20.5 Photorespiration and the C4 and CAM Pathways in Chapter 20 Photosynthesis and Carbohydrate Synthesis in Plants

20.5 Photorespiration and the $C_{4}$ $bold upper C bold 4$ and CAM Pathways

As we have seen, photosynthetic cells produce $O_{2}$ $upper O Subscript 2$ (by the splitting of $H_{2} O$ $upper H Subscript 2 Baseline upper O$ ) during the light-driven reactions and use ${CO}_{2}$ $CO Subscript 2$ during the light-independent processes, so the net gaseous change during photosynthesis is the uptake of ${CO}_{2}$ $CO Subscript 2$ and release of $O_{2}$ $upper O Subscript 2$ :

{CO}_{2} + H_{2} O \to O_{2} + ({CH}_{2} O)

$upper C upper O 2 plus normal upper H 2 normal upper O right-arrow upper O Subscript 2 Baseline plus left-parenthesis upper C upper H 2 normal upper O right-parenthesis$

In the dark, plants also carry out mitochondrial respiration, the oxidation of substrates to ${CO}_{2}$ $CO Subscript 2$ and the conversion of $O_{2}$ $upper O Subscript 2$ to $H_{2} O$ $upper H Subscript 2 Baseline upper O$ . And there is another process in plants that, like mitochondrial respiration, consumes $O_{2}$ $upper O Subscript 2$ and produces ${CO}_{2}$ $CO Subscript 2$ and, like photosynthesis, is driven by light. This process, photorespiration, is a costly side reaction of photosynthesis, a result of the lack of specificity of the enzyme rubisco. In this section we describe this side reaction and the strategies plants use to minimize its metabolic consequences.

Photorespiration Results from Rubisco’s Oxygenase Activity

Rubisco is not absolutely specific for ${CO}_{2}$ $CO Subscript 2$ as a substrate. Molecular oxygen $(O_{2})$ $left-parenthesis upper O Subscript 2 Baseline right-parenthesis$ competes with ${CO}_{2}$ $CO Subscript 2$ at the active site, and about once in every three or four turnovers, rubisco catalyzes the condensation of $O_{2}$ $upper O Subscript 2$ with ribulose 1,5-bisphosphate to form 3-phosphoglycerate and 2-phosphoglycolate (Fig. 20-38), a metabolically unneeded product. This is the oxygenase activity referred to in the full name of rubisco: ribulose 1,5-bisphosphate carboxylase/oxygenase. The reaction with $O_{2}$ $upper O Subscript 2$ results in no fixation of ${CO}_{2}$ $CO Subscript 2$ and is presumably a net liability to the cell; salvaging the carbons from 2-phosphoglycolate (by the pathway outlined below) consumes significant amounts of cellular energy and releases some previously fixed ${CO}_{2}$ $CO Subscript 2$ .

A figure shows the oxygenase activity of rubisco. — FIGURE 20-38 Oxygenase activity of rubisco. Rubisco can incorporate $O_{2}$ $upper O Subscript 2$ rather than ${CO}_{2}$ $CO Subscript 2$ into ribulose 1,5-bisphosphate. The unstable intermediate thus formed splits into 2-phosphoglycolate (recycled as described in Fig. 20-39) and 3-phosphoglycerate, which can reenter the Calvin cycle.

Ribulose 1,5-bisphosphate is shown as a vertical five carbon chain with C 1 bonded to 2 H and to O bonded to a circle labeled P; C 2 double bonded to O; C 3 and C 4 bonded to H on the left and O H on the right; and C 5 bonded to 2 H and to O bonded to a circle labeled P. Upward- and downward-pointing arrows indicate a reversible reaction. This yields the enediol form, which is similar except that C 2 is bonded to O H and double bonded to C 3, which is also bonded to O H. An arrow points down and is joined by a curved line showing the addition of red highlighted O 2. This produces an enzyme-bound intermediate shown in brackets. It is a five-carbon chain with C 1 bonded to 2 H and to O bonded to a circle labeled P; C 2 bonded to red highlighted O on the left further bonded to red highlighted O bonded to H and bonded to O H on the right; C 3 double bonded to O; C 4 bonded to H on the left and O H on the right; and C 5 bonded to 2 H and to O bonded to a circle labeled P. An arrow points down accompanied by a curved arrow showing the addition of O H minus with O highlighted in blue and the loss of H 2 O with the O highlighted in red. This yields two products. 2-phosphoglycerate has a top C bonded to 2 H, bonded to O on the right bonded to a circle labeled P, and bonded to C below bonded to O minus to the lower left and double bonded to O to the lower right. 3-phosphoglycerate has a top C bonded to blue highlighted O minus to the upper left, double bonded to O to the upper right, and bonded to C below bonded to H to the left, O H to the right, and C below bonded to 2 H and to O bonded to a circle labeled P.

