Photosynthesis, Carbon Fixation and

Virtually all life on Earth ultimately depends on the light-driven fixation of carbon dioxide (CO₂) according to the following equation: 6CO₂ + 6H₂ O → C₆ H₁₂ O₆ (glucose) + 6O₂

Photosynthesis takes place in subcellular membrane-bound compartments called chloroplasts . As radiotracers such as carbon-14 became available to researchers following World War II (1939-45), one application was to define the biochemistry of photosynthetic CO₂ fixation. Major class divisions in the plant kingdom are based on how CO₂ is fixed.

C₃ Photosynthesis.

Many important biological processes are sustained by cycles that continuously consume and renew one or more key intermediates while producing some other major product. Photosynthesis is sustained by the Calvin-Benson cycle.

The C₃ photosynthetic mechanism is so named because the carbon atom of a molecule of CO₂ taken up by an illuminated leaf is first detected in the three-carbon compound 3-phosphoglyceric acid (PGA). The vast majority of higher plants and algae are C₃ species. PGA is formed when CO₂ combines with a 5-carbon sugar, ribulose biphosphate (RuBP). The reaction is catalyzed by the enzyme RuBP carboxylase/oxygenase, an abundant protein in all green tissues. This multifunctional enzyme has come to be called rubisco.

During each turn of the Calvin-Benson cycle, two molecules of PGA (a total of six carbon atoms) undergo a complex series of enzyme-catalyzed transformations in which the carbon atoms pass through metabolite pools consisting of three-, four-, five-, six-, and seven-carbon sugar phosphates. These reactions regenerate RuBP, which then combines with CO₂ to form two PGAs and complete the cycle. So, of the six (2 H 3) original carbon atoms in PGA, five give rise to RuBP and the one remaining appears as one of the six carbon atoms in the sugar glucose-6-phosphate (G6P). Therefore, for every six CO₂ molecules fixed, one G6P leaves the Calvin-Benson cycle for synthesis of starch, sucrose, cellulose, and ultimately all of the organic constituents of the plant.

In terms of pure chemistry, the conversion of CO₂ to carbohydrate is an example of reduction, in which a source of energy-rich electrons is required. As the term photosynthesis suggests, the energy for the reductive reactions of the Calvin-Benson cycle comes from visible light. An extensive membrane system in the chloroplast harbors the pigments (chlorophylls and carotenoids) that transfer light packets (quanta) to specialized pigment-protein sites where they energize individual electrons extracted from molecules of water (H₂ O). The oxygen atoms in the water are released as O₂. Each high-energy electron consumes the energy of two quanta. Two electrons are used to convert a compound called nicotinamide adenosine dinucleotide phosphate from its oxidized form (NADP⁺) to its reduced form (NADPH). The sequence of electron transport from H₂ O to NADP⁺ also fuels the phosphorylation of adenosine diphosphate (ADP) to high-energy adenosine triphosphate (ATP ). Both NADPH and ATP interact directly with the enzymes of the Calvin-Benson cycle during fixation of CO₂. Two molecules of NADPH and three molecules of ATP are required to fix each molecule of CO₂ during C₃ photosynthesis.

Photorespiration.

C₃ plants also engage in an active CO₂-releasing process called photorespiration that operates concurrently with normal photosyn-thesis in the light. Photorespiration drains away useful energy, and is thus a wasteful process. Since the CO₂ formed by photorespiration is rapidly re-fixed by photosynthesis it is difficult to measure directly, and its existence was not suspected until the 1950s. Since then, biochemists and physiologists have elucidated the mechanism, but have not come to agreement on its purpose, if any. It is important to note that photorespiration is not the same as the ubiquitous respiratory CO₂ released from mitochondria in all eukaryotic cells, including animal and plant tissues.

