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relies upon the cooperation and enforcement of good management practices by growers, and can fail if not implemented rigorously on all farms. Another strategy to prevent or delay the acquisition of resistance by insects is the inclusion of several unrelated toxin genes in a transgenic crop, but this so-called ‘‘gene pyramiding’’ will be a much more expensive and much longer term option. A potential problem with any pest control agent is that it might inadvertently affect harmless or even benign organisms. On this score, the Bt toxins appear to perform relatively well, and certainly better than many synthetic pesticides. In 1999, Losey and coworkers reported possible adverse effects of Bt-containing corn pollen on monarch butterfly (Danaus plexippus) larvae. Although this finding generated a great deal of controversy at the time, subsequent detailed studies have shown that there is no significant impact of Bt corn pollen on monarch butterfly larvae.
List of Technical Nomenclature Refugia
Refuges where pest populations are tolerated in order to reduce the possibility of them developing resistance to control agents, such as pesticides or pest resistant GM crops.
See also: Diseases: Breeding for Disease Resistance. Integrated Pest Management: Disease Prediction Models; Practice; Principles. Weeds: Herbicide Resistance.
Further Reading Carozzi N and Koziel M (eds) (1997) Advances in Insect Control: the role of transgenic plants. London: Taylor & Francis. de Maagd RA, Bravo A, and Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 17: 193–199. Flexner JL and Belnavis DL (1999) Microbial insecticides. In: Rechcigl JE and Rechcigl NA (eds) Biological and Biotechnological Control of Insect Pests, pp. 35–62. Boca Raton, FL: CRC Press. Gatehouse AM, Ferry N, and Raemaekers RJ (2002) The case of the monarch butterfly: a verdict is returned. Trends in Genetics 18: 249–251. Kaiser J (1996) Pests overwhelm Bt cotton crop. Science 272: 423. Losey JE, Rayor LS, and Carter ME (1999) Transgenic pollen harms monarch larvae. Nature 399: 214. McGaughey WH, Gould F, and Gelernter W (1998) Bt resistance management. Nature Biotechnology 16: 144–146. Shelton AM and Sears MK (2003) The monarch butterfly controversy: scientific interpretations of a phenomenon. Plant Journal 27: 483–488.
PHOTOSYNTHESIS AND PARTITIONING Contents
C3 Plants C4 Plants CAM Plants Photorespiration Photoinhibition Primary Products of Photosynthesis, Sucrose and other Soluble Carbohydrates Sources and Sinks
C3 Plants A S Raghavendra, University of Hyderabad, Hyderabad, India Copyright 2003, Elsevier Ltd. All Rights Reserved.
Summary The basic route of carbon assimilation in plants is the Calvin cycle, where the first product of
carboxylation is 3-phosphoglycerate (PGA), a 3carbon compound. There are two variants of carbon assimilation, where plants use the C4-pathway or CAM, as a CO2 concentrating mechanism, to raise the level of CO2 at the vicinity of rubisco in chloroplasts. Plants possessing only the Calvin cycle are called C3 plants, and these constitute almost 90% of the plant kingdom. The Calvin cycle consists of three phases: (1) formation of PGA from ribulose1,5-bisphosphate (RuBP) and CO2 (carboxylation
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phase); (2) reduction of PGA to triose-P (reduction phase); and (3) regeneration of the CO2 acceptor, RuBP, from triose-P (regeneration phase). The Calvin cycle is regulated in several ways: autocatalysis, flux control, light-activation of key enzymes, and through regulatory metabolites. Intense efforts are being made to genetically manipulate enzymes/ proteins related to the Calvin cycle, such as rubisco, rubisco activase, fructose-1,6-bisphosphatase and phosphate translocator.
Introduction Carbon assimilation by plants and other photosynthetic organisms is a very important event in the global carbon cycle. Plants fix carbon primarily into 3-phosphoglycerate (PGA, a 3-carbon compound) and hence the process is named the C3 photosynthesis or C3 pathway, or Calvin cycle (named after Nobel laureate, Melvin Calvin). The other two variants of photosynthetic carbon assimilation are C4 photosynthesis (or C4 pathway) and crassulacean acid metabolism (CAM). However, the carbon from C4 acids formed initially during these two pathways has to be refixed ultimately through the C3 or Calvin or Benson–Calvin cycle. Thus, the C3 photosynthesis is the basic route of carbon assimilation while the C4 pathway and CAM function as carbon accumulating mechanisms and form adjuncts of the Calvin cycle. Plants possessing only the Benson–Calvin cycle are
Biochemistry/Reactions The path of carbon assimilation was mapped quite elegantly by Professor Melvin Calvin and his group during the early 1950s, by the combined use of twodimensional paper chromatography and radioisotopic carbon (14C); therefore, this pathway is called the Calvin cycle. It is also called the reductive pentose phosphate (RPP) pathway, or photosynthetic carbon reduction cycle (PCRC). C3 photosynthesis has three principal phases: carboxylation, reduction, and regeneration. During the first phase of carboxylation, carbon dioxide is accepted by ribulose-1,5-bisphosphate (RuBP) to give two molecules of PGA. During the next phase of reduction, PGA is reduced to triose phosphate (triose-P), by using assimilatory force or assimilatory power (generated in light reactions) of ATP and NADPH. The last phase is the regeneration of the primary acceptor of CO2, RuBP, from triose phosphate through a series of reactions. For each molecule of CO2 fixed, three ATP and two NADPH molecules are required. The first step of C3 photosynthesis is catalyzed by the enzyme ribulose 1,5-bisphosphate carboxylase/ oxygenase (rubisco), which prefers CO2 to the dissolved form of carbon (bicarbonate) as the substrate (Figure 1). Since the concentration of CO2
3-Phosphoglycerate (PGA)
H2O CO2
called C3 plants, while the other two categories include C4 plants and CAM plants.
Rubisco
ADP
PGA kinase
Ribulose 1,5bisphosphate (RuBP)
1,3-Bisphosphoglycerate (BPGA)
NADPH + H+
ADP
Phosphoribulokinase
ATP
Phosphopentose epimerase
GAP dehydrogenase NADP + Pi
Triose -P Xylulose 5-P Dihydroxyacetone-P isomerase Glyceraldehyde
Ribulose 5-P (Ru5P) Phosphopentose isomerase
ATP
(Xu5P)
(DHAP)
3-P (GAP)
Transketolase Aldolase
Ribose 5-P (R5P)
Sedoheptulose 7-P (S7P)
SBPase
Fructose 1,6bisphosphate (FBP) H2O
Pi
FBPase
Pi H2O
Fructose 6-P (F6P)
Sedoheptulose 1,7bisphosphate (SBP) Aldolase
Transketolase
Erythrose 4-P (E4P)
Figure 1 Schematic representation of the Calvin cycle or C3 photosynthesis. The individual enzymes are as follows: rubisco (EC 4.1.1.39); PGA kinase (EC 2.7.2.3); GAP dehydrogenase (EC 1.2.1.13); triose-P isomerase (EC 5.3.1.1); aldolase (EC 4.1.2.13); FBPase (EC 3.1.3.11); SBPase (EC 3.1.3.37); transketolase (EC 2.2.1.1); phosphopentose epimerase (EC 5.1.3.1); phosphopentose isomerase (EC 5.3.1.6); and phosphoribulokinase (EC 2.7.1.19). The major product of the cycle is GAP, which is either exported out of the chloroplast to form sucrose or used within the chloroplast to form starch. A part of the GAP is used for the autocatalysis of the cycle.
