Key Concept Question concerning the Calvin Cycle: How do sugarcane plants and saguaro cacti avoid the wasteful side effects of photorespiration?

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1 Bioc Dr. Miesfeld Spring 2008 Calvin Cycle and C4/CAM Pathways Supplemental Reading Key Concepts - Three stages of the Calvin Cycle Stage 1: Fixation of CO2 to form 3-phosphoglycerate Stage 2: Reduction of 3-phosphoglycerate to from hexose sugars Stage 3: Regeneration of ribulose 1,5-bisphosphate - Regulation of the Calvin Cycle - The C4 and CAM pathways reduce photorespiration in hot climates Key Concept Question concerning the Calvin Cycle: How do sugarcane plants and saguaro cacti avoid the wasteful side effects of photorespiration? Biochemical Applications of the Calvin Cycle: Photorespiration is a wasteful side reaction catalyzed by the enzyme ribulose-1-5-bisphosphate carboxylase/oxygenase (rubisco) which reduces crop yields by competing for the C3 carbon dioxide fixation pathway. The C4 pathway in corn plants leads to reduced rates of photorespiration because CO2 is concentrated in the chloroplast stroma and out competes O2 for rubisco binding. In an effort to increase crop yields in C3 plants such as rice, transgenic plants are being developed that contain corn genes encoding multiple enzymes in the C4 pathway. Three stages of the Calvin cycle Plants store light energy in the form of carbohydrate, primarily starch and sucrose. The carbon and oxygen for this process comes from CO2, and the energy for carbon fixation is derived from the ATP and NADPH made during Figure 1. photosynthesis. The conversion of CO2 to carbohydrate is called the Calvin cycle and is named after Melvin Calvin who discovered it. This cycle requires the enzyme ribulose 1,5-bisphosphate carboxylase commonly called rubisco. As summarized in figure 1, the Calvin cycle generates the triose phosphates 3phosphoglycerate, glyceraldehyde-3p (GAP) and dihydroxyacetone phosphate, all of which are used to synthesize the hexose phosphates fructose-1,6bisphosphate and fructose 6-phosphate. Hexose phosphates produced by the Calvin cycle are converted to 1) sucrose for transport to other plant tissues, 2) starch for energy stores within the cell, 3) cellulose for cell wall synthesis, and 4) pentose phosphates for metabolic intermediates. 1 of 10 pages

2 Bioc Dr. Miesfeld Spring 2008 The basic scheme of the Calvin Figure 2. cycle is illustrated in figure 2 where it can be seen that it consists of three stages; Fixation, Reduction and Regeneration. Note that three turns of the cycle results in the fixation of three molecules of CO 2 into the triose phosphate 3- phosphoglycerate through a reaction catalyzed by the rubisco enzyme. This key reaction in stage 1 combines three molecules of ribulose-1,5-bisphosphate (RuBP), a five carbon (C 5 ) compound, with three molecules of CO 2 to form six molecules of the C 3 compound 3- phosphoglycerate. Using energy available from ATP hydrolysis and NADPH oxidation, the six molecules of 3- phosphoglycerate are converted intoto six molecules of glyceraldehydes-3p, of which one is used to synthesize glucose (stage 2), and the other five are recycled to generate RuBP (stage 3). The Calvin cycle is sometimes called the Dark Reactions, but do not be fooled by this name - the Calvin Cycle is the most active during the daylight hours when ATP and NADPH are plentiful. The net reaction of three turns of the Calvin cycle shown in figure 2 can be written as: 3 CO RuBP + 6 NADPH + 9 ATP + 6 H 2 O ---> 1 GAP + 3 RuBP + 6 NADP ADP + 9 P i If we just look at the fate of the carbons coming from CO 2 (C 1 ) in this reaction, we see that one net C 3 compound (glyceraldehydes-3p) is formed and three C 5 molecules (RuBP) are regenerated: 3 C C 5 ---> 1 C C 5 Stage 1: Fixation of CO 2 to form 3-phosphoglycerate When Calvin began his experiments in the 1940s it was known that CO 2 in plant chloroplasts was converted to hexose phosphates, however, it wasn't clear how this was accomplished. To identify the metabolic intermediates in this process, Calvin and his colleagues used radioactive labeling with 14 CO 2 to follow carbon fixation in photosynthetic algae cells grown in culture. As shown in figure 4, they found that within a few seconds of adding 14 CO 2 to the culture, the cells accumulated 14 C-labeled 3-phosophoglycerate, suggesting Figure 4. that this was the first product of the carboxylation reaction. Within a minute of adding 14 CO 2 to the culture, they found numerous compounds labeled with 14 C, many of which were later identified as Calvin cycle intermediates. 2 of 10 pages

