The Path from C3 to C4 Photosynthesis

Abstract

The C4 photosynthetic carbon cycle is an elaborated addition to the C3 photosynthetic pathway. It evolved as an adaptation to high light intensities, high temperatures, and dryness. Therefore, C4 plants dominate grassland floras and biomass production in the warmer climates of the tropical and subtropical regions (Edwards et al., 2010). In all plants CO2 is fixed by the enzyme Rubisco. It catalyzes the carboxylation of ribulose-1,5-bisphosphate, leading to two molecules of 3-phosphoglycerate. Instead of CO2, Rubisco can also add oxygen to ribulose-1,5-bisphosphate, resulting in one molecule each of 3-phosphoglycerate and 2-phosphoglycolate. Phosphoglycolate has no known metabolic purpose and in higher concentrations it is toxic for the plant (Anderson, 1971). It therefore has to be processed in a metabolic pathway called photorespiration. Photorespiration is not only energy demanding, but furthermore leads to a net loss of CO2. Thus the efficiency of photosynthesis can be decreased by 40% under unfavorable conditions including high temperatures and dryness (Ehleringer et al., 1991). The unfavorable oxygenase reaction of Rubisco can be explained as a relict of the evolutionary history of this enzyme, which evolved more than 3 billion years ago when atmospheric CO2 concentrations were high and oxygen concentrations low. Apparently, later on, it was impossible to alter the enzyme’s properties or to exchange Rubisco by another carboxylase. Nevertheless, plants developed different ways to cope with this problem. Perhaps the most successful solution was C4 photosynthesis. The establishment of C4 photosynthesis includes several biochemical and anatomical modifications that allow plants with this photosynthetic pathway to concentrate CO2 at the site of Rubisco. Thereby its oxygenase reaction and the following photorespiratory pathway are largely repressed in C4 plants. In most C4 plants the CO2 concentration mechanism is achieved by a division of labor between two distinct, specialized leaf cell types, the mesophyll and the bundle sheath cells, although in some species C4 photosynthesis functions within individual cells (Edwards et al., 2004). Since Rubisco can operate under high CO2 concentrations in the bundle sheath cells, it works more efficiently than in C3 plants. Consequently C4 plants need less of this enzyme, which is by far the most abundant protein in leaves of C3 plants. This leads to a better nitrogen-use efficiency of C4 compared to C3 plants, since the rate of photosynthesis per unit nitrogen in the leaf is increased (Oaks, 1994). Additionally C4 plants exhibit better water-use efficiency than C3 plants. Because of the CO2 concentration mechanism they can acquire enough CO2 even when keeping their stomata more closed. Thus water loss by transpiration is reduced (Long, 1999). In the mesophyll cells of C4 plants CO2 is converted to bicarbonate by carbonic anhydrase and initially fixed by phosphoenolpyruvate (PEP) carboxylase (PEPC) using PEP as CO2 acceptor. The resulting oxaloacetate is composed of four carbon atoms, which is the basis for the name of this metabolic pathway. Oxaloacetate is rapidly converted to the more stable C4 acids malate or Asp that diffuse to the bundle sheath cells. Here, CO2 is released by one of three different decarboxylating enzymes, which define the three basic biochemical subtypes of C4 photosynthesis, NADP-dependent malic enzyme (NADP-ME), NAD-dependent ME (NAD-ME), and PEP carboxykinase (PEPCK). The released CO2 is refixed by Rubisco, which exclusively operates in the bundle sheath cells in C4 plants. The three-carbon compound resulting from CO2 release diffuses back to the mesophyll cells where the primary CO2 acceptor PEP is regenerated by pyruvate orthophosphate dikinase by the consumption of, at the end, two molecules of ATP (Hatch, 1987). Figure 1 shows a scheme of the NADP-ME subtype of C4 photosynthesis. Here malate is the dominant transport metabolite while Asp can be used in parallel. The synthesis of malate occurs in the mesophyll chloroplasts, the decarboxylation by NADP-ME in the bundle sheath chloroplasts. NADP-ME type of C4 photosynthesis. 