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within the plant cell is quite low (about 10 mmol l 1), the activities of rubisco are rather low and are limited at the normal atmospheric CO2. However, this phenomenon is compensated by the presence of large amounts of rubisco protein, which could be up to 50% of leaf protein. Apart from carboxylation, rubisco also catalyzes oxygenase activity, where O2 reacts with RuBP to form phosphoglycolate, in addition to PGA. The affinity of rubisco for CO2 and O2, at typical in vivo concentrations, is quite similar, and as a result the carboxylase and oxygenase reactions are unavoidable in the present atmospheric levels of O2 and CO2. The next phase of reduction of PGA to triose-P involves three steps: activation of PGA to 1,3bisphosphoglycerate or BPGA (catalyzed by PGA kinase); (2) reduction of BPGA to glyceraldehyde 3-phosphate (GAP) with the help of NADPH (catalyzed by GAP dehydrogenase or GAPDH); and (3) interconversion of GAP and dihydroxyacetone phosphate (DHAP), by the enzyme triose-P isomerase (Figure 1). Both GAP and DHAP are termed triose-P. During the third phase, RuBP (the CO2 acceptor) is regenerated from triose-P through a series of reactions involving condensation and rearrangement. The triose phosphates, GAP and DHAP, are condensed by aldolase to form fructose 1,6-bisphosphate (FBP). This six-carbon sugar is then irreversibly hydrolyzed to fructose 6-phosphate (F6P) by fructose-1,6-bisphosphatase (FBPase). A two-carbon entity is transferred from F6P to GAP by the enzyme transketolase to form xylulose 5-phosphate (Xu5P) and erythrose-4-phosphate (E4P). Then, E4P is combined with DHAP to form sedoheptulose 1,7bisphosphate (SBP), by the enzyme aldolase. This 7-carbon product, SBP, is hydrolyzed by sedoheptulose-1,7-bisphosphatase (SBPase) to yield sedoheptulose 7-phosphate (S7P). Two carbons from S7P are transferred to GAP by transketolase producing Xu5P and ribose-5-phosphate (R5P). The resulting R5P is converted to ribulose-5-P (Ru5P) by phosphopentose isomerase. The two molecules of Xu5P are converted into Ru5P by phosphopentose epimerase. The final step of the regeneration phase is the irreversible conversion (using ATP) of Ru5P to RuBP by phosphoribulokinase (PRK). An important feature of the Calvin cycle is its autocatalysis, where plants are able to reach steadystate rates of photosynthetic conversion of CO2 after a short delay, as the intermediates build up to the appropriate levels. Autocatalysis of the Calvin cycle is facilitated by the cyclic operation of the pathway as well as the controlled removal of the intermediates from the pathway for other purposes.
Products The major product of the Calvin cycle is triose-P (GAP and DHAP) and further metabolism of triose-P is the branching point of the Calvin cycle. The available triose-P is used for conversion into two major products of photosynthesis: (1) starch (a glucose polymer), which accumulates during the day inside the chloroplast; and (2) sucrose, which is formed in the cytosol. Triose phosphate is exported from the chloroplast via the phosphate translocator (on the inner chloroplast envelope membrane) and is further metabolized to sucrose in the cytosol. Sucrose is largely exported from the leaf to different sink tissues, e.g., roots, developing leaves, reproductive organs, and other heterotrophic tissues. Sucrose may also accumulate in the vacuole during the day. Besides the conversion into starch and sucrose, a significant portion of the triose-P is also converted within the chloroplasts to amino acids and fatty acids. Only 1/6 of triose phosphate is utilized/ exported out for various biosynthetic processes, while the other 5/6 is needed to regenerate RuBP.
Rubisco Rubisco is a unique and interesting enzyme, mediating the key reaction of photosynthetic CO2 assimilation: conversion of one molecule of RuBP and one of CO2 into two molecules of PGA (Figure 2). Besides the carboxylation reaction, Rubisco reacts with hv
hv CA1P
RUBISCO Inactive ATP ADP + Pi Rubisco activase
Rubisco activase
Inactive
pH Mg2+ Metabolites
Active CA1P
RUBISCO Active RuBP
Carboxylase
PGA + PGA CO2 O2
P-glycolate + PGA
Oxygenase
Figure 2 The reactions and regulation of rubisco. The enzyme functions as both a carboxylase and an oxygenase. The product of the carboxylase reaction, PGA, is metabolized in the Calvin cycle. One of the products of oxygenase, P-glycolate, is metabolized through photorespiration. Carboxyarabinitol 1-phosphate (CA1P) binds tightly to rubisco and makes it inactive (e.g., in darkness). Rubisco is activated in light in two ways: (1) by rubisco activase, which itself is activated in light through ATP; and (2) changes in the stromal microenvironment (increase in pH, Mg2 þ levels, and Calvin cycle metabolites). Both the processes facilitate the release of CA1P and activation of rubisco.
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oxygen to form one molecule of 2-phosphoglycolate and one of PGA. The plants try to retrieve part of the carbon diverted into phosphoglycolate through the process of photorespiration or C2 cycle. Photorespiration also helps in the recycling of nitrogen and dissipation of excess energy and reductants (ATP and NADPH) in the chloroplasts. A major drawback of rubisco is its very slow catalytic capacity. The turnover number of the enzyme is extremely small, and such unfavorable kinetic behavior requires large amounts of enzyme (up to 50% of the total soluble protein) to ensure the carbon flux. Rubisco of higher plants is a complex protein, with eight large subunits (mass of about 58 kDa) and eight small subunits (mass of about 18 kDa). The large subunits have the catalytic centers, while the small subunits seem to stabilize the rubisco complex. In cyanobacteria, rubisco is also made up of eight large and eight small subunits, while in purple bacteria, rubisco is a dimer of two large subunits only. The large subunits are encoded by the plastid genome and the small subunits in the nucleus. The regulation of rubisco is described below.
Regulation C3 photosynthesis is regulated in multiple ways, but is primarily initiated by illumination. One of the most important phenomena in the pathway is the light activation of key enzymes, through either dithiol-reduction of cysteines on protein or through changes in the microenvironment (e.g., magnesium or pH levels) of the stroma. Then, changes in substrate availability and metabolite flux rapidly set in motion an efficient autocatalysis of the Calvin cycle. In a long-term mode, the levels and turnover of rubisco protein also modulate the capacity of carbon fixation in the Calvin cycle. For example, limitation of nitrogen availability decreases the levels of rubisco protein and restricts the photosynthetic carbon assimilation in the leaves. Regulation of Rubisco
The activity of rubisco, the first enzyme in the Calvin cycle, is highly regulated. Rubisco is inactive in the dark and is converted to an active form on illumination, which catalyzes fixation of CO2. Activation of rubisco is the result of carbamylation, which involves the binding of CO2 and Mg2 þ to a lysine residue near the catalytic site. Rubisco is active only when lysine-201 reacts with CO2 near the catalytic site to form a carbamate and allows the binding of the Mg2 þ ion. Carbamylation changes the conformation of the large subunit and activates the enzyme, while the active conformation
is stabilized by the formation of a complex with Mg2 þ . Carbamylation is essential for rubisco activation, as the noncarbamylated rubisco binds RuBP too tightly to allow catalysis. Another protein, rubisco activase, is also involved in mediating the light activation of rubisco. On illumination, rubisco activase releases the inhibitor compounds, such as 2-carboxyarabinitol 1-phosphate or CA1P, which are bound to the active site of rubisco; otherwise, for example in darkness, these inhibitors prevent activation (carbamylation) of the enzyme. Rubisco activase itself is activated in light by utilizing ATP produced from photosynthetic electron transport. Rubisco activase and rubisco activation provide another mechanism of strong regulation by light of carbon assimilating reactions of photosynthesis. CA1P, which occurs naturally in the leaves of several plants, is a strong inhibitor of rubisco. The affinity of rubisco for CA1P is much stronger than that for RuBP, the substrate. As a result, CAP, which accumulates in leaves during the night, inactivates rubisco by blocking the binding sites. During the day (or on illumination), the bound CA1P is released from rubisco, and this process is further accelerated by rubisco activase. However, the physiological role of CA1P is still debated, as it is not found in all plant species.