Phosphoglycolate Is Salvaged in a Costly Set of Reactions in $C_{3}$ $bold upper C bold 3$ Plants

The glycolate pathway converts two molecules of 2-phosphoglycolate to a molecule of serine (three carbons) and a molecule of ${CO}_{2}$ $CO Subscript 2$ (Fig. 20-39). In the chloroplast, a phosphatase converts 2-phosphoglycolate to glycolate, which is exported to the peroxisome. There, glycolate is oxidized by molecular oxygen, and the resulting aldehyde (glyoxylate) undergoes transamination to glycine. The hydrogen peroxide formed as a side product of glycolate oxidation is rendered harmless by peroxidases in the peroxisome. Glycine passes from the peroxisome to the mitochondrial matrix, where it undergoes oxidative decarboxylation by the glycine decarboxylase complex, an enzyme similar in structure and mechanism to two mitochondrial complexes we have already encountered: the pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex (Chapter 16). The glycine decarboxylase complex oxidizes glycine to ${CO}_{2}$ $CO Subscript 2$ and ${NH}_{3}$ $NH Subscript 3$ , with the concomitant reduction of ${NAD}^{+}$ $NAD Superscript plus$ to NADH and transfer of the remaining carbon from glycine to the cofactor tetrahydrofolate. The one-carbon unit carried on tetrahydrofolate is then transferred to a second glycine by serine hydroxymethyltransferase, producing serine. The net reaction catalyzed by the glycine decarboxylase complex and serine hydroxymethyltransferase is

2 Glycine + {NAD}^{+} + H_{2} O \to serine + {CO}_{2} + {NH}_{3} + NADH + H^{+}

$2 upper G l y c i n e plus upper N upper A upper D Superscript plus Baseline plus normal upper H 2 normal upper O right-arrow s e r i n e plus upper C upper O 2 plus upper N upper H 3 plus upper N upper A upper D upper H plus normal upper H Superscript plus$

The serine is converted to hydroxypyruvate, then to glycerate, and finally to 3-phosphoglycerate, which is used to regenerate ribulose 1,5-bisphosphate, completing the long, expensive cycle (Fig. 20-39).

A figure shows the glycolate pathway, which converts 2-phosphoglycolate to serine and then to 3-phosphoglycerate. — FIGURE 20-39 Glycolate pathway. This pathway, which salvages 2-phosphoglycolate (shaded light red) by converting it to serine and, eventually, to 3-phosphoglycerate, involves three cellular compartments. Glycolate formed by dephosphorylation of 2-phosphoglycolate in chloroplasts is oxidized to glyoxylate and transaminated to glycine in peroxisomes. In mitochondria, two glycine molecules condense to form serine and ${CO}_{2}$ $CO Subscript 2$ , released in photorespiration. This reaction is catalyzed by glycine decarboxylase, an enzyme present at very high levels in the mitochondria of $C_{3}$ $upper C Subscript 3$ plants. The serine is converted to hydroxypyruvate and then to glycerate in peroxisomes; glycerate reenters the chloroplasts to be phosphorylated, rejoining the Calvin cycle. Oxygen is consumed at two steps during photorespiration.