3 Figure 5. Bioc Dr. Miesfeld Spring 2008 The work of Calvin and others led to the identification and characterization of rubisco as the key enzyme in the carbon fixation reaction. As shown in figure 5, the rubisco reaction can be broken down into four basic steps beginning with the formation of an enediolate intermediate of RuBP which requires the participation of a carbamoylated lysine residue (Lys 201) in the enzyme active site. Carboxylation then occurs in step 2 by nucleophilic attack on the CO 2 to form an unstable C 6 intermediate (2-carboxy-3-keto D-arabinitol 1,5-bisphosphate) that is hydrated in step 3. Aldol cleavage in step 4 leads to the formation of two molecules of 3-phosphoglycerate. The rubisco reaction is very exergonic ( ΔG o ' = kj/mol) with the aldol cleavage step being a major contributor to the favorable change in free energy. Rubisco is a multisubunit enzyme consisting of eight identical catalytic subunits at the core surrounded by eight smaller subunits that function to stabilize the complex and presumably enhance enzyme activity. The molecular structure of the spinach plant rubisco enzyme is shown in figure 6. Because of its low catalytic efficiency, a mere three carboxylation reactions per second (k cat = Figure 6. ~3/sec), chloroplasts contain large amounts of the enzyme in order to meet the demands of the cell. In fact, 50% of the total protein in a spinach leaf is rubisco, and the concentration of rubisco in chloroplast stroma is a staggering 250 mg/ml. Considering that rubisco plays a central role in all photosynthetic autotrophic organisms on earth, of which ~85% are photosynthetic plants and microorganisms that inhabit the oceans, rubisco is arguably the most abundant enzyme, and perhaps the most abundant protein, on this planet. Stage 2: Reduction of 3-phosphoglycerate to from hexose sugars The 3-phosphoglycerate product of the rubisco reaction is converted to glyceraldehyde-3- phosphate (GAP) by two isozymes of phosphoglycerate kinase and glyceraldehyde-3p dehydrogenase as shown in figure 7. In the Calvin cycle, 3-phosphoglycerate kinase uses ATP in a phosphoryl transfer reaction to generate 1,3-bisphosphoglycerate which is then reduced to GAP by glyceraldehyde-3p dehydrogenase. This stromal enzyme is similar to the cytosolic version except it uses NADPH instead of NADH as the electron pair donor. Remember that for every 3 CO2 that are fixed by carboxylation of 3 RuBP molecules, six moles of 3-phosphoglycerate are generated by aldol cleavage. Therefore, 6 ATP and 6 NADPH are required for every 3 CO2 that are converted to one net glyceraldehyde-3p. An additional 3 ATP are used in stage 3 to 3 of 10 pages

4 Figure 8. Bioc Dr. Miesfeld Spring 2008 regenerate these 3 RuBP molecules. One of the six GAP molecules is isomerized to dihydroxyacetone phosphate (DHAP) by another glycolytic enzyme, triose phosphate isomerase, and used for the net synthesis of hexose sugars. The triose phosphates GAP and DHAP are used to generate fructose-1,6-bisphosphate by a condensation reaction catalyzed by the enzyme transaldolase which leads to Figure 7. starch synthesis in the chloroplast and sucrose synthesis in the cytosol through interconversion of the hexose phosphates fructose 6- phosphate, glucose 6-phosphate and glucose 1- phosphate. Since the chloroplast membrane is not permeable to triose phosphates, GAP and DHAP must be exported to the cytosol through a P i -triose phosphate antiporter which also serves to import P i into the stroma. Stage 3: Regeneration of ribulose 1,5-bisphosphate In this final stage of the Calvin cycle, a series of enzyme reactions convert five C 3 molecules (GAP or DHAP) into three C 5 molecules (RuBP) to replenish supplies of this CO 2 acceptor molecule which is required in the rubisco reaction. Two of the primary enzymes in this carbon shuffle are transketolase and transaldolase which are involved in interconverting C 3, C 4, C 6 and C 7 molecules as shown in figure 8. The transketolase reaction transfers a C 2 group from a ketose to an aldose and requires thiamine pyrophosphate as a coenzyme in the reaction. In one case, transketolase transfers the C 2 unit (CO-CH 2 OH) from a C 6 molecule (fructose-6p) to a C 3 molecule (GAP), forming C 4 (erythrose-4p) and C 5 (xyulose-5p) molecules. In a second transketolase reaction, a C 2 unit is transferred from a C 7 molecule (sedoheptulose-7p) to a C 3 molecule (GAP), forming two different C 5 molecules (ribose-5p and xylulose-5p). As we have already seen in stage 2 (figure 7), transaldolase catalyzes the formation of a C 6 molecule (fructose-1,6-bp) from two C 3 molecules 4 of 10 pages