Photorespiration starts with the formation of a two-carbon phosphoglycolic acid molecule during photosynthesis. Since this is a potent inhibitor of the Calvin-Benson cycle, its metabolism to nontoxic derivatives is essential. First, the phosphate group is removed (by action of an enzyme called a phosphatase) to yield glycolate. The following series of conversions: 2 glycolate → 2 glyoxylate → 2 glycine → serine → hydroxypyruvate → PGA results in the formation of a Calvin-Benson cycle intermediate (PGA) that is used to make RuBP. Notice that the four glycolate carbon atoms ultimately appear as one molecule of PGA (three carbon atoms). The fourth atom of carbon is released as CO₂ during the glycine to serine conversion, and this is the source of CO₂ released in photorespiration. Additional photosynthetic energy (i.e., NADPH and ATP) is consumed during metabolism of photorespiratory PGA, refixation of CO₂, and reassimilation of ammonia released during the glycine → serine step. Hence, photorespiration drains energy away from productive photosynthesis.

Photorespiration can be observed by a number of means. When a stream of CO₂-free air is passed over a C₃ leaf, release of CO₂ by the leaf results in an elevated concentration of this gas in the downstream flow. This release rate is highly dependent upon illumination of the leaf and will be depressed severalfold by darkening. Another method relies on the fact that the rate of photosynthetic fixation of CO₂ is directly dependent on the concentration of CO₂ at low levels of this component. Hence, sealing a leaf in a small transparent vessel under illumination will cause the concentration of CO₂ inside to fall until the rate of uptake equals the rate of evolution due to photorespiration. The final equilibrium concentration of CO₂ (called the CO₂ compensation point) is highly dependent on the concentration of O₂ in the gas and is commonly employed as a robust, although indirect, measure of photorespiration. But the most direct indicator of photorespiration is based on comparison of rates of CO₂ uptake at high and low levels of O₂ in the surrounding atmosphere. Lowering the O₂ concentration from the normal 21 percent to 1 to 2 percent can result in an instantaneous 30 percent increase in photosynthetic rate (see below). This response of photosynthesis to O₂ is attributed to photorespiration and is called the Warburg Effect for its discoverer Otto Warburg.

Although the source of phosphoglycolic acid for photorespiration was for some time a controversial subject, it is now widely accepted that it originates at the site of CO₂ fixation. Specifically, when RuBP binds to rubisco its structure is perturbed, rendering it vulnerable to attack by either CO₂ or O₂. Reaction of RuBP with CO₂ yields two PGAs while reaction with O₂ results in formation of one PGA molecule and one phosphoglycolic acid molecule. The probability that a bound RuBP will react with either CO₂ or O₂ is governed by the relative concentrations of these gases in the aqueous environment of the chloroplast. Hence, CO₂ and O₂ are considered to compete for the bound RuBP. This competition accounts for the increase in photorespiration at high O₂ concentration, and the fact that photorespiration can be almost completely suppressed by high concentrations of CO₂ even in the presence of O₂. Measurements with purified rubisco in the laboratory indicate that the rate of photorespiratory release of CO₂ is about 20 percent of the total rate of CO₂ uptake for a healthy C₃ leaf in air at 25°C. Photorespiration increases considerably with temperature, however. Photorespiration is most significant when temperatures are high and plants must close stomata to prevent water loss. Without access to fresh CO₂ from the atmosphere, photorespiration becomes the major reaction catalyzed by rubisco.

The role of photorespiration in plant metabolism is the subject of debate. It has been suggested to be a means of disposal of excess photosynthetic energy. Also, it may provide a way to protect the leaf from damaging effects of light that could occur if CO₂ levels inside the leaf were to fall below some critical threshold. Still, there may be no essential role for photorespiration. It is probably an anomaly of the rubisco mechanism that appeared on this planet before O₂ was present in the atmosphere. Later, as O₂ levels in the atmosphere rose due to photosynthesis, this vulnerability to O₂ affected photosynthesis and growth. Interestingly, some plants have evolved means to suppress photorespiration while retaining rubisco and the Calvin-Benson cycle.

C₄ Photosynthesis.

Familiar species possessing the C₄ photosynthesis mechanism are maize, sorghum, sugarcane, and several common weeds. The defining feature of CO₂ fixation in this case is involvement of two distinct cell types that shuttle metabolites back and forth to complete a modified photosynthetic cycle. Microscopic examination of leaf sections reveals two chloroplast-containing cell types in an arrangement termed Kranz anatomy. Bundle sheath cells form a cylindrical layer one cell deep around each leaf vein. These cells are typically enlarged, thick walled, and densely packed with chloroplasts. At least two layers of loosely packed mesophyll cells separate adjacent bundle sheath strands. Although mesophyll cells resemble those observed in C₃ leaves, they function much differently.