The figure shows a vertical series of three organelles: a chloroplast, a peroxisome, and a mitochondrion. The chloroplast has a light green background labeled chloroplast stroma and three stacks of green ovals. The Calvin cycle is shown in the upper center overlapping the edges of two stacks of green ovals. It has ribulose 1,5-bisphosphate at the top, a clockwise arrow pointing down to 3-phosphoglycerate the bottom, and a series of three arrows pointing back up to ribulose 1,5-bisphosphate. A curved arrow from a blue square labeled O 2 meets the downward arrow from ribulose 1,5-bisphosphate to 3-phosphoglycerate and then curves away to a light red box labeled 2-phosphoglycerate. 2-phosphoglycerate is shown as C bonded to 2 H, to O further bonded to a circle labeled P, and to C below that forms C O O minus. An arrow points down from 2-phosphoglycolate to glycolate with an arrow branching off to show the loss of P subscript i end subscript. Glycolate is shown as C bonded to 2 H and O H and to C below that forms C O O minus. An arrow points down to glycolate in the peroxisome, shown with the same structure. An arrow labeled glycolic acid oxidase points downward and is accompanied by a curved arrow that shows the addition of a blue box labeled O 2 and loss of H 2 O 2. This reaction yields glyoxylate, shown as a two-carbon chain of a top C forming C H O and a bottom C forming C O O minus. An arrow points down from glyoxylate to glycine and is joined by an arrow from serine to the right to a second arrow labeled transamination [further bonded N H 2]. Glycine is shown as a top C bonded to 2 H, to N H 3 with a positive charge on H, and to C below that forms C O O minus. An arrow points down from glycine to 2 glycine in the mitochondrion, shown with the same structure. An arrow labeled glycine decarboxylase curves down from 2 glycine and is met by a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H plus H plus before continuing to branch to show the loss of N H 3 and to a green box labeled C O 2 released in photorespiration. The main arrow meets a short arrow to serine, shown as C H 2 bonded to O H above and to C H to the right bonded to N H 3 above with a positive charge on H and to C O O minus to the right. An arrow points up from serine to serine in the peroxisome, shown with the same structure. An arrow up from serine branches to a curved arrow labeled transamination that yields glycine and to hydroxypyruvate above. Hydroxypyruvate is shown as C H 2 bonded to O H above and to C to the right double bonded to O above and bonded to C O O minus to the right. An arrow labeled alpha-hydroxy acid reductase points up to glycerate accompanied by a curved arrow showing the addition of an orange oval labeled N A D H plus H plus and loss of N A D plus. Glycerate is shown as C H 2 bonded to O H above and to C H to the right bonded to O H above and to C O O minus to the right. An arrow points up from glycerate in the peroxisome to glycerate in the chloroplast stroma, which is shown with the same shape. An arrow points up to 3-phosphoglycerate at the bottom of the Calvin cycle and is accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P.

In bright sunlight, the carbon flux through the glycolate salvage pathway can be very high, producing about five times more ${CO}_{2}$ $CO Subscript 2$ than is typically produced by all the oxidations of the citric acid cycle. To generate this large flux, mitochondria contain prodigious amounts of the glycine decarboxylase complex: the four proteins of the complex make up half of all the protein in the mitochondrial matrix in the leaves of pea and spinach plants. In nonphotosynthetic parts of a plant, such as potato tubers, mitochondria have very low concentrations of the glycine decarboxylase complex.

The practical effects of this inefficiency are large and costly. The average yield of soybeans and wheat in the United States is reduced by an estimated 36% and 20%, respectively, by the necessity of recycling glycolate from photorespiration.

The combined activity of the rubisco oxygenase and the glycolate salvage pathway consumes $O_{2}$ $upper O Subscript 2$ and produces ${CO}_{2}$ $CO Subscript 2$ — hence the name photorespiration. Unlike mitochondrial respiration, photorespiration does not conserve energy and actually inhibits net biomass formation. This inefficiency has led to evolutionary adaptations in the ${CO}_{2}$ $CO Subscript 2$ -assimilation processes, particularly in plants that have evolved in warm climates. The apparent inefficiency of rubisco, and its effect in limiting biomass production, has inspired efforts to genetically engineer a “better” rubisco, but this goal is not, as yet, within reach (Box 20-1).

BOX 20-1

Will Genetic Engineering of Photosynthetic Organisms Increase Their Efficiency?

Three pressing world problems have prompted serious attention to the possibility of engineering plants to be more efficient in converting sunlight into biomass: the greenhouse effect of increasing levels of atmospheric ${CO}_{2}$ $CO Subscript 2$ on climate change, the finite supply of oil for generating energy, and the need for more and better food for the world’s growing population.

The concentration of ${CO}_{2}$ $CO Subscript 2$ in the earth’s atmosphere has risen steadily over the past 50 years (Fig. 1), a combined effect of the use of fossil fuels for energy and the clearing and burning of tropical forests to allow use of the land for agriculture. As atmospheric ${CO}_{2}$ $CO Subscript 2$ increases, the atmosphere absorbs more heat radiated from the earth’s surface and reradiates more heat toward the surface of the planet (and in all other directions). Retention of heat raises the temperature at the surface of the earth; this is the greenhouse effect. One way to limit the increase in atmospheric ${CO}_{2}$ $CO Subscript 2$ would be to engineer plants or microorganisms with a greater capacity for sequestering ${CO}_{2}$ $CO Subscript 2$ .