3-PGA, 3-Phosphoglyceric acid; AspAT, Asp aminotransferase; AlaAT, Ala aminotransferase; CA, carbonic anhydrase; MDH, malate dehydrogenase; OAA, oxaloacetate; PPDK, pyruvate orthophosphate dikinase; TP, triosephosphate. The two other biochemical subtypes differ from the NADP-ME type by the transport metabolites used and the subcellular localization of the decarboxylation reaction. In NAD-ME plants Asp, which is synthesized in the mesophyll cytosol, is used as transport metabolite. After deamination and reduction, the resulting malate is decarboxylated by NAD-ME in the bundle sheath mitochondria. Plants of the PEPCK type use Asp as well as malate as transport metabolites. Asp is synthesized in the cytosol of mesophyll cells and decarboxylated in the cytosol of bundle sheath cells by the combined action of Asp amino transferase and PEPCK. As in NADP-ME-type C4 species, malate is synthesized in the mesophyll chloroplasts but decarboxylated by NAD-ME in the mitochondria of bundle sheath cells. This reaction produces NADH that is used in the mitochondria to produce the ATP needed to drive the PEPCK reaction (Hatch, 1987). If Asp is used as transport metabolite, usually the three-carbon decarboxylation product, pyruvate, is partially transported back to the mesophyll cells in the form of Ala to maintain the ammonia balance between the two cell types (Hatch, 1987). Compared to C3 plants the bundle sheath cells of C4 plants have expanded physiological functions. This is reflected by the enlargement and a higher organelle content of these cells in most C4 species. For the efficient function of the C4 pathway a close contact between mesophyll and bundle sheath cells is indispensable and they are tightly interconnected to each other by high numbers of plasmodesmata (Dengler and Nelson, 1999). To ensure a direct contact between bundle sheath and mesophyll cells, C4 plants possess a characteristic leaf anatomy. The bundle sheath cells enclose the vascular bundles and are themselves surrounded by the mesophyll cells. The high vein density in the leaves of C4 plants leads to a nearly one-to-one ratio of the volumes of mesophyll and bundle sheath tissues. The internal anatomy of a C4 leaf is often composed of a repeating pattern of vein-bundle sheath-mesophyll-mesophyll-bundle sheath-vein. Because of its wreath-like structure this type of leaf anatomy was termed Kranz anatomy by the German botanist G. Haberlandt (1904). Kranz anatomy is found with more or less considerable variations in nearly all monocotyledonous and dicotyledonous lineages that use the two-cell mode of C4 photosynthesis. While the above differences are directly related to the CO2 concentration mechanism, there are many further modifications known that evolved to integrate the C4 pathway optimally into the plant’s metabolism. For instance, C4 species of the NADP-ME subtype are depleted in PSII in their bundle sheath cells to lower oxygen production in these cells. Accordingly, the production of reduction equivalents in the bundle sheath cells is reduced and the reduction phase of the Calvin-Benson cycle, i.e. the conversion of 3-phosphoglycerate to triose phosphate, has been at least partially shifted to the mesophyll cells (Fig. 1). There is another adaptation in C4 plants that affects the light reactions of photosynthesis. Compared to C3 photosynthesis the C4 pathway consumes one (PEPCK type) or two (NADP-ME and NAD-ME type) additional molecules of ATP per fixed CO2 without the need of additional reduction equivalents. This increase in ATP-to-NADPH ratio is compensated for in some C4 plants by enhancing cyclic electron flow around PSI, which provides additional ATP without concomitantly producing NADPH. Large-scale trancriptomic and proteomic approaches also revealed that other metabolic pathways such as amino acid synthesis, nitrogen or sulfur assimilation, and lipid metabolism are compartmentalized between mesophyll and bundle sheath cells in at least some C4 plants (Majeran and van Wijk, 2009). C3 angiosperms evolved more than 50 times independently into C4 plants (Muhaidat et al., 2007). Most of the C4 species occur in the grasses (approximately 4,600) and sedges (approximately 1,600). Only a total of about 1,600 C4 species are found in the dicots where they are spread over 16 families with 75% of them clustering in the four families Chenopodiaceae, Amaranthaceae, Euphorbiaceae, and Asteraceae (Muhaidat et al., 2007). C4 grasses probably evolved in the early Oligocene about 30 million years ago, while C4 dicots appeared later, less than 20 million years ago (Sage, 2004). The polyphyletic origin of C4 photosynthesis indicates that only relatively small evolutionary changes were required for the establishment of this photosynthetic pathway. It can be assumed that C4 evolution must have been easy in genetic terms. This raises the question of whether we can use the information about the genetic architecture and evolution of this pathway and introduce modules of C4-ness into present C3 plant and thereby