5 Bioc Dr. Miesfeld Spring 2008 (GAP and DHAP), and here in stage 3, the same enzyme generates a C 7 molecule (sedoheptulose-1,7bp) from C 3 (DHAP) and C 4 molecules (erythrose-4p). Figure 9 summarizes these carbon shuffle reactions showing how five C 3 molecules of GAP are converted to three C 5 molecules consisting of two xylulose-5p and one ribose-5p. The two xylulose-5p and one ribose- 5P molecules are first converted to three ribulose-5p molecules by the enzymes ribulose-5p epimerase and ribose-5p isomerase, respectively, and then the enzyme ribulose-5p kinase catalyzes a phosphoryl transfer involving three ATP to generate the final three molecules of RuBP. The regeneration of the three RuBP molecules used to fix 3 CO 2 requires an additional 3 ATP. Figure 9. Let's review the stoichiometry of the Calvin cycle reactions by summarizing what is required to synthesize one molecule of glucose from six CO 2. We can break the net reaction of the Calvin cycle down into two components, 1) the synthesis of one glucose molecule from six CO 2 using 12 ATP and 12 NADPH, and 2) regeneration of six RuBP using six ATP : 5 of 10 pages

6 Bioc Dr. Miesfeld Spring 2008 Glucose synthesis 6 CO RuBP + 12 NADPH + 12 ATP + 10 H 2 O ---> 4 GAP + 2 DHAP + Fructose-6P + Glucose + 12 NADP ADP + 16 P i Regeneration of RuBP 4 GAP + 2 DHAP + Fructose-6P + 6 ATP + 2 H 2 O ---> 6 RuBP + 6 ADP + 2 P i Net reaction from six turns of the Calvin cycle 6 CO NADPH + 18 ATP + 12 H 2 O ---> Glucose + 12 NADP ADP + 18 P i Regulation of the Calvin Cycle At night, plant cells rely on glycolysis and mitochondrial aerobic respiration to generate ATP for cellular processes. Since photophosphorylation and NADPH production by the photosynthetic electron transport system is shut down in the dark, it is crucial that the Calvin cycle only be active in the light. Otherwise, if glycolysis, the pentose phosphate pathway and the Calvin cycle were all active at the same time, then simultaneous starch degradation and carbohydrate biosynthesis would quickly deplete the ATP and NADPH pools in the stroma. Light stimulates the activity of Calvin cycle enzymes by two mechanisms: 1. Calvin cycle enzymes are activated by elevated ph and by increased Mg2+ concentrations in the stroma. Light activation of the photosynthetic electron transport system causes stromal ph to increase from ph 7 to ph 8 as a result of proton pumping Figure 10. into the thylakoid lumen. As shown in figure 10, this influx of H+ into the lumen causes an efflux of Mg 2+ to the stroma to balance the charge (the thylakoid membrane, unlike the mitochondrial inner membrane, is permeable to ion movement). Since the Calvin cycle enzymes rubisco and fructose-1,6-bisphosphatase are both activated by elevated ph and Mg 2+, light activation of photosynthetic electron transport indirectly stimulates metabolic flux through the Calvin cycle. 2. Calvin cycle enzymes are activated thioredoxin-mediated reduction of disulfide bridges. Thioredoxin is a small protein of 12 kda that is found throughout nature and functions as a redox protein that can interconvert disulfide bridges and sulfhydrals in cysteine residues of target proteins. In the dark, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatese, RuBP kinase and glyceradehyde-3p dehydrogenase, all contain disulfide bridges that inhibit enzyme activity. Light activation of the photosynthetic electron transport system leads to reduction of thioredoxin present in the stroma, which in turn reduces disulfide bridges in these enzymes resulting in their activation. As shown in figure 11, photon absorption by the PSI reaction center leads to increased levels of reduced ferredoxin which passes electrons one at a time to the enzyme ferredoxin-thioredoxin reductase when NADP + becomes limiting (high NADPH levels in 6 of 10 pages