FIGURE 1 The concentration of ${CO}_{2}$ $CO Subscript 2$ in the atmosphere measured at the Mauna Loa Observatory in Hawaii. [Data from the National Oceanic and Atmospheric Administration and the Scripps Institution of Oceanography ${CO}_{2}$ $CO Subscript 2$ Program.]

The estimated amount of total carbon in all terrestrial systems (atmosphere, soil, biomass) is about 3,200 gigatons (GT), or 3,200 billion metric tons. The atmosphere contains another 760 GT of ${CO}_{2}$ $CO Subscript 2$ .

The flux of carbon through these terrestrial reservoirs (Fig. 2) is largely due to the photosynthetic activities of plants and the degradative activities of microorganisms. Plants fix some 123 GT of carbon annually, then immediately release about half of that to the atmosphere as they respire. Much of the remainder is gradually released to the atmosphere by microbial action on dead plant materials, but biomass is sequestered in woody plants and trees for decades or centuries. Anthropogenic carbon flux, the amount of ${CO}_{2}$ $CO Subscript 2$ released into the atmosphere by human activities, is 9 GT per year — small compared with total biomass, but enough to tip the balance toward increased ${CO}_{2}$ $CO Subscript 2$ in the atmosphere. Estimates indicate that the forests of North America sequester 0.7 GT of carbon annually, which represents about a tenth of the annual global production of ${CO}_{2}$ $CO Subscript 2$ from fossil fuels. Clearly, preservation of forests and reforestation are effective ways to limit the flow of ${CO}_{2}$ $CO Subscript 2$ back into the atmosphere.

FIGURE 2 The terrestrial carbon cycle. Carbon stocks (boxed text) are shown as gigatons (GT), and fluxes (arrows) are shown in GT per year. Animal biomass is negligible here — less than 0.5 GT. [Information from C. Jansson et al., BioScience 60:683, 2010, Fig. 1.]

Brown soil is shown across the bottom of the illustration with green hills around the sides and background and with a lake in the middle. The sun is shown at the upper left. A box at the top center reads, Atmosphere, 760. At the upper right, an arrow points toward the center and it light on the right but becomes blue toward the arrowhead and is labeled 9, net anthropogenic flux. An arrow pointing downward to the right and just below the sun is blue at the top and green toward the arrowhead and is labeled 123, photosynthesis. An arrow to its right pointing upward is green at the bottom and blue at the arrowhead and is labeled, 60 plant respiration. A green box against a green hill is labeled plant biomass, 560. A gray box in the soil is labeled microbial biomass, 110. A blue arrow points upward from the soil to the air, is light blue at the bottom and dark blue at the arrowhead, and is labeled 60, microbial respiration and decomposition. A peach box in the soil is labeled soil, 2,500. An arrow pointing from above the soil into the soil is blue at the top and brown below and is labeled 3, net carbon sequestration as soil carbon.

A second approach to limiting the increase of atmospheric ${CO}_{2}$ $CO Subscript 2$ , while also addressing the need to replace dwindling fossil fuels, is to use renewable biomass as a source of ethanol to replace fossil fuels in internal combustion engines. This reduces the unidirectional movement of carbon from fossil fuels into the atmospheric pool of ${CO}_{2}$ $CO Subscript 2$ , replacing it with the cyclic flow of ${CO}_{2}$ $CO Subscript 2$ from ethanol to ${CO}_{2}$ $CO Subscript 2$ and back to biomass. When maize, wheat, or switchgrass is fermented to ethanol for fuel, every increase in biomass production brought about by more efficient photosynthesis should result in a corresponding decrease in the use of fossil fuels.

Finally, engineering of food crops to yield more food per acre of land, or per hour of work, could improve human nutrition worldwide.

In principle, these goals might be accomplished by developing a rubisco that didn’t also catalyze the wasteful reaction with $O_{2}$ $upper O Subscript 2$ , or by increasing the turnover number for rubisco, or by increasing the level of rubisco or other enzymes in the pathway for ${CO}_{2}$ $CO Subscript 2$ fixation. Rubisco, as we have noted, is an unusually inefficient enzyme, with a turnover number of $3 s^{- 1}$ $3 s Superscript negative 1$ at 25 °C; most enzymes have turnover numbers orders of magnitude larger. It also catalyzes the wasteful reaction with oxygen, which further reduces its efficiency in fixing ${CO}_{2}$ $CO Subscript 2$ and producing biomass. If rubisco could be genetically engineered to turn over faster or to be more selective for ${CO}_{2}$ $CO Subscript 2$ relative to $O_{2}$ $upper O Subscript 2$ , would the effect be greater photosynthetic production of biomass and thus greater sequestration of ${CO}_{2}$ $CO Subscript 2$ , greater production of nonfossil fuel, and improved nutrition?