transform them into C3-C4 intermediate or even C4-like plants (Sheehy et al., 2007). The currently most widely accepted model of C4 evolution proposes a stepwise sequence of changes leading from C3 to C4 plants (Fig. 2). Each of these changes on its own is leading to a distinct evolutionary benefit for the resulting species independent of whether it will progress toward the full expression of the C4 syndrome. This scenario explains why the evolution of this complex trait could occur so many times independently. The model is mainly based on comparative analyses of extant C3, C4, and especially C3-C4 intermediate species, and a detailed elaboration can be found in Sage (2004). Here, we only present a short summary and elucidate how the evolutionary changes might have been realized through modifications at the molecular/genetic level. Stepwise evolution of C4 photosynthesis. It is thought that the existence of many redundant genes in the genomes of the relevant organisms and species was a general prerequisite for C4 evolution (Monson, 2003). These gene redundancies have been acquired by duplications of whole genomes, genome segments, or only single genes. Multiple copies of a gene allow evolutionary modifications of one copy without losing the original function of the gene itself. Thus redundant gene copies prevent deleterious consequences of evolutionary changes that alter or switch off the specific function of a certain gene. In further steps, leaves have been altered toward Kranz anatomy, a photorespiratory CO2 pump was established, and finally a C4 cycle was created. All these steps were accompanied by massive changes in gene regulation. Also the kinetic properties of enzymes, involved in metabolic pathways that were affected by these evolutionary changes, were adjusted to the new requirements (Fig. 2). The first step toward C4 evolution was the development of the Kranz anatomy. To establish a mechanism that efficiently concentrates CO2 in bundle sheath cells the mean distance of a mesophyll cell to the next bundle sheath cell must be as short as possible. Ideally each mesophyll cell should be directly adjacent to at least one bundle sheath cell. Therefore, in planar leaves the vein density had to be enhanced. A higher vein density increased also the mechanical integrity of the leaves, which could be beneficial in windy habitats, or improved the water supply of leaves in dry and hot biotopes (Sage, 2004). In succulent terete or semiterete leaves, evolution of C4 occurred in some dicots with development of a single Kranz unit surrounding the vascular and water storage tissue (Edwards et al., 2004). A comparative analysis of the leaf development in both monocot and dicot C3 and C4 species revealed that the close vein spacing in leaves of C4 plants is due to changes in the initiation frequency and pattering of the minor and not the major veins (Ueno et al., 2006; McKown and Dengler, 2009). In Arabidopsis (Arabidopsis thaliana) the formation of veins from ground tissue is triggered by polar flow by the transport to cells and later on into vascular bundles et al., modifications of production and modifications of the of ground tissue cell to cells are for the vein density in C4 compared to C3 leaves and Dengler, 2009). Since the the initiation of veins are not even in C3 model plants, it is to the changes that to the C4 leaf anatomy. The of bundle sheath enlargement of these cells and the increase in the of in this tissue might be a of the higher vein the bundle sheath cells of C3 plants possess only a chloroplasts, and the photosynthetic is low. higher vein also the ratio of bundle sheath to mesophyll cells Since only the mesophyll cells high photosynthetic this that the photosynthetic of a leaf with a The evolutionary to maintain the photosynthetic could have to an increase of the of chloroplasts in the bundle sheath cells. to the to the photorespiratory in bundle sheath cells the increase of numbers also an increase in the numbers of mitochondria and in these cells. C3-C4 intermediate species possess a photorespiratory that CO2 into the bundle sheath cells 2010). This is achieved by the decarboxylation reaction to the bundle sheath all by in the mesophyll has to be to the bundle sheath cells for further The affects photosynthetic CO2 in two All photorespiratory CO2 is the leaf far from the it has to diffuse through several cell it could from the This the plant’s of the CO2 and the loss of carbon due to photorespiration. In some C3-C4 intermediate species this is by the of the within the bundle sheath since the mitochondria concentrate adjacent to