7 Figure 12. Figure 13. Bioc Dr. Miesfeld Spring 2008 the stroma). Ferredoxin-thioredoxin reductase is a soluble enzyme that contains a 4Fe-4S cluster and is able to Figure 11. donate two electrons to oxidized thioredoxin causing reduction of its disulfide bridge. Reduced thioredoxin then uses these electrons to reduce the disulfide bridges in target enzymes. As long as reduced thioredoxin is present in the stroma, these Calvin cycle enzymes are maintained in the active state, however, when the sun goes down, spontaneous oxidation leads to their inactivation. The C4 and CAM pathways reduce photorespiration in hot climates Rubisco also catalyzes a oxygenase reaction that combines RuBP with O 2 to generate one molecule of 3- phosphoglycerate (C 3 ) and one molecule of 2- phosphoglycolate (C 2 ) as shown in figure 12. It is thought that this "wasteful" reaction belies the ancient history of the rubisco enzyme which has been around since before O 2 levels in the atmosphere were as high as they are today. The 2-phosphoglycolate is metabolized by the glycolate pathway which generates CO 2 and requires a significant amount of cellular energy to salvage the C 2 group for reincorporation into the Calvin cycle. As shown in figure 13, the 2-phosphoglycolate is converted to glycolate which is exported to peroxisomes to make glyoxylate and glycine which is then exported to mitochondria where two molecules of glycine are converted to one molecule of serine. Oxygenation of RuBP, and metabolism of 2- phosphoglycolate by the glycolate pathway, is collectively called photorespiration because O 2 is consumed and CO 2 is released. However, unlike mitochondrial respiration, photorespiration requires energy input and is therefore considered a wasteful pathway in photosynthetic cells. 7 of 10 pages

8 Bioc Dr. Miesfeld Spring 2008 Plants in hot, sunny, climates are especially susceptible to photorespiration due to high O2:CO2 ratios under these conditions (O2 is more solulble at higher temperatures). In the 1960s, Marshall Hatch and Roger Slack, plant biochemists at the Colonial Sugar Refining Company in Brisbane, Australia, used 14CO2 labeling experiments to determine what the initial products were in the carbon fixation reactions of sugarcane plants. To their surprise, they found that malate was more quickly labeled with 14C than was 3-phosphoglycerate. Follow up work showed that plants such as sugarcane and corn, and weeds like crabgrass, thrive under high temperature conditions by having very low levels of photorespiration. Rather than accumulating mutations in rubisco that increase the efficiency of carbon fixation, they Figure 14. found that these plants evolved a way to dramatically increase the concentration of CO2 inside the stroma, thereby preventing significant O2 binding to the rubisco active site. The mechanism involves the carboxylation of phosphoenolpyruvate (PEP) by the enzyme PEP carboxylase to form oxaloacetate (OAA), a four carbon (C4) intermediate that serves as a transient CO2 carrier molecule. Two variations of the "Hatch-Slack" pathway have been described; 1) the C4 pathway in tropical plants such as sugarcane that utilize two separate cell types, one for CO2 uptake, and the other for rubisco-mediated carboxylation, and 2) the CAM pathway found in desert succulents such as the giant saguaro cactus which captures CO2 at night when transpiration rates are low (figure 14). Gas exchange in land plants occurs through a special cell structure called a stomate consisting of two guard cells that change shape to open and close the stomate in response to physiological cues. Sugarcane grows in a moist hot climate and the stomata (plural for stomate) open during the day to capture CO2 when photosynthetic electron transport rates are high due to intense sunlight. In contrast, saguaro cacti live in a dry hot climate and only open their stomata at night, therefore they must rely on the captured CO2 to drive the Calvin cycle reactions during the day when sunlight is available and the stomata are closed. Figure 15. The C4 pathway in sugarcane is shown in figure 15 where it can be seen that mesophyll cells are responsible for CO2 capture, whereas, interior bundle sheath cells (further away from atmospheric O2), use CO2 released from the C4 intermediate malate to carry out the Calvin cycle reactions. The C4 and C3 pathways are physically separated in the two cell types with malate functioning as the CO2 transporter molecule into the bundle sheath cell, and pyruvate serving to recycle the carbons back to the mesophyll cell. This "separation of labor" between the two cell types essentially eliminates the oxygenase reaction in rubisco and thereby blocks photorespiration. Note that the C4 pathway has a price in that two high energy phosphate bonds (AMP + PPi) are required to convert pyruvate to phosphoenolpyruvate in mesophyll cells. 8 of 10 pages