When CO₂ enters the leaf it is first fixed in the mesophyll cells by the enzyme phosphoenolpyruvate (PEP) carboxylase. The carbon atom from CO₂ is first detected in the four-carbon organic acid oxaloacetic acid (OAA), hence the name C₄ photosynthesis. The OAA is then reduced to malic acid or converted to the amino acid aspartic acid depending on species. Malate and aspartate are transported to bundle sheath cells where they are decarboxylated, thereby releasing CO₂. This newly formed CO₂ is refixed by rubisco and metabolized by the Calvin-Benson cycle present in the bundle sheath chloroplasts. The remaining three carbon atoms derived from the malate and aspartate are transported back to the mesophyll cells as pyruvic acid to regenerate the three-carbon PEP.

The characteristic carboxylation/decarboxylation sequence of C₄ photosynthesis pumps CO₂ from mesophyll to bundle sheath cells, thereby accomplishing one desirable end. The concentration of CO₂ in bundle sheath cells of C₄ plants is severalfold higher than in leaf cells of C₃ species. Hence, photorespiration is virtually absent in C₄ leaves. Since none of the other enzyme-catalyzed reactions is sensitive to O₂, the Warburg effect is not observed and the CO₂ compensation point (a reliable indicator of photorespiratory capacity, see above) is very low for C₄ leaves. Somewhat more light energy is required to fix each molecule of CO₂ using the C₄ pathway since PEP regeneration requires ATP. Although 2 NADPH are consumed as in C₃ plants, the ATP requirement for C₄ photosynthesis is four to five per CO₂ fixed.

Crassulacean Acid Metabolism (CAM).

The crassulacean acid metabolism (CAM) mode of photosynthesis was discovered first in plants of the family Crassulaceae but familiar species include pineapple and cacti. It is considered an adaptation to life in arid environments. CAM photosynthesis resembles C₄ photosynthesis in terms of the pathway of fixation of carbon. The prominent difference, however, is that CAM plants take up CO₂ from the atmosphere at night and synthesize malic acid via PEP carboxylase. During the daytime the leaf pores (stomata) that admit CO₂ close to conserve water. Malic acid is decarboxylated and the CO₂ is refixed by the Calvin-Benson cycle. Some of the starch accumulated during daytime is converted to PEP at night to support CO₂ fixation. Also, unlike C₄ photosyn-thesis, all of the CAM reactions take place in each leaf cell.

Significance of Carbon Fixation Reactions

The choice of CO₂ fixation pathway has profound implications for how a plant responds to the innumerable combinations of light intensity, leaf internal CO₂ concentration, temperature, and water status in the natural environment. As discussed above, at normal atmospheric CO₂ levels photosynthesis is lower by at least 25 percent in C₃ plants than it would be if photorespiration were absent. The generally higher rates of photosynthesis in C₄ plants are attributable to both suppression of photorespiration in these species and the superior ability of PEP carboxylase to fix CO₂ at the very low concentrations of this gas that can occur inside leaf tissue. These differences are most pronounced at high light intensity. Photosynthesis in C₃ leaves attain maximal rates at light levels of about 50 percent of full sunlight. However, C₄ photosynthesis continues to increase with light intensity even in full sunlight. It is little wonder that the highest yielding crop species use the C₄ mechanism. Conversely, C₃ plants are capable of more efficient use of light quanta when light levels are low, as would be the case for shaded conditions. Also, high temperatures favor C₄ plants because the number of molecules of H₂ O lost to evaporation via the stomata (transpi-ration) per CO₂ fixed is much lower for these species compared to C₃ species. However, C₃ plants tend to be more competitive in cool environments. Finally, although projected increases in global atmospheric CO₂ levels during the twenty-first century should enhance photosynthesis in all species, associated changes in distribution of temperature and rainfall will also exert great influence on the composition and characteristics of Earth's flora.

see also Atmosphere and Plants; Cactus; Calvin, Melvin; Chloroplast; de Saussure, Nicholas; Ingenhousz, Jan; Photosynthesis, Light Reactions and.

Richard B. Peterson

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