Light Activation of Enzymes Light directly activates five enzymes of the Calvin cycle, namely rubisco, GAPDH, FBPase, SBPase, and PRK. Among these, rubisco is activated both indirectly (due to changes in the microenvironment of the chloroplast stroma) and directly (through rubisco activase). The other four enzymes are activated as a result of the reduction of dithiols on the enzyme. Interestingly, four of the five light-activated enzymes (rubisco, FBPase, SBPase, and PRK) catalyze irreversible reactions. Although the dark activities of these enzymes may vary, the Calvin cycle is essentially inactive until illumination takes place. The Calvin cycle enzymes are activated as a result of the reduction of dithiols on the protein, mediated by a ferredoxin–thioredoxin system. On illumination, ferredoxin is reduced by the thylakoids, which in turn reduce thioredoxin. Reduced thioredoxin can reduce the dithiols at or near the active site of FBPase, SBPase, and PRK, thus activating the enzymes of the Calvin cycle. The thioredoxin– ferredoxin system (reduced in light) also activates two other chloroplastic enzymes, F1-ATP synthase and NADP-malate dehydrogenase. Light not only activates several enzymes, but also inactivates the key enzymes of the oxidative pentose
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phosphate pathway, again through thioredoxin mediated reduction of enzymes. Glucose-6-phosphate dehydrogenase is one such enzyme, which is inactivated on illumination to ensure that the oxidative pentose phosphate pathway does not operate simultaneously with the reductive pentose phosphate pathway, thus avoiding a futile cycle. Light causes a marked change in the microenvironment of the chloroplast stroma. The photosynthetic electron transport chain in the thylakoid membrane decreases the proton concentration, raises the pH, and increases the Mg2 þ concentration in the stroma. Rubisco, FBPase, and SBPase respond to such changes in pH and Mg2 þ concentrations. These enzymes are almost inactive at pH 7.2 and low Mg2 þ , and their activity increases with the rise in pH and Mg2 þ concentration. The enzymes are also under metabolite control. The stromal enzymes FBPase and SBPase are activated by their substrates and allosterically inhibited by their products. High levels of FBP or SBP activate these enzymes, while F6P or S7P inhibit the activity of the corresponding enzyme, to avoid excessive product accumulation and sequestration of phosphate. Similarly, PRK is inhibited by 3-PGA and ADP. As both these metabolites inhibit PRK, phophorylation by PGA kinase can proceed even under limiting ATP supply, thus preventing the accumulation of PGA. Multiprotein Complexes
The occurrence and operation of multienzyme complexes involving Calvin cycle enzymes have been reported. Such complexes could be an important mode of regulation. For example, GAP and PRK interact with a small nuclear encoded chloroplast protein, CP12, and form a complex, which may be an additional mechanism for light regulation of PPK in vivo. Complexes involving different combinations of C3-enzymes have been found, and these may facilitate rapid channeling of intermediates between enzymes, improving the efficiency of the cycle. Multienzyme complexes of phosphoribose isomerase, PRK, GAPDH, and SBPase have been located on the thylakoid membrane by immunoelectron microscopy. A complex of sucrose phosphate synthase and sucrose phosphate phosphatase has also been reported. Flux Control
Flux control is an important concept, and the relative importance of individual enzymes in controlling the flux of carbon fixation through the Calvin cycle has been examined. Studies of transgenic plants with altered levels of individual enzymes in the C3 cycle
have revealed that no single enzyme has complete control of carbon fixation. Control is shared among a number of enzymes, with rubisco, SBPase, and aldolase having the most prominent roles. Enzymes with highly regulated activity, such as PRK, do not seem to have strong control on carbon fixation, whereas aldolase, which catalyzes a reversible reaction, strongly influences the rate of carbon fixation.
C3 Plants: Limitations and Prospects for Improvement C3-photosynthesis is a feature of even the primitive lower groups and appears to have evolved much earlier than the CAM or C4 pathway. Of about 300 000 plants known on earth, B90% are C3 plants, while the CAM and C4-species constitute about 10% and 1%, respectively. Furthermore, most crops (particularly cereals, legumes, and oilseed crops) are of the C3-type; therefore, C3 plants have attracted the attention of several scientists. The performance and productivity of C3 plants is restricted by at least three major factors: high photorespiration (a nonavoidable consequence of oxygenase activity of rubisco), a high water requirement, and a preference for temperate regions. Attempts were made to evolve varieties with reduced photorespiration or high photosynthetic rates, but single features were not able to improve the productivity of plants. Another approach was to introduce a set of C4 traits into C3 plants, but hybridization of C3 and C4 species of Atriplex resulted in only C3-type plants. Mutants deficient in one or more enzymes of photorespiration were unable to grow at atmospheric levels of CO2, indicating that the process of photorespiration (as well as rubisco oxygenase activity) was an adaptation to the present atmospheric levels of CO2/O2. However, a very well known factor that could improve the photosynthetic performance and productivity of C3 plants is elevated CO2.
Molecular Biology and Biotechnology The research before the 1950s involved studies mainly on the physiology (e.g., Blackman’s law of limiting factors) and biochemistry (e.g., Calvin cycle) of C3-photosynthesis. By the end of the twentieth century, focus had shifted onto the molecular biology of chloroplasts (and their components) and biotechnology to manipulate some of the proteins/enzymes. Genes
In the past decade, enormous progress has been made in identifying the genes involved in not only
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photosynthesis but also other metabolic processes. The genomes of plants such as Arabidopsis and rice (Oryza sativa) have been completely sequenced. With reference to photosynthesis, the initial work was carried out with genes encoding the components of photosynthetic electron transport, but later covered most of the genes encoding the enzymes of the Calvin cycle and related processes. The genes encoding the enzymes of the Calvin cycle, with the exception of the large subunit of rubisco, are located in the nuclear genome; the large subunit of rubisco is encoded by genes in the chloroplast. The preproteins are synthesized in the cytosol with N-terminal extensions called transit sequences, which direct them to the chloroplasts, where they are cleaved to form mature proteins. In recent years, cloning and analysis of the genes encoding the enzymes of the Calvin cycle have facilitated further studies on the control of their expression. Regulation of Gene Expression
There is a perfect coordination of mRNA synthesis and protein accumulation during leaf development and chloroplast maturation. Much of the control has been found at the level of gene transcription. Light has a central role in regulating the biogenesis of the photosynthetic apparatus during chloroplast development, including switching on the expression of genes encoding enzymes of C3 photosynthesis. These
processes appear to be complex, involving many regulatory proteins and cross-talk between the different photoreceptor signaling pathways. The expression of C3 photosynthesis genes in leaves is also modulated by environmental and metabolic signals. High levels of glucose or sucrose reduce levels of a number of Calvin cycle mRNAs, including those encoding the small subunit of rubisco, SBPase, and FBPase. This feedback mechanism, acting at the level of transcription, might be important for source–sink regulation in the plant. Photosynthesis genes respond remarkably to nutrient status, particularly nitrogen and phosphorus levels, and this is influenced again by the carbohydrate status. Thus, an interaction between carbohydrate and nutrient status may be a long-term strategy to control carbon metabolism. Genetic Manipulation
Several enzymes of the Calvin cycle have been genetically engineered in plants (Table 1). Attempts have been made to either overexpress the target enzyme or suppress the enzyme levels. The overexpression of these enzymes causes only a small improvement in the overall performance of the plant. However, the suppression of photosynthetic enzymes invariably led to a marked decrease in photosynthetic rate. A reduction in the levels of rubisco, rubisco activase, and NADP-GAPDH resulted in a significant
Table 1 Examples of genetic manipulation of enzymes or proteins involved in photosynthesis of C3 plants; the effect (suppression or overexpression) of the target enzyme/protein, on the test plant and the consequence of such genetic manipulation are indicated Target enzyme or protein/test plant
Effect
Consequence
Rubisco/tobacco
Suppression
Rubisco activase/tobacco
Suppression
GAP dehydrogenase/tobacco
Suppression
Aldolase/potato (Solanum tuberosum) Transketolase/tobacco
Suppression
Marginal effect on photosynthesis at high N and moderate light; significant limitation on photosynthesis at limiting N and high light Reduction in activation/carbamylation of rubisco in light; photosynthesis decreased at both low and high CO2 No significant reduction in growth; increase in PGA indicating the export and reduction of PGA in cytosol Strong reduction in growth
Sucrose-P synthase/ Arabidopsis Phosphate translocator/potato
Suppression Suppression Suppression
Small decrease in transketolase led to marked decrease in growth, photosynthesis, and phenyl propanoid metabolism Decrease in sucrose synthesis as well as photosynthetic rate No effect on overall rate of carbon fixation, but increased starch accumulation in leaves in light Enhanced photosynthesis and growth; increased allocation of carbon into sucrose
Single cyanobacterial FBP/ SBPase/tobacco
Overexpression
Mutated form of sucrose 6-P synthase (to avoid downregulation)/tobacco
Overexpression
High sucrose-P synthase activity and stimulation of sucrose synthesis
Rat liver 6-phosphofructo-2kinase/tobacco
Overexpression
Higher level of fructose-2,6-bisphosphate; sucrose synthesis reduced at the beginning of light period
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decrease in photosynthesis, particularly at high light intensity. The manipulation of stromal FBPase or triose-P/Pi translocator affected the growth pattern only marginally, but significantly changed the pattern of assimilate translocation. The above genetically modified plants were obviously trying to compensate for the deficit of each enzyme/protein; such is the adaptive capacity of plants, due to their metabolic flexibility. In plants, FBPase and SBPase are separate enzymes, while in cyanobacteria, a single enzyme (FBP/SBPase) performs both of their functions. Overexpression of the single cyanobacterial enzyme from Synechococcus enhanced the photosynthesis and growth of tobacco (Nicotiana tabacum) plants.
aralocaspica, where CO2 concentrating mechanisms or C4 photosynthesis operates within a single cell. It would be quite exciting to analyze these plants and exploit the extracted information. With the introduction of an efficient CO2 concentrating mechanism, the performance of C3 plants is bound to improve.