the vascular bundles et al., the the CO2 concentration within the bundle sheath cells. As a the carboxylation of Rubisco in the bundle sheath cells while its oxygenase reaction is 2010). It is assumed that the establishment of such a photorespiratory CO2 pump is an intermediate step on the toward C4 photosynthesis. A photorespiratory CO2 pump can be at the level. The expression of only one a of the had to be to the bundle sheath cells. This might have been achieved through relatively changes in the that the expression of these genes with et al., 2007). In where several with different leaf expression in the C3 species this might also have the of that are not bundle sheath In the C3-C4 intermediate species for only the of is to the bundle Since the enzyme is without this be decarboxylated in the mesophyll et al., For other C3-C4 from the and it was found that also the other genes were or at least in the bundle sheath cells et al., It that Kranz anatomy and bundle sheath cells with increased of were established, a photorespiratory CO2 pump could be achieved in genetic terms. The photorespiratory CO2 pump and the resulting CO2 content in the bundle sheath cells might have to a further increase in organelle numbers in these cells (Sage, 2004). The next step toward C4 photosynthesis might have been an increase in the of carbonic anhydrase and in the cytosol of the mesophyll cells. This have in the photorespiratory CO2 that from the bundle sheath into the mesophyll cells. Also this evolutionary step is reflected by C3-C4 intermediate species of the which higher in and protein as compared to C3 species but not exhibit C4 cycle et al., et al., 2003). To establish a C4 cycle the C4 cycle must have been at this It is known that even in C3 plants the of the decarboxylating NADP-ME and NAD-ME is increased in vascular and Thus the expression of the related genes must have been shifted to the bundle sheath cells. To the C4 cycle the expression of pyruvate orthophosphate dikinase must have been to allow an efficient PEP Plants in this phase of C4 evolution exhibit high of C4 cycle enzymes, but high Rubisco in the mesophyll cells. CO2 is only partially fixed through the C4 pathway. The step in C4 photosynthesis and to integrate the C4 pathway and the Calvin-Benson cycle was the of the two carboxylation was to the mesophyll and Rubisco to the bundle sheath cells. This step when the C4 cycle increased to such a that CO2 by the as by Rubisco and the C4 and the Calvin-Benson cycle for CO2 and ATP (Monson, 1999). the of the CO2 initially through the C4 cycle it was fixed by Rubisco. The C4 pathway was further by other of both the C4 and Calvin-Benson by the light reaction of photosynthesis and by carbonic anhydrase in the cytosol of mesophyll cells. The C4 photosynthetic pathway is by the of metabolites between the and the cytosol within mesophyll and bundle sheath cells, The evolution of this required also the establishment of the transport In plants of the NADP-ME for for molecule of CO2 one molecule of pyruvate and oxaloacetate each have to be transported into the mesophyll chloroplasts and in a PEP and malate have to be to the In bundle sheath cells, on the other malate has to and pyruvate has to the the rate of CO2 Large-scale and analyses that also other pathways related to and carbon metabolism were with to or to sheath et al., et al., This was most due to differences in the supply of energy and reduction equivalents in the different and to the of the metabolic The evolution of C4 photosynthesis was accompanied by massive changes in gene the of leaves of the C4 plant and the related C3 species were compared by a gene expression et al., of the in their between the two species. As to be the expression of genes involved in the C4 cycle, the photorespiratory and the photosynthetic light reactions several other pathways in their For instance, the C4 reduced in with one carbon compound the and amino acid metabolism et al., Most genes of the and protein synthesis are in the C4 species. in the C4 leaf are for genes involved in synthesis, and nitrogen metabolism et al., C4 evolution required changes in the gene expression et that in about of the genes are between mesophyll and bundle sheath cells. to the et al., only less than of the genes of are between the mesophyll and bundle sheath cells of this C3 This indicates that the establishment of C4 photosynthesis involved a and of leaf functions. Most of the evolutionary leading to the and changes in gene are not at the and only a have been in These that appeared to have been in the i.e. different genes were altered in different