The traditional view of metabolic pathways held that one step in any pathway was the slowest and therefore the limiting factor in material flow through the pathway. However, efforts to engineer cells or organisms to produce more of the “limiting” enzyme in a pathway have often given discouraging results; the organisms often show little or no change in the flux through that pathway. The Calvin cycle is an instructive case in point. Increasing the amount of rubisco in plant cells through genetic engineering has little or no effect on the rate of ${CO}_{2}$ $CO Subscript 2$ conversion into carbohydrate. Similarly, changes in the levels of enzymes known to be regulated by light and therefore suspected of playing key roles in the regulation of the ${CO}_{2}$ $CO Subscript 2$ -assimilation pathway (fructose 1,6-bisphosphatase, 3-phosphoglycerate kinase, and glyceraldehyde 3-phosphate dehydrogenase) also produce little or no significant improvement in photosynthetic rate. This should probably not be surprising; in the living organism, pathways can be limited by more than one enzymatic step, because every change in one step results in compensating changes in other steps. Metabolic control analysis is the science of measuring, understanding, and eventually altering the factors that govern the overall flux through a pathway. Its application will be essential to the success of engineering plants for higher efficiency or greater yield.

In $C_{4}$ $bold upper C bold 4$ Plants, ${CO}_{2}$ $bold upper C upper O bold 2$ Fixation and Rubisco Activity Are Spatially Separated

In many plants that grow in the tropics (and in temperate-zone crop plants native to the tropics, such as maize, sugarcane, and sorghum) a mechanism has evolved to circumvent the problem of wasteful photorespiration. The step in which ${CO}_{2}$ $CO Subscript 2$ is fixed into a three-carbon product, 3-phosphoglycerate, is preceded by several steps, one of which is temporary fixation of ${CO}_{2}$ $CO Subscript 2$ into oxaloacetate, a four-carbon compound. Plants that use this process are referred to as $C_{4}$ $bold upper C bold 4$ plants, and the assimilation process is known as the $C_{4}$ $bold upper C bold 4$ pathway, by comparison to the $C_{3}$ $upper C Subscript 3$ pathway in which ${CO}_{2}$ $CO Subscript 2$ is first fixed in the three-carbon compound 3-phosphoglycerate.

The $C_{4}$ $upper C Subscript 4$ plants, which typically grow at high light intensity and high temperatures, have several important characteristics: high photosynthetic rates, high growth rates, low photorespiration rates, low rates of water loss, and a specialized leaf structure. Photosynthesis in the leaves of $C_{4}$ $upper C Subscript 4$ plants involves two cell types: mesophyll and bundle-sheath cells (Fig. 20-40a).

A two-part figure shows how C subscript 4 end subscript plants assimilate carbon with a micrograph in part a and a diagram showing the pathway in part b. — FIGURE 20-40 ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation in $C_{4}$ $bold upper C bold 4$ plants. The $C_{4}$ $upper C Subscript 4$ pathway, involving mesophyll cells and bundle-sheath cells, predominates in plants of tropical origin. (a) Electron micrograph showing chloroplasts of adjacent mesophyll and bundle-sheath cells. The bundle-sheath cell contains starch granules. Plasmodesmata connecting the two cells are visible. (b) The $C_{4}$ $upper C Subscript 4$ pathway of ${CO}_{2}$ $CO Subscript 2$ assimilation, which occurs through a four-carbon intermediate.

FIGURE 20-40 ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation in $C_{4}$ $bold upper C bold 4$ plants. The $C_{4}$ $upper C Subscript 4$ pathway, involving mesophyll cells and bundle-sheath cells, predominates in plants of tropical origin. (a) Electron micrograph showing chloroplasts of adjacent mesophyll and bundle-sheath cells. The bundle-sheath cell contains starch granules. Plasmodesmata connecting the two cells are visible. (b) The $C_{4}$ $upper C Subscript 4$ pathway of ${CO}_{2}$ $CO Subscript 2$ assimilation, which occurs through a four-carbon intermediate.