Future Outlook
List of Technical Nomenclature
The performance of C3 plants improves considerably when the atmospheric CO2 level is elevated. Thus, under conditions of global warming with its elevated CO2 levels, the use and cultivation of C3 plants offer much greater potential than that of C4 plants. However, the C3 plants may require additional nutrients under high CO2. This aspect needs to be studied further so that the C3 plants can be exploited effectively under elevated CO2. Despite detailed studies, the functional mechanism of rubisco continues to be a marvel and a mystery. Further experiments are warranted to understand the conditions that would ensure the activated state of rubisco. Specific alterations in the amino acid residues with the help of site-directed mutagenesis may be of interest to achieve this target. An increase in the ratio of carboxylase to oxygenase of rubisco would be of immense potential to increase the photosynthetic efficiency. However, neither extensive screening of higher plant species and their cultivars, nor the use of different metabolites/inhibitors helped to alter the ratio of carboxylase/oxygenase activity. However, some of the plants from lower groups (particularly red algae) have been found to possess rubisco with a much higher carboxylase to oxygenase ratio than that of higher plants. These forms of rubisco are termed ‘‘super rubiscos,’’ in view of their vast potential. Attempts are currently being made to incorporate ‘‘super rubisco’’ into higher plants. Another possibility of improving the performance of C3 plants is to supplement with CO2 concentrating mechanisms. Nature has already achieved this in C4 and CAM plants. An encouraging and important step in this direction is the success of expressing corn (Zea mays; maize) phosphoenolpyruvate carboxylase (PEPC) in rice leaves to achieve 4100-fold increase in the activity of PEPC. There are plants, such as Hydrilla verticillata, Egeria densa, and Borszczowia
Carbon assimilation
Conversion of simple inorganic carbon dioxide into complex organic carbon compounds, enriched with energy, such as sugars.
Chloroplast stroma
Soluble compartment of chloroplasts, enclosed by the envelope membranes, and contains several biosynthetic enzymes, metabolites, nucleic acids and lipid granules.
Gene transcription
The passing of genetic message contained in DNA to RNA, facilitating the translation of information into assembly of a specific chain of amino acids and biosynthesis of proteins.
Sink
The tissues which import and utilize the assimilated organic compounds, e.g. young developing parts, growing fruits, or storage organs, such as tubers.
Source
The organ which makes and supplies the energy-rich organic compounds, usually the leaf.
Acknowledgments The preparation of this manuscript and current work on photosynthesis in our laboratory is supported by grant No. SP/SO/A12/98 from the Department of Science and Technology, New Delhi.
See also: Genetic Modification of Primary Metabolism: Photosynthesis. Photosynthesis and Partitioning: C4 Plants; CAM Plants; Photoinhibition.
Further Reading Blankenship RE (2002) Molecular Mechanisms of Photosynthesis. London: Blackwell Science. Grossman AR (2000) Chlamydomonas reinhardtii and photosynthesis: Genetics to genomics. Current Opinion in Plant Biology 3: 132–137. Gutteridge S and Jordan DB (2001) Dynamics of photosynthetic CO2 fixation: Control, regulation and productivity. In: Aro E-M and Andersson B (eds) Regulation of Photosynthesis, pp. 297–312. Dordrecht: Kluwer Academic Publishers.
680 PHOTOSYNTHESIS AND PARTITIONING / C4 Plants Heldt HW (1997) Plant Biochemistry and Molecular Biology. Oxford: Oxford University Press. Lawlor DW (2001) Photosynthesis: Molecular, Physiological and Environmental Processes, 3rd edn. Berlin: Springer Verlag. Leegood RC (1999) Photosynthesis in C3 plants: The Benson–Calvin cycle and photorespiration. In: Lea PJ and Leegood RC (eds) Plant Biochemistry and Molecular Biology, 2nd edn, pp. 29–50. Chichester: John Wiley & Sons. Malkin R and Niyogi K (2000) Photosynthesis. In: Buchanan BB, Gruissem W, and Jones RL (eds) Biochemistry and Molecular Biology of Plants, pp. 568–628. Rockville, MD: American Society of Plant Physiologists. Martin W, Scheibe R, and Schnarrenberger C (2000) The Calvin cycle and its regulation. In: Leegood RC, Sharkey TD, and Von Caemmerer S (eds) Photosynthesis: Physiology and Metabolism, pp. 9–51. Dordrecht: Kluwer Academic Publishers. Quick WP and Neuhaus HE (1997) The regulation and control of photosynthetic carbon assimilation. In: Foyer CH and Quick WP (eds) A Molecular Approach to Primary Metabolism in Higher Plants, pp. 41–62. London: Taylor and Francis. Rao KK and Hall DO (1999) Photosynthesis, 6th edn. Cambridge: Cambridge University Press. Sharkey TD (1998) Photosynthetic carbon reduction. In: Raghavendra AS (ed.) Photosynthesis: A Comprehensive Treatise, pp. 111–122. Cambridge: Cambridge University Press. Spreitzer RJ and Salvucci ME (2002) Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme. Annual Review of Plant Physiology and Plant Molecular Biology 53: 449–475. Stitt M (1996) Metabolic regulation of photosynthesis. In: Baker NR (ed.) Photosynthesis and the Environment, pp. 151–190. Dordrecht: Kluwer Academic Publishers. Von Caemmerer S and Quick WP (2000) Rubisco: physiology in vivo. In: Leegood RC, Sharkey TD, and Von Caemmerer S (eds) Photosynthesis: Physiology and Metabolism, pp. 85–113. Dordrecht: Kluwer Academic Publishers.
C4 Plants R C Leegood, University of Sheffield, Sheffield, UK Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Plants with the C4 pathway of photosynthesis, typically grasses from warm habitats, are responsible for about a quarter of global terrestrial productivity. The C4 mechanism involves a division of labor
between two different photosynthetic cell types, the mesophyll and the bundle sheath. The CO2-concentrating function of C4 photosynthesis concentrates CO2 at the site of Rubisco in the bundle sheath, providing a mechanism that overcomes the photorespiration incurred by the oxygenase activity of Rubisco, and which accounts for losses of around a quarter of the recently fixed carbon in C3 plants, particularly at higher temperatures. The C4 pathway also results in improved water and nitrogen-use efficiency compared with C3 plants. In a sense, C4 plants were discovered, but not recognized as such, in the nineteenth century, when Haberlandt described their distinctive Kranz (German, ‘‘wreath’’) anatomy: a distinct layer of cells rich in chloroplasts surrounding the vascular bundle (Figure 1). The true nature of these plants did not become apparent until the 1960s, from work by a Russian, Karpilov, on corn (Zea mays, maize), and by Kortschak and colleagues on sugar cane (Saccharum officinarum) in Hawaii, who reported that the first products of photosynthesis were malate and aspartate, and not glycerate 3-P, as would be expected from CO2 fixation via the C3 Benson– Calvin cycle. In the mid-1960s, Hatch and Slack, in Australia, elucidated the path of carbon in C4 photosynthesis.
Agricultural and Ecological Importance Although the number of terrestrial plant species that are C4 is quite modest (about 3%), about 50% of the grasses are C4. Corn is one of the three principal cereal crops, together with wheat (Triticum aestivum) and rice (Oryza sativa) (both C3 plants), each of which constitutes about 30% of total global cereal production (Table 1). Other important C4 grass crops include millet (Panicum miliaceum), Sorghum spp., sugar cane, and pasture grasses (Poaceae). C4 plants have two primary requirements for success: a warm growing season and high light, although aridity may also influence their distribution. Fourteen out of 18 of the world’s worst weeds are C4. C4 grasses are the dominant (490%) components of warm temperate and tropical grasslands, such as prairies and savannas. In prairies, C3 grasses tend to be active during the spring, whereas C4 grasses tend to be active during the summer, when temperatures are higher and soil water lower. Similarly, decreases in temperature with altitude tend to favor C3 grasses. Other biomes with a high representation of C4 plants, particularly at low (tropical) latitudes, include deserts and semideserts, arid steppes and salt deserts, beach dunes and saltmarshes, wetlands, and disturbed ground in arid regions.
relies upon the cooperation and enforcement of good management practices by growers, and can fail if not implemented rigorously on all farms. Another strategy to prevent or delay the acquisition of resistance by insects is the inclusion of several unrelated toxin genes in a transgenic crop, but this so-called ‘‘gene pyramiding’’ will be a much more expensive and much longer term option. A potential problem with any pest control agent is that it might inadvertently affect harmless or even benign organisms. On this score, the Bt toxins appear to perform relatively well, and certainly better than many synthetic pesticides. In 1999, Losey and coworkers reported possible adverse effects of Bt-containing corn pollen on monarch butterfly (Danaus plexippus) larvae. Although this finding generated a great deal of controversy at the time, subsequent detailed studies have shown that there is no significant impact of Bt corn pollen on monarch butterfly larvae.
