing the performanceof support the use of such creative of the students who had learned studentson questionson the finalexam that related to the methods in the classroom. As about the Calvin cycle through Calvincycle. one of my senior undergraduate the standard methods of review TAs bemoaned, "Perhaps if I had Thetwo groups were those who were taught the Calvin cycle through with textbook diagrams when I learned about the Calvin [C]ycle in my standardcoverage, and those who were taught the Calvin cycle did not use this musical this way, I would be more inter- Downloaded from http://online.ucpress.edu/abt/article-pdf/69/6/364/54241/4452179.pdf by guest on 24 September 2021 classes (a total of 111 students throughthe musical with a reviewof the topic using standard mate- ested in learning about plants." classes) to those who rial.SEM = StandardError ofthe Mean. from six In conclusion, this is a sim- had been additionally exposed StudentGroup CalvinCycle Story StandardCoverage ple, inexpensive way to involve to this in-class activity (a total SampleSize 22 111 students in learning the biochem- of 22 students from one class). MeanScore % 90 71 istry of carbon fixation in plants. I used the general linear model SEM 3.3 3.5 I consider this to be an interest- (GLM) using the statistical soft- (%) Minimum% 50 0 ing and stimulating alternative, or ware MINITAB(MINITAB Inc.) to Maximum(%) 100 100 at least supplement, to the tradi- compare their performance on the Median 100 100 tional methods of passive learn- that dealt (%) final exam questions ing where the instructor simply the Calvin cycle; these ques- with describes the biochemical steDs. tions were similar to the review questions listed previously. This exercise can be easily implemented in both high school shown in Table 1, the mean score of the group of students As and introductory college courses with adequate detail, and without exposure to this activity was significantly lower than should add both entertainment and substance to the course. that of the group who had been taught using this activity at the a = 0.05 level (p = 0.023). The gender and academic year (fresh- man, sophomore, junior, and senior) of the students were also Acknowledgments in the analysis and were found not to be significant considered I thank my students for participating in the musicals and = for and p = 0.78 for academic year). Age of the (p 0.61 gender, other biological plays I have incorporated in my teaching, espe- students was not known and not considered in this analysis. cially the story of the Calvin cycle. I also thank the Department However, as Denison students are traditional students, the age of Biology at Denison University for the funds to purchase the range is very narrow and corresponds in general to academic few arts and crafts supplies needed for this activity, and the Another factor that may have been interesting would year. Department of Theatre at Denison University for lending us a have been the overall GPA of the students. These data were few costumes for the play. not available for this study. Given my limited time at Denison University, this was a one-time assessment on the effectiveness of this teaching activity. References Campbell, N. & Reece, J.B. (2005). Biology, 7th Edition. San Francisco, Conclusions CA: Pearson Education Inc., Benjamin Cummings. Chinnici, J.P., Yue, J.W. & Kierom, M.T. (2004). Students as "human One of my goals in using such plays is to stimulate enthu- chromosomes" in role-playing mitosis and meiosis. The American siasm about biology and its interactions with the world and to BiologyTeacher, 66(1), 35-39. move the students away from the conception of science as dull Graham, L.E., Graham,J.M. & Wilcox, L.W. (2006). Plant Biology,2nd and impossibly difficult. Students generally have positive reac- Edition. San Francisco, CA: Pearson Education Inc., Benjamin tions to these biological plays and consider them an indication Cummings. of my enthusiasm for biology. I have found these plays, together Mauseth, J.D. (2003). : An Introduction to Plant Biology, 3rd with a review of the steps of the Calvin cycle and the importance Edition. Sudbury, MA:Jones and Bartlett Publishers. of each step, to be a helpful strategy to engage the students Rost, T.L., Barbour,M.G., Stocking, C.R. & Murphy, T.M. (2006). Plant in the learning process and to impress upon the students the Biology,2nd Edition.Toronto, Canada: Thomson Learning, Inc. importance of the biochemical steps involved and the interrela- Stavroulakis, A.M. (2005). Meio-socks & other genetic yarns. The tionship between anatomy (the open stomata and the internal AmericanBiology Teacher, 67(4), 233-238. leaf structure) and (gas exchange and carbon fixa- Taiz, L. & Zeiger, E. (2002). Plant Physiology,3rd Edition. Sunderland, tion). In addition, the interrelationship between the photochem- MA:Sinauer Associates, Inc. as without ical reactions and biochemical reactions is evident, Tobin, Aj. & Dusheck,J. (2005). AskingAbout Life,3rd Edition.Toronto, NADPH and ATP,the musical play will come to a halt! Canada:Thomson Learning, Inc.

CALVINCYCLE 367