Part a is a micrograph that shows a mesophyll on top that has several square to rectangular stacks of horizontally aligned membranes with many membranes in between and several large black circles. There is a pale space between the mesophyll cell and a bundle sheath cell below. A series of seven vertical dark bands connecting the two cells are labeled plasmodesmata. The bundle sheath cell has layers of membranes that are more spread out and that do not form stacks. Part b shows a light red box labeled C O 2 (in air) with an arrow pointing down to H C O 3 minus accompanied by a curved arrow showing the addition of H 2 O and loss of H plus. An arrow points down from H C O 3 minus to join a cyclic series of reactions that begins in the mesophyll cell. This arrow joins at the twelve o’clock position. The arrow from H C O 3 minus meets an arrow labeled P E P carboxylase that runs from P E P at the ten o’clock position to oxaloacetate at the two o’clock position with an arrow branching away at the one o’clock position to show the loss of P subscript i end subscript. An arrow labeled malate dehydrogenase points down from oxaloacetate to malate and is accompanied by a curved arrow showing the addition of an orange oval labeled N A D P H plus H plus and loss of N A D P plus. An arrow points down from malate through a connection between the mesophyll cell to the bundle sheath cell below to another molecule of malate. The membranes between the plasmodesmata are labeled as plasma membranes. A curved arrow labeled malic enzyme points from malate to pyruvate at the eight o’clock position, just beneath the left-hand opening to the mesophyll cell above. This arrow is accompanied by a curved arrow showing the addition of N A D P plus and loss of an orange oval labeled N A D P plus H plus. A the bottom of the curved arrow, an arrow branches to show the loss of a light red box labeled C O 2 that joins with a curved arrow below labeled rubisco that curves from ribulose 1,5-bisphosphate on the left to 3-phosphoglycerate on the right. Another arrow curves from 3-phosphoglycerate back to ribulose 1,5-bisphosphate with an arrow branching off at the six o’clock position to show the loss of triose phosphates. Pyruvate at the upper left of the bundle sheath cell moves through the channel above into the bundle sheath cell, from which an arrow labeled pyruvate phosphate dikinase points up to P E P and is accompanied by an arrow showing the addition of an orange oval labeled A T P plus P lowercase i and loss of A M P plus P P subscript i end subscript. All data are approximate.

The fixation of ${CO}_{2}$ $CO Subscript 2$ into the four-carbon oxaloacetate occurs in the cytosol of leaf mesophyll cells. The reaction is catalyzed by phosphoenolpyruvate (PEP) carboxylase, for which the substrate is ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ , not ${CO}_{2}$ $CO Subscript 2$ . The oxaloacetate thus formed is either reduced to malate at the expense of NADPH (as shown in Fig. 20-40b) or converted to aspartate by transamination:

Oxaloacetate + α -amino acid \to L-aspartate + α -keto acid

$upper O x a l o a c e t a t e plus alpha hyphen a m i n o a c i d right-arrow upper L hyphen a s p a r t a t e plus alpha hyphen k e t o a c i d$

The malate or aspartate formed in the mesophyll cells then passes into neighboring bundle-sheath cells through plasmodesmata, protein-lined channels that connect two plant cells and provide a path for movement of metabolites and even small proteins between cells. In the bundle-sheath cells, malate is oxidized and decarboxylated to yield pyruvate and ${CO}_{2}$ $CO Subscript 2$ by the action of malic enzyme, reducing ${NADP}^{+}$ $NADP Superscript plus$ . In plants that use aspartate as the ${CO}_{2}$ $CO Subscript 2$ carrier, aspartate arriving in bundle-sheath cells is transaminated to form oxaloacetate and reduced to malate, then the ${CO}_{2}$ $CO Subscript 2$ is released by malic enzyme or PEP carboxykinase. Labeling experiments show that the free ${CO}_{2}$ $CO Subscript 2$ released in the bundle-sheath cells is the same ${CO}_{2}$ $CO Subscript 2$ molecule originally fixed into oxaloacetate in the mesophyll cells. This ${CO}_{2}$ $CO Subscript 2$ is now fixed again, this time by rubisco, in exactly the same reaction that occurs in $C_{3}$ $upper C Subscript 3$ plants: incorporation of ${CO}_{2}$ $CO Subscript 2$ into C-1 of 3-phosphoglycerate.

The pyruvate formed by decarboxylation of malate in bundle-sheath cells is transferred back to the mesophyll cells, where it is converted to PEP by an unusual enzymatic reaction catalyzed by pyruvate phosphate dikinase (Fig. 20-40b). This enzyme is called a dikinase because two different molecules are simultaneously phosphorylated by one molecule of ATP: pyruvate to PEP, and phosphate to pyrophosphate. The pyrophosphate is subsequently hydrolyzed to phosphate, so two high-energy phosphate groups of ATP are used in regenerating PEP. The PEP is now ready to receive another molecule of ${CO}_{2}$ $CO Subscript 2$ in the mesophyll cell.