List of Technical Nomenclature Refugia
Refuges where pest populations are tolerated in order to reduce the possibility of them developing resistance to control agents, such as pesticides or pest resistant GM crops.
See also: Diseases: Breeding for Disease Resistance. Integrated Pest Management: Disease Prediction Models; Practice; Principles. Weeds: Herbicide Resistance.
Further Reading Carozzi N and Koziel M (eds) (1997) Advances in Insect Control: the role of transgenic plants. London: Taylor & Francis. de Maagd RA, Bravo A, and Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 17: 193–199. Flexner JL and Belnavis DL (1999) Microbial insecticides. In: Rechcigl JE and Rechcigl NA (eds) Biological and Biotechnological Control of Insect Pests, pp. 35–62. Boca Raton, FL: CRC Press. Gatehouse AM, Ferry N, and Raemaekers RJ (2002) The case of the monarch butterfly: a verdict is returned. Trends in Genetics 18: 249–251. Kaiser J (1996) Pests overwhelm Bt cotton crop. Science 272: 423. Losey JE, Rayor LS, and Carter ME (1999) Transgenic pollen harms monarch larvae. Nature 399: 214. McGaughey WH, Gould F, and Gelernter W (1998) Bt resistance management. Nature Biotechnology 16: 144–146. Shelton AM and Sears MK (2003) The monarch butterfly controversy: scientific interpretations of a phenomenon. Plant Journal 27: 483–488.
PHOTOSYNTHESIS AND PARTITIONING Contents
C3 Plants C4 Plants CAM Plants Photorespiration Photoinhibition Primary Products of Photosynthesis, Sucrose and other Soluble Carbohydrates Sources and Sinks
C3 Plants A S Raghavendra, University of Hyderabad, Hyderabad, India Copyright 2003, Elsevier Ltd. All Rights Reserved.
Summary The basic route of carbon assimilation in plants is the Calvin cycle, where the first product of
carboxylation is 3-phosphoglycerate (PGA), a 3carbon compound. There are two variants of carbon assimilation, where plants use the C4-pathway or CAM, as a CO2 concentrating mechanism, to raise the level of CO2 at the vicinity of rubisco in chloroplasts. Plants possessing only the Calvin cycle are called C3 plants, and these constitute almost 90% of the plant kingdom. The Calvin cycle consists of three phases: (1) formation of PGA from ribulose1,5-bisphosphate (RuBP) and CO2 (carboxylation
674 PHOTOSYNTHESIS AND PARTITIONING / C3 Plants
phase); (2) reduction of PGA to triose-P (reduction phase); and (3) regeneration of the CO2 acceptor, RuBP, from triose-P (regeneration phase). The Calvin cycle is regulated in several ways: autocatalysis, flux control, light-activation of key enzymes, and through regulatory metabolites. Intense efforts are being made to genetically manipulate enzymes/ proteins related to the Calvin cycle, such as rubisco, rubisco activase, fructose-1,6-bisphosphatase and phosphate translocator.
Introduction Carbon assimilation by plants and other photosynthetic organisms is a very important event in the global carbon cycle. Plants fix carbon primarily into 3-phosphoglycerate (PGA, a 3-carbon compound) and hence the process is named the C3 photosynthesis or C3 pathway, or Calvin cycle (named after Nobel laureate, Melvin Calvin). The other two variants of photosynthetic carbon assimilation are C4 photosynthesis (or C4 pathway) and crassulacean acid metabolism (CAM). However, the carbon from C4 acids formed initially during these two pathways has to be refixed ultimately through the C3 or Calvin or Benson–Calvin cycle. Thus, the C3 photosynthesis is the basic route of carbon assimilation while the C4 pathway and CAM function as carbon accumulating mechanisms and form adjuncts of the Calvin cycle. Plants possessing only the Benson–Calvin cycle are
Biochemistry/Reactions The path of carbon assimilation was mapped quite elegantly by Professor Melvin Calvin and his group during the early 1950s, by the combined use of twodimensional paper chromatography and radioisotopic carbon (14C); therefore, this pathway is called the Calvin cycle. It is also called the reductive pentose phosphate (RPP) pathway, or photosynthetic carbon reduction cycle (PCRC). C3 photosynthesis has three principal phases: carboxylation, reduction, and regeneration. During the first phase of carboxylation, carbon dioxide is accepted by ribulose-1,5-bisphosphate (RuBP) to give two molecules of PGA. During the next phase of reduction, PGA is reduced to triose phosphate (triose-P), by using assimilatory force or assimilatory power (generated in light reactions) of ATP and NADPH. The last phase is the regeneration of the primary acceptor of CO2, RuBP, from triose phosphate through a series of reactions. For each molecule of CO2 fixed, three ATP and two NADPH molecules are required. The first step of C3 photosynthesis is catalyzed by the enzyme ribulose 1,5-bisphosphate carboxylase/ oxygenase (rubisco), which prefers CO2 to the dissolved form of carbon (bicarbonate) as the substrate (Figure 1). Since the concentration of CO2
3-Phosphoglycerate (PGA)
H2O CO2
called C3 plants, while the other two categories include C4 plants and CAM plants.
Rubisco
ADP
PGA kinase
Ribulose 1,5bisphosphate (RuBP)
1,3-Bisphosphoglycerate (BPGA)
NADPH + H+
ADP
Phosphoribulokinase
ATP
Phosphopentose epimerase
GAP dehydrogenase NADP + Pi
Triose -P Xylulose 5-P Dihydroxyacetone-P isomerase Glyceraldehyde
Ribulose 5-P (Ru5P) Phosphopentose isomerase
ATP
(Xu5P)
(DHAP)
3-P (GAP)
Transketolase Aldolase
Ribose 5-P (R5P)
Sedoheptulose 7-P (S7P)
SBPase
Fructose 1,6bisphosphate (FBP) H2O
Pi
FBPase
Pi H2O
Fructose 6-P (F6P)
Sedoheptulose 1,7bisphosphate (SBP) Aldolase
Transketolase
Erythrose 4-P (E4P)
Figure 1 Schematic representation of the Calvin cycle or C3 photosynthesis. The individual enzymes are as follows: rubisco (EC 4.1.1.39); PGA kinase (EC 2.7.2.3); GAP dehydrogenase (EC 1.2.1.13); triose-P isomerase (EC 5.3.1.1); aldolase (EC 4.1.2.13); FBPase (EC 3.1.3.11); SBPase (EC 3.1.3.37); transketolase (EC 2.2.1.1); phosphopentose epimerase (EC 5.1.3.1); phosphopentose isomerase (EC 5.3.1.6); and phosphoribulokinase (EC 2.7.1.19). The major product of the cycle is GAP, which is either exported out of the chloroplast to form sucrose or used within the chloroplast to form starch. A part of the GAP is used for the autocatalysis of the cycle.