9 Bioc Dr. Miesfeld Spring 2008 The CAM pathway was first discovered in succulent plants of the Crassulaceae family and is therefore called Crassulacean Acid Metabolism (CAM) pathway. Just like the C 4 pathway in sugarcane, the CAM pathway functions to concentrate CO 2 levels in the chloroplast stroma to limit the oxygenase activity of rubisco. The main difference is that instead of using two cell types to physically separate atmospheric O 2 from rubisco as we saw in C 4 plants, CAM plants like the saguaro cactus use a temporal separation. As shown in figure 16, during the night when the stomata are open, CO 2 is captured by the mesophyll cells and incorporated into OAA by PEP carboxylase which is then reduced by the enzyme NAD-malate dehydrogenase to form malate. The malate is stored inside vacuoles overnight until sunlight activates the Calvin cycle enzymes the next morning at which time malate is oxidized and decarboxylated by NADP-malic enzyme to generate CO 2 and pyruvate. Unlike in C 4 plants in which the carbon skeleton of pyruvate is recycled directly to PEP, the pyruvate is stored as starch during the day. At night, the starch granules are degraded to form pyruvate which is converted to PEP, thereby allowing PEP carboxylase reaction to capture CO 2 when the stomata are open. Photorespiration is kept to a minimum during the day when the stomata are closed and CO 2 levels in the chloroplast are high due to the decarboxylation of malate. Moreover, by keeping the stomates closed, water loss by transpiration (evaporation) is inhibited at times of the day when temperatures can reach 40ºC in the Sonoran desert. Given the increased efficiency of the C 4 pathway compared to the C 3 pathway at elevated temperatures, crop scientists have begun to develop strategies to try and convert C 3 crops into pseudo C 4 plants. One focus of this research has been rice which is a staple food crop that can be grown in tropical areas where water is plentiful and daytime temperatures can be high. Traditional rice breeding experiments using C 4 plants such as corn are not Figure 16. feasible, and therefore, plant researchers have recently turned to gene transfer techniques to try and "build" the C 4 pathway into the rice genome using recombinant DNA methods. Introduction of single genes from the C 4 pathway, such as PEP carboxylase, do show biochemical activity in the mesophyll cells of rice, however, photosynthetic efficiency is not significantly altered. Current strategies are now aimed at introducing multiple genes into rice, for example, PEP carboxylase, NADP-malate dehydrogenase and NADP-malic enzyme, which together would reconstitute the key reactions in the C 4 pathway. A major challenge in these type of "metabolic engineering" approaches is being able to regulate the expression and biochemical activity of multiple foreign genes in ways that mimic the finely-tuned control of metabolic pathways that have evolved over time. 9 of 10 pages

10 Bioc Dr. Miesfeld Spring 2008 ANSWER TO KEY CONCEPT QUESTIon Concerning the CaLVIN CYCLE: Sugarcane plants and saguaro cacti avoid the wasteful side effects of photorespiration by separating the process of gas exchange through the stroma, from the carbon dioxide fixation reaction mediated by the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). In addition to carbon dioxide fixation, the rubisco also catalyzes an oxygenase reaction that combines ribulose-1,5-bisphosphate (RuBP) with O 2 to generate one molecule of 3- phosphoglycerate and one molecule of 2-phosphoglycolate. This wasteful side reaction, called photorespiration, not only diminishes net conversion of CO 2 to carbohydrate, but also requires that the Glycolate pathway consume ATP in order to recover the two carbons from 2- phosophochlycholate. Sugarcane plants use the C 4 pathway to reduce photorespiration by separating gas exchange and CO 2 fixation into two different cell types (mesophyll and bundle sheath cells). In contrast, saguaro cacti, use the CAM pathway which limits photorespiration by capturing CO 2 at night and then recovering it from the C 4 intermediate in the same cells during the day when it is too hot to open the stomates for gas exchange. 10 of 10 pages