The PEP carboxylase of mesophyll cells has a high affinity for ${HCO}_{3}^{-}$ $HCO Subscript 3 Superscript minus$ (which is favored relative to ${CO}_{2}$ $CO Subscript 2$ in aqueous solution) and can fix ${CO}_{2}$ $CO Subscript 2$ more efficiently than can rubisco. Unlike rubisco, it does not use $O_{2}$ $upper O Subscript 2$ as an alternative substrate, so there is no competition between ${CO}_{2}$ $CO Subscript 2$ and $O_{2}$ $upper O Subscript 2$ . The PEP carboxylase reaction, then, serves to fix and concentrate ${CO}_{2}$ $CO Subscript 2$ in the form of malate. Release of ${CO}_{2}$ $CO Subscript 2$ from malate in the bundle-sheath cells yields a sufficiently high local concentration of ${CO}_{2}$ $CO Subscript 2$ for rubisco to function near its maximal rate, and for suppression of the enzyme’s oxygenase activity.

Once ${CO}_{2}$ $CO Subscript 2$ is fixed into 3-phosphoglycerate in the bundle-sheath cells, the other reactions of the Calvin cycle take place exactly as described earlier. Thus in $C_{4}$ $upper C Subscript 4$ plants, mesophyll cells carry out ${CO}_{2}$ $CO Subscript 2$ assimilation by the $C_{4}$ $upper C Subscript 4$ pathway and bundle-sheath cells synthesize starch and sucrose by the $C_{3}$ $upper C Subscript 3$ pathway.

Three enzymes of the $C_{4}$ $upper C Subscript 4$ pathway are regulated by light, becoming more active in daylight. Malate dehydrogenase is activated by the thioredoxin-dependent reduction mechanism shown in Figure 20-37; PEP carboxylase is activated by phosphorylation of a Ser residue; and pyruvate phosphate dikinase is activated by dephosphorylation.

The pathway of ${CO}_{2}$ $CO Subscript 2$ assimilation has a greater energy cost in $C_{4}$ $upper C Subscript 4$ plants than in $C_{3}$ $upper C Subscript 3$ plants. For each molecule of ${CO}_{2}$ $CO Subscript 2$ assimilated in the $C_{4}$ $upper C Subscript 4$ pathway, a molecule of PEP must be regenerated at the expense of two phosphoanhydride bonds in ATP. Thus $C_{4}$ $upper C Subscript 4$ plants need five ATP molecules to assimilate one molecule of ${CO}_{2}$ $CO Subscript 2$ , whereas $C_{3}$ $upper C Subscript 3$ plants need only three (nine per triose phosphate). As the temperature increases (and the affinity of rubisco for ${CO}_{2}$ $CO Subscript 2$ decreases, as noted above), a point is reached, at about 28 to 30 °C, at which the gain in efficiency from the elimination of photorespiration more than compensates for this energetic cost. $C_{4}$ $upper C Subscript 4$ plants (crabgrass, for example) outgrow most $C_{3}$ $upper C Subscript 3$ plants during the summer, as any experienced gardener can attest.

In CAM Plants, ${CO}_{2}$ $bold upper C upper O bold 2$ Capture and Rubisco Action Are Temporally Separated

Succulent plants such as cactus and pineapple, which are native to very hot, very dry environments, have another variation on photosynthetic ${CO}_{2}$ $CO Subscript 2$ fixation, which reduces loss of water vapor through the pores (stomata) by which ${CO}_{2}$ $CO Subscript 2$ and $O_{2}$ $upper O Subscript 2$ must enter leaf tissue. Instead of separating the initial trapping of ${CO}_{2}$ $CO Subscript 2$ and its fixation by rubisco across space (as do the $C_{4}$ $upper C Subscript 4$ plants), they separate these two events over time. At night, when the air is cooler and moister, the stomata open to allow entry of ${CO}_{2}$ $CO Subscript 2$ , which is then fixed into oxaloacetate by PEP carboxylase. The oxaloacetate is reduced to malate and stored in the vacuoles, to protect cytosolic and plastid enzymes from the low pH produced by malic acid dissociation. During the day the stomata close, preventing the water loss that would result from high daytime temperatures, and the ${CO}_{2}$ $CO Subscript 2$ trapped overnight in malate is released as ${CO}_{2}$ $CO Subscript 2$ by the NADP-linked malic enzyme. This ${CO}_{2}$ $CO Subscript 2$ is now assimilated by the action of rubisco and the Calvin cycle enzymes. Because this method of ${CO}_{2}$ $CO Subscript 2$ fixation was first discovered in stonecrops, perennial flowering plants of the family Crassulaceae, it is called crassulacean acid metabolism, and the plants are called CAM plants. Table 20-1 compares characteristics of $C_{3}$ $upper C Subscript 3$ , $C_{4}$ $upper C Subscript 4$ , and CAM plants.