PHOTOSYNTHESIS AND PARTITIONING / C3 Plants 675
within the plant cell is quite low (about 10 mmol l 1), the activities of rubisco are rather low and are limited at the normal atmospheric CO2. However, this phenomenon is compensated by the presence of large amounts of rubisco protein, which could be up to 50% of leaf protein. Apart from carboxylation, rubisco also catalyzes oxygenase activity, where O2 reacts with RuBP to form phosphoglycolate, in addition to PGA. The affinity of rubisco for CO2 and O2, at typical in vivo concentrations, is quite similar, and as a result the carboxylase and oxygenase reactions are unavoidable in the present atmospheric levels of O2 and CO2. The next phase of reduction of PGA to triose-P involves three steps: activation of PGA to 1,3bisphosphoglycerate or BPGA (catalyzed by PGA kinase); (2) reduction of BPGA to glyceraldehyde 3-phosphate (GAP) with the help of NADPH (catalyzed by GAP dehydrogenase or GAPDH); and (3) interconversion of GAP and dihydroxyacetone phosphate (DHAP), by the enzyme triose-P isomerase (Figure 1). Both GAP and DHAP are termed triose-P. During the third phase, RuBP (the CO2 acceptor) is regenerated from triose-P through a series of reactions involving condensation and rearrangement. The triose phosphates, GAP and DHAP, are condensed by aldolase to form fructose 1,6-bisphosphate (FBP). This six-carbon sugar is then irreversibly hydrolyzed to fructose 6-phosphate (F6P) by fructose-1,6-bisphosphatase (FBPase). A two-carbon entity is transferred from F6P to GAP by the enzyme transketolase to form xylulose 5-phosphate (Xu5P) and erythrose-4-phosphate (E4P). Then, E4P is combined with DHAP to form sedoheptulose 1,7bisphosphate (SBP), by the enzyme aldolase. This 7-carbon product, SBP, is hydrolyzed by sedoheptulose-1,7-bisphosphatase (SBPase) to yield sedoheptulose 7-phosphate (S7P). Two carbons from S7P are transferred to GAP by transketolase producing Xu5P and ribose-5-phosphate (R5P). The resulting R5P is converted to ribulose-5-P (Ru5P) by phosphopentose isomerase. The two molecules of Xu5P are converted into Ru5P by phosphopentose epimerase. The final step of the regeneration phase is the irreversible conversion (using ATP) of Ru5P to RuBP by phosphoribulokinase (PRK). An important feature of the Calvin cycle is its autocatalysis, where plants are able to reach steadystate rates of photosynthetic conversion of CO2 after a short delay, as the intermediates build up to the appropriate levels. Autocatalysis of the Calvin cycle is facilitated by the cyclic operation of the pathway as well as the controlled removal of the intermediates from the pathway for other purposes.
Products The major product of the Calvin cycle is triose-P (GAP and DHAP) and further metabolism of triose-P is the branching point of the Calvin cycle. The available triose-P is used for conversion into two major products of photosynthesis: (1) starch (a glucose polymer), which accumulates during the day inside the chloroplast; and (2) sucrose, which is formed in the cytosol. Triose phosphate is exported from the chloroplast via the phosphate translocator (on the inner chloroplast envelope membrane) and is further metabolized to sucrose in the cytosol. Sucrose is largely exported from the leaf to different sink tissues, e.g., roots, developing leaves, reproductive organs, and other heterotrophic tissues. Sucrose may also accumulate in the vacuole during the day. Besides the conversion into starch and sucrose, a significant portion of the triose-P is also converted within the chloroplasts to amino acids and fatty acids. Only 1/6 of triose phosphate is utilized/ exported out for various biosynthetic processes, while the other 5/6 is needed to regenerate RuBP.
Rubisco Rubisco is a unique and interesting enzyme, mediating the key reaction of photosynthetic CO2 assimilation: conversion of one molecule of RuBP and one of CO2 into two molecules of PGA (Figure 2). Besides the carboxylation reaction, Rubisco reacts with hv
hv CA1P
RUBISCO Inactive ATP ADP + Pi Rubisco activase
Rubisco activase
Inactive
pH Mg2+ Metabolites
Active CA1P
RUBISCO Active RuBP
Carboxylase
PGA + PGA CO2 O2
P-glycolate + PGA
Oxygenase
Figure 2 The reactions and regulation of rubisco. The enzyme functions as both a carboxylase and an oxygenase. The product of the carboxylase reaction, PGA, is metabolized in the Calvin cycle. One of the products of oxygenase, P-glycolate, is metabolized through photorespiration. Carboxyarabinitol 1-phosphate (CA1P) binds tightly to rubisco and makes it inactive (e.g., in darkness). Rubisco is activated in light in two ways: (1) by rubisco activase, which itself is activated in light through ATP; and (2) changes in the stromal microenvironment (increase in pH, Mg2 þ levels, and Calvin cycle metabolites). Both the processes facilitate the release of CA1P and activation of rubisco.
676 PHOTOSYNTHESIS AND PARTITIONING / C3 Plants
oxygen to form one molecule of 2-phosphoglycolate and one of PGA. The plants try to retrieve part of the carbon diverted into phosphoglycolate through the process of photorespiration or C2 cycle. Photorespiration also helps in the recycling of nitrogen and dissipation of excess energy and reductants (ATP and NADPH) in the chloroplasts. A major drawback of rubisco is its very slow catalytic capacity. The turnover number of the enzyme is extremely small, and such unfavorable kinetic behavior requires large amounts of enzyme (up to 50% of the total soluble protein) to ensure the carbon flux. Rubisco of higher plants is a complex protein, with eight large subunits (mass of about 58 kDa) and eight small subunits (mass of about 18 kDa). The large subunits have the catalytic centers, while the small subunits seem to stabilize the rubisco complex. In cyanobacteria, rubisco is also made up of eight large and eight small subunits, while in purple bacteria, rubisco is a dimer of two large subunits only. The large subunits are encoded by the plastid genome and the small subunits in the nucleus. The regulation of rubisco is described below.
Regulation C3 photosynthesis is regulated in multiple ways, but is primarily initiated by illumination. One of the most important phenomena in the pathway is the light activation of key enzymes, through either dithiol-reduction of cysteines on protein or through changes in the microenvironment (e.g., magnesium or pH levels) of the stroma. Then, changes in substrate availability and metabolite flux rapidly set in motion an efficient autocatalysis of the Calvin cycle. In a long-term mode, the levels and turnover of rubisco protein also modulate the capacity of carbon fixation in the Calvin cycle. For example, limitation of nitrogen availability decreases the levels of rubisco protein and restricts the photosynthetic carbon assimilation in the leaves. Regulation of Rubisco
The activity of rubisco, the first enzyme in the Calvin cycle, is highly regulated. Rubisco is inactive in the dark and is converted to an active form on illumination, which catalyzes fixation of CO2. Activation of rubisco is the result of carbamylation, which involves the binding of CO2 and Mg2 þ to a lysine residue near the catalytic site. Rubisco is active only when lysine-201 reacts with CO2 near the catalytic site to form a carbamate and allows the binding of the Mg2 þ ion. Carbamylation changes the conformation of the large subunit and activates the enzyme, while the active conformation
is stabilized by the formation of a complex with Mg2 þ . Carbamylation is essential for rubisco activation, as the noncarbamylated rubisco binds RuBP too tightly to allow catalysis. Another protein, rubisco activase, is also involved in mediating the light activation of rubisco. On illumination, rubisco activase releases the inhibitor compounds, such as 2-carboxyarabinitol 1-phosphate or CA1P, which are bound to the active site of rubisco; otherwise, for example in darkness, these inhibitors prevent activation (carbamylation) of the enzyme. Rubisco activase itself is activated in light by utilizing ATP produced from photosynthetic electron transport. Rubisco activase and rubisco activation provide another mechanism of strong regulation by light of carbon assimilating reactions of photosynthesis. CA1P, which occurs naturally in the leaves of several plants, is a strong inhibitor of rubisco. The affinity of rubisco for CA1P is much stronger than that for RuBP, the substrate. As a result, CAP, which accumulates in leaves during the night, inactivates rubisco by blocking the binding sites. During the day (or on illumination), the bound CA1P is released from rubisco, and this process is further accelerated by rubisco activase. However, the physiological role of CA1P is still debated, as it is not found in all plant species.
Light Activation of Enzymes Light directly activates five enzymes of the Calvin cycle, namely rubisco, GAPDH, FBPase, SBPase, and PRK. Among these, rubisco is activated both indirectly (due to changes in the microenvironment of the chloroplast stroma) and directly (through rubisco activase). The other four enzymes are activated as a result of the reduction of dithiols on the enzyme. Interestingly, four of the five light-activated enzymes (rubisco, FBPase, SBPase, and PRK) catalyze irreversible reactions. Although the dark activities of these enzymes may vary, the Calvin cycle is essentially inactive until illumination takes place. The Calvin cycle enzymes are activated as a result of the reduction of dithiols on the protein, mediated by a ferredoxin–thioredoxin system. On illumination, ferredoxin is reduced by the thylakoids, which in turn reduce thioredoxin. Reduced thioredoxin can reduce the dithiols at or near the active site of FBPase, SBPase, and PRK, thus activating the enzymes of the Calvin cycle. The thioredoxin– ferredoxin system (reduced in light) also activates two other chloroplastic enzymes, F1-ATP synthase and NADP-malate dehydrogenase. Light not only activates several enzymes, but also inactivates the key enzymes of the oxidative pentose
PHOTOSYNTHESIS AND PARTITIONING / C3 Plants 677
phosphate pathway, again through thioredoxin mediated reduction of enzymes. Glucose-6-phosphate dehydrogenase is one such enzyme, which is inactivated on illumination to ensure that the oxidative pentose phosphate pathway does not operate simultaneously with the reductive pentose phosphate pathway, thus avoiding a futile cycle. Light causes a marked change in the microenvironment of the chloroplast stroma. The photosynthetic electron transport chain in the thylakoid membrane decreases the proton concentration, raises the pH, and increases the Mg2 þ concentration in the stroma. Rubisco, FBPase, and SBPase respond to such changes in pH and Mg2 þ concentrations. These enzymes are almost inactive at pH 7.2 and low Mg2 þ , and their activity increases with the rise in pH and Mg2 þ concentration. The enzymes are also under metabolite control. The stromal enzymes FBPase and SBPase are activated by their substrates and allosterically inhibited by their products. High levels of FBP or SBP activate these enzymes, while F6P or S7P inhibit the activity of the corresponding enzyme, to avoid excessive product accumulation and sequestration of phosphate. Similarly, PRK is inhibited by 3-PGA and ADP. As both these metabolites inhibit PRK, phophorylation by PGA kinase can proceed even under limiting ATP supply, thus preventing the accumulation of PGA. Multiprotein Complexes
The occurrence and operation of multienzyme complexes involving Calvin cycle enzymes have been reported. Such complexes could be an important mode of regulation. For example, GAP and PRK interact with a small nuclear encoded chloroplast protein, CP12, and form a complex, which may be an additional mechanism for light regulation of PPK in vivo. Complexes involving different combinations of C3-enzymes have been found, and these may facilitate rapid channeling of intermediates between enzymes, improving the efficiency of the cycle. Multienzyme complexes of phosphoribose isomerase, PRK, GAPDH, and SBPase have been located on the thylakoid membrane by immunoelectron microscopy. A complex of sucrose phosphate synthase and sucrose phosphate phosphatase has also been reported. Flux Control
Flux control is an important concept, and the relative importance of individual enzymes in controlling the flux of carbon fixation through the Calvin cycle has been examined. Studies of transgenic plants with altered levels of individual enzymes in the C3 cycle
have revealed that no single enzyme has complete control of carbon fixation. Control is shared among a number of enzymes, with rubisco, SBPase, and aldolase having the most prominent roles. Enzymes with highly regulated activity, such as PRK, do not seem to have strong control on carbon fixation, whereas aldolase, which catalyzes a reversible reaction, strongly influences the rate of carbon fixation.
C3 Plants: Limitations and Prospects for Improvement C3-photosynthesis is a feature of even the primitive lower groups and appears to have evolved much earlier than the CAM or C4 pathway. Of about 300 000 plants known on earth, B90% are C3 plants, while the CAM and C4-species constitute about 10% and 1%, respectively. Furthermore, most crops (particularly cereals, legumes, and oilseed crops) are of the C3-type; therefore, C3 plants have attracted the attention of several scientists. The performance and productivity of C3 plants is restricted by at least three major factors: high photorespiration (a nonavoidable consequence of oxygenase activity of rubisco), a high water requirement, and a preference for temperate regions. Attempts were made to evolve varieties with reduced photorespiration or high photosynthetic rates, but single features were not able to improve the productivity of plants. Another approach was to introduce a set of C4 traits into C3 plants, but hybridization of C3 and C4 species of Atriplex resulted in only C3-type plants. Mutants deficient in one or more enzymes of photorespiration were unable to grow at atmospheric levels of CO2, indicating that the process of photorespiration (as well as rubisco oxygenase activity) was an adaptation to the present atmospheric levels of CO2/O2. However, a very well known factor that could improve the photosynthetic performance and productivity of C3 plants is elevated CO2.
Molecular Biology and Biotechnology The research before the 1950s involved studies mainly on the physiology (e.g., Blackman’s law of limiting factors) and biochemistry (e.g., Calvin cycle) of C3-photosynthesis. By the end of the twentieth century, focus had shifted onto the molecular biology of chloroplasts (and their components) and biotechnology to manipulate some of the proteins/enzymes. Genes
In the past decade, enormous progress has been made in identifying the genes involved in not only
678 PHOTOSYNTHESIS AND PARTITIONING / C3 Plants
photosynthesis but also other metabolic processes. The genomes of plants such as Arabidopsis and rice (Oryza sativa) have been completely sequenced. With reference to photosynthesis, the initial work was carried out with genes encoding the components of photosynthetic electron transport, but later covered most of the genes encoding the enzymes of the Calvin cycle and related processes. The genes encoding the enzymes of the Calvin cycle, with the exception of the large subunit of rubisco, are located in the nuclear genome; the large subunit of rubisco is encoded by genes in the chloroplast. The preproteins are synthesized in the cytosol with N-terminal extensions called transit sequences, which direct them to the chloroplasts, where they are cleaved to form mature proteins. In recent years, cloning and analysis of the genes encoding the enzymes of the Calvin cycle have facilitated further studies on the control of their expression. Regulation of Gene Expression
There is a perfect coordination of mRNA synthesis and protein accumulation during leaf development and chloroplast maturation. Much of the control has been found at the level of gene transcription. Light has a central role in regulating the biogenesis of the photosynthetic apparatus during chloroplast development, including switching on the expression of genes encoding enzymes of C3 photosynthesis. These
processes appear to be complex, involving many regulatory proteins and cross-talk between the different photoreceptor signaling pathways. The expression of C3 photosynthesis genes in leaves is also modulated by environmental and metabolic signals. High levels of glucose or sucrose reduce levels of a number of Calvin cycle mRNAs, including those encoding the small subunit of rubisco, SBPase, and FBPase. This feedback mechanism, acting at the level of transcription, might be important for source–sink regulation in the plant. Photosynthesis genes respond remarkably to nutrient status, particularly nitrogen and phosphorus levels, and this is influenced again by the carbohydrate status. Thus, an interaction between carbohydrate and nutrient status may be a long-term strategy to control carbon metabolism. Genetic Manipulation
Several enzymes of the Calvin cycle have been genetically engineered in plants (Table 1). Attempts have been made to either overexpress the target enzyme or suppress the enzyme levels. The overexpression of these enzymes causes only a small improvement in the overall performance of the plant. However, the suppression of photosynthetic enzymes invariably led to a marked decrease in photosynthetic rate. A reduction in the levels of rubisco, rubisco activase, and NADP-GAPDH resulted in a significant
Table 1 Examples of genetic manipulation of enzymes or proteins involved in photosynthesis of C3 plants; the effect (suppression or overexpression) of the target enzyme/protein, on the test plant and the consequence of such genetic manipulation are indicated Target enzyme or protein/test plant
Effect
Consequence
Rubisco/tobacco
Suppression
Rubisco activase/tobacco
Suppression
GAP dehydrogenase/tobacco
Suppression
Aldolase/potato (Solanum tuberosum) Transketolase/tobacco
Suppression
Marginal effect on photosynthesis at high N and moderate light; significant limitation on photosynthesis at limiting N and high light Reduction in activation/carbamylation of rubisco in light; photosynthesis decreased at both low and high CO2 No significant reduction in growth; increase in PGA indicating the export and reduction of PGA in cytosol Strong reduction in growth
Sucrose-P synthase/ Arabidopsis Phosphate translocator/potato
Suppression Suppression Suppression
Small decrease in transketolase led to marked decrease in growth, photosynthesis, and phenyl propanoid metabolism Decrease in sucrose synthesis as well as photosynthetic rate No effect on overall rate of carbon fixation, but increased starch accumulation in leaves in light Enhanced photosynthesis and growth; increased allocation of carbon into sucrose
Single cyanobacterial FBP/ SBPase/tobacco
Overexpression
Mutated form of sucrose 6-P synthase (to avoid downregulation)/tobacco
Overexpression
High sucrose-P synthase activity and stimulation of sucrose synthesis
Rat liver 6-phosphofructo-2kinase/tobacco
Overexpression
Higher level of fructose-2,6-bisphosphate; sucrose synthesis reduced at the beginning of light period
PHOTOSYNTHESIS AND PARTITIONING / C3 Plants 679
decrease in photosynthesis, particularly at high light intensity. The manipulation of stromal FBPase or triose-P/Pi translocator affected the growth pattern only marginally, but significantly changed the pattern of assimilate translocation. The above genetically modified plants were obviously trying to compensate for the deficit of each enzyme/protein; such is the adaptive capacity of plants, due to their metabolic flexibility. In plants, FBPase and SBPase are separate enzymes, while in cyanobacteria, a single enzyme (FBP/SBPase) performs both of their functions. Overexpression of the single cyanobacterial enzyme from Synechococcus enhanced the photosynthesis and growth of tobacco (Nicotiana tabacum) plants.
aralocaspica, where CO2 concentrating mechanisms or C4 photosynthesis operates within a single cell. It would be quite exciting to analyze these plants and exploit the extracted information. With the introduction of an efficient CO2 concentrating mechanism, the performance of C3 plants is bound to improve.