TABLE 20-1 Comparison of $C_{3}, C_{4}$ $bold upper C bold 3 comma bold upper C bold 4$ , and CAM Plants
	$C_{3}$ $bold upper C bold 3$ Plants	$C_{4}$ $bold upper C bold 4$ Plants	CAM Plants
Examples	Spinach, pea, rice, wheat, beans, most trees	Maize (corn), sugarcane, crabgrass	Cactus, prickly pear, orchid, pineapple
Most efficient environment	15 to 25 °C	Hot and dry; 30 to 47 °C	Extremely dry; 35 °C
Path of ${CO}_{2}$ $CO Subscript 2$ fixation	$C_{3}$ $upper C Subscript 3$ photosynthesis only	Sequential $C_{4}$ $upper C Subscript 4$ and $C_{3}$ $upper C Subscript 3$ cycles spatially separated: $C_{4}$ $upper C Subscript 4$ in mesophyll cells followed by $C_{3}$ $upper C Subscript 3$ in bundle-sheath cells	$C_{3}$ $upper C Subscript 3$ and $C_{4}$ $upper C Subscript 4$ cycles, separated spatially and temporally
Cell type involved	Mesophyll cells	$C_{4}$ $upper C Subscript 4$ in mesophyll cells, $C_{3}$ $upper C Subscript 3$ in bundle-sheath cells	$C_{3}$ $upper C Subscript 3$ and $C_{4}$ $upper C Subscript 4$ in the same mesophyll cells
Light conditions	Light	Light	$C_{3}$ $upper C Subscript 3$ in light; $C_{4}$ $upper C Subscript 4$ in dark
Initial ${CO}_{2}$ $CO Subscript 2$ acceptor	Ribulose 1,5-bisphosphate	Phosphoenolpyruvate	Ribulose 1,5-bisphosphate in light; phosphoenolpyruvate in dark
${CO}_{2}$ $CO Subscript 2$ -fixing enzyme	Rubisco	PEP carboxylase, then rubisco	Rubisco in light; PEP carboxylase at night
First stable product of ${CO}_{2}$ $CO Subscript 2$ fixation	3-Phosphoglycerate	Oxaloacetate in $C_{4}$ $upper C Subscript 4$ cycle	3-Phosphoglycerate in light; oxaloacetate in dark
Energy needed for complete reduction of one molecule of ${CO}_{2}$ $CO Subscript 2$	3 ATP, 2 NADPH	5 ATP, 2 NADPH	6.5 ATP, 2 NADPH
Photorespiration	Present	Absent or suppressed	Absent or suppressed

SUMMARY 20.5 Photorespiration and the $C_{4}$ $bold-italic upper C bold 4$ and CAM Pathways

Rubisco is not completely specific for ${CO}_{2}$ $CO Subscript 2$ as its substrate; it can also use $O_{2}$ $upper O Subscript 2$ , producing 2-phosphoglycolate, which must be disposed of in an oxygen-dependent pathway. The result is increased consumption of $O_{2}$ $upper O Subscript 2$ — photorespiration.
The 2-phosphoglycolate is converted to glyoxylate, to glycine, and then to serine in a pathway that involves enzymes in the chloroplast stroma, peroxisomes, and mitochondria.
In $C_{4}$ $upper C Subscript 4$ plants, the ${CO}_{2}$ $CO Subscript 2$ -assimilation pathway minimizes photorespiration: ${CO}_{2}$ $CO Subscript 2$ is first fixed in mesophyll cells into a four-carbon compound, which passes into bundle-sheath cells and releases ${CO}_{2}$ $CO Subscript 2$ in high concentrations. The released ${CO}_{2}$ $CO Subscript 2$ is fixed by rubisco, and the remaining reactions of the Calvin cycle occur as in $C_{3}$ $upper C Subscript 3$ plants.
In CAM plants, ${CO}_{2}$ $CO Subscript 2$ is fixed into malate in the dark and stored in vacuoles until daylight, when the stomata are closed (minimizing water loss), and the stored malate serves as a source of ${CO}_{2}$ $CO Subscript 2$ for rubisco.