Future Outlook
List of Technical Nomenclature
The performance of C3 plants improves considerably when the atmospheric CO2 level is elevated. Thus, under conditions of global warming with its elevated CO2 levels, the use and cultivation of C3 plants offer much greater potential than that of C4 plants. However, the C3 plants may require additional nutrients under high CO2. This aspect needs to be studied further so that the C3 plants can be exploited effectively under elevated CO2. Despite detailed studies, the functional mechanism of rubisco continues to be a marvel and a mystery. Further experiments are warranted to understand the conditions that would ensure the activated state of rubisco. Specific alterations in the amino acid residues with the help of site-directed mutagenesis may be of interest to achieve this target. An increase in the ratio of carboxylase to oxygenase of rubisco would be of immense potential to increase the photosynthetic efficiency. However, neither extensive screening of higher plant species and their cultivars, nor the use of different metabolites/inhibitors helped to alter the ratio of carboxylase/oxygenase activity. However, some of the plants from lower groups (particularly red algae) have been found to possess rubisco with a much higher carboxylase to oxygenase ratio than that of higher plants. These forms of rubisco are termed ‘‘super rubiscos,’’ in view of their vast potential. Attempts are currently being made to incorporate ‘‘super rubisco’’ into higher plants. Another possibility of improving the performance of C3 plants is to supplement with CO2 concentrating mechanisms. Nature has already achieved this in C4 and CAM plants. An encouraging and important step in this direction is the success of expressing corn (Zea mays; maize) phosphoenolpyruvate carboxylase (PEPC) in rice leaves to achieve 4100-fold increase in the activity of PEPC. There are plants, such as Hydrilla verticillata, Egeria densa, and Borszczowia
Carbon assimilation
Conversion of simple inorganic carbon dioxide into complex organic carbon compounds, enriched with energy, such as sugars.
Chloroplast stroma
Soluble compartment of chloroplasts, enclosed by the envelope membranes, and contains several biosynthetic enzymes, metabolites, nucleic acids and lipid granules.
Gene transcription
The passing of genetic message contained in DNA to RNA, facilitating the translation of information into assembly of a specific chain of amino acids and biosynthesis of proteins.
Sink
The tissues which import and utilize the assimilated organic compounds, e.g. young developing parts, growing fruits, or storage organs, such as tubers.
Source
The organ which makes and supplies the energy-rich organic compounds, usually the leaf.
Acknowledgments The preparation of this manuscript and current work on photosynthesis in our laboratory is supported by grant No. SP/SO/A12/98 from the Department of Science and Technology, New Delhi.
See also: Genetic Modification of Primary Metabolism: Photosynthesis. Photosynthesis and Partitioning: C4 Plants; CAM Plants; Photoinhibition.
Further Reading Blankenship RE (2002) Molecular Mechanisms of Photosynthesis. London: Blackwell Science. Grossman AR (2000) Chlamydomonas reinhardtii and photosynthesis: Genetics to genomics. Current Opinion in Plant Biology 3: 132–137. Gutteridge S and Jordan DB (2001) Dynamics of photosynthetic CO2 fixation: Control, regulation and productivity. In: Aro E-M and Andersson B (eds) Regulation of Photosynthesis, pp. 297–312. Dordrecht: Kluwer Academic Publishers.
680 PHOTOSYNTHESIS AND PARTITIONING / C4 Plants Heldt HW (1997) Plant Biochemistry and Molecular Biology. Oxford: Oxford University Press. Lawlor DW (2001) Photosynthesis: Molecular, Physiological and Environmental Processes, 3rd edn. Berlin: Springer Verlag. Leegood RC (1999) Photosynthesis in C3 plants: The Benson–Calvin cycle and photorespiration. In: Lea PJ and Leegood RC (eds) Plant Biochemistry and Molecular Biology, 2nd edn, pp. 29–50. Chichester: John Wiley & Sons. Malkin R and Niyogi K (2000) Photosynthesis. In: Buchanan BB, Gruissem W, and Jones RL (eds) Biochemistry and Molecular Biology of Plants, pp. 568–628. Rockville, MD: American Society of Plant Physiologists. Martin W, Scheibe R, and Schnarrenberger C (2000) The Calvin cycle and its regulation. In: Leegood RC, Sharkey TD, and Von Caemmerer S (eds) Photosynthesis: Physiology and Metabolism, pp. 9–51. Dordrecht: Kluwer Academic Publishers. Quick WP and Neuhaus HE (1997) The regulation and control of photosynthetic carbon assimilation. In: Foyer CH and Quick WP (eds) A Molecular Approach to Primary Metabolism in Higher Plants, pp. 41–62. London: Taylor and Francis. Rao KK and Hall DO (1999) Photosynthesis, 6th edn. Cambridge: Cambridge University Press. Sharkey TD (1998) Photosynthetic carbon reduction. In: Raghavendra AS (ed.) Photosynthesis: A Comprehensive Treatise, pp. 111–122. Cambridge: Cambridge University Press. Spreitzer RJ and Salvucci ME (2002) Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme. Annual Review of Plant Physiology and Plant Molecular Biology 53: 449–475. Stitt M (1996) Metabolic regulation of photosynthesis. In: Baker NR (ed.) Photosynthesis and the Environment, pp. 151–190. Dordrecht: Kluwer Academic Publishers. Von Caemmerer S and Quick WP (2000) Rubisco: physiology in vivo. In: Leegood RC, Sharkey TD, and Von Caemmerer S (eds) Photosynthesis: Physiology and Metabolism, pp. 85–113. Dordrecht: Kluwer Academic Publishers.
C4 Plants R C Leegood, University of Sheffield, Sheffield, UK Copyright 2003, Elsevier Ltd. All Rights Reserved.
Introduction Plants with the C4 pathway of photosynthesis, typically grasses from warm habitats, are responsible for about a quarter of global terrestrial productivity. The C4 mechanism involves a division of labor
between two different photosynthetic cell types, the mesophyll and the bundle sheath. The CO2-concentrating function of C4 photosynthesis concentrates CO2 at the site of Rubisco in the bundle sheath, providing a mechanism that overcomes the photorespiration incurred by the oxygenase activity of Rubisco, and which accounts for losses of around a quarter of the recently fixed carbon in C3 plants, particularly at higher temperatures. The C4 pathway also results in improved water and nitrogen-use efficiency compared with C3 plants. In a sense, C4 plants were discovered, but not recognized as such, in the nineteenth century, when Haberlandt described their distinctive Kranz (German, ‘‘wreath’’) anatomy: a distinct layer of cells rich in chloroplasts surrounding the vascular bundle (Figure 1). The true nature of these plants did not become apparent until the 1960s, from work by a Russian, Karpilov, on corn (Zea mays, maize), and by Kortschak and colleagues on sugar cane (Saccharum officinarum) in Hawaii, who reported that the first products of photosynthesis were malate and aspartate, and not glycerate 3-P, as would be expected from CO2 fixation via the C3 Benson– Calvin cycle. In the mid-1960s, Hatch and Slack, in Australia, elucidated the path of carbon in C4 photosynthesis.
Agricultural and Ecological Importance Although the number of terrestrial plant species that are C4 is quite modest (about 3%), about 50% of the grasses are C4. Corn is one of the three principal cereal crops, together with wheat (Triticum aestivum) and rice (Oryza sativa) (both C3 plants), each of which constitutes about 30% of total global cereal production (Table 1). Other important C4 grass crops include millet (Panicum miliaceum), Sorghum spp., sugar cane, and pasture grasses (Poaceae). C4 plants have two primary requirements for success: a warm growing season and high light, although aridity may also influence their distribution. Fourteen out of 18 of the world’s worst weeds are C4. C4 grasses are the dominant (490%) components of warm temperate and tropical grasslands, such as prairies and savannas. In prairies, C3 grasses tend to be active during the spring, whereas C4 grasses tend to be active during the summer, when temperatures are higher and soil water lower. Similarly, decreases in temperature with altitude tend to favor C3 grasses. Other biomes with a high representation of C4 plants, particularly at low (tropical) latitudes, include deserts and semideserts, arid steppes and salt deserts, beach dunes and saltmarshes, wetlands, and disturbed ground in arid regions.