ways to them for their function in the C4 pathway and 2010). gene expression can be achieved by For instance, the expression of the photosynthetic of the C4 plant on a the which is about of the site et al., 2004). A was also found in the of the genes from C3 these the to direct mesophyll Accordingly, modifications within a were to a gene with no expression into a gene et al., 2007). In the bundle expression of one of the genes the small of Rubisco in the C4 plant was to be mainly at the et al., Most the are stable in mesophyll and bundle sheath cells. This is by that are in the and regions of the et al., The massive changes in gene expression the from C3 to C4 photosynthesis combined with the that C4 evolution must have been easy in genetic that gene in C3 plants were probably the for evolutionary changes toward C4 photosynthesis with In C3 plants gene that a of genes involved in photosynthesis and related metabolic pathways and or bundle gene expression in C4 species maintain their cell of expression in C3 species et al., et al., that the gene the development and of mesophyll and bundle sheath cells of C4 plants are not different from of C3 species. for and metabolic in C3 angiosperms and could as a for the establishment of C4 leaf anatomy and metabolism. of gene the development and anatomy of a leaf of a C3 is the above we about the of that gene expression in the mesophyll and bundle sheath cells of both C3 and C4 plants. The and are the only This of occurs in all plants. In Arabidopsis the are largely redundant and the expression of more than genes that are mainly with photosynthesis. In the mesophyll and bundle sheath of the C4 species the two genes are with only development in the bundle sheath cells and 2009). Thus the to be an of the gene of sheath in the C4 plant All C4 cycle evolved from To ensure high through the C4 the concentration of and metabolites is as compared to the original metabolic in the C3 species. Accordingly, the evolution of the C4 involved changes in their kinetic and The C4 of is the for these evolutionary and 2010). C4 PEP with a lower than the while their to the other i.e. is The C4 are more toward the Asp and malate and are more affected by the or These differences in properties were achieved by relatively small changes in primary enzyme The of from and amino acid and was used as an to some of the evolutionary changes at the amino acid of and 2004). The changes to be to certain that are by the enzyme’s The lower for the PEP is related to an Ala to exchange in the of the enzyme et al., This amino acid exchange is found in all C4 so far but not in or acid metabolism and 2010). the C4 of the grasses these to be even more C4 evolved at least times independently within the the resulting a high of A was found for amino acid et al., 2007). Only two of the amino acid that are under in are also for the evolution of dicot C4 This could requirements for C4 when compared to dicot C4 this might also the that most of the dicot C4 lineages are compared to the first of C4 photosynthesis within the grasses (Ehleringer et al., 2004). that the C4 of the are more for their in C4 photosynthesis than their dicot This might the higher of within the photosynthetic of the The C4 NADP-ME also acquired kinetic and properties their evolution from enzyme regions could be that are involved in an altered by malate and differences in of the enzyme et al., 2007). of C4 to the new metabolic of the C4 pathway could also a in the of the The photosynthetic carbonic anhydrase gene of is a for this The gene is in the mesophyll cells et al., and evolved from an gene that a carbonic to a in the of the enzyme, the C4 a enzyme et al., 2009). this carbonic anhydrase gene was in leaves, that the localization of the protein was of minor and altered It is not so far to which other enzymes, which are not directly related to the C4 were C4 The of the will massive in the energy from plant biomass is developed to energy and might with production for and in the It will be a to increase production in a both in of and total C4 plants exhibit high photosynthetic and efficient use of nitrogen and water have an in years and the of C4 photosynthesis into C3 is (Sheehy et al., 2007). there are under to a concentration pathway into the most for to about the genetic architecture of C4 photosynthesis and the gene is a prerequisite to be successful in this To elucidate these different approaches are with and as well as approaches are to genes that are related to C4 a reduced CO2 high vein or bundle sheath cells. The analysis of the and of different of C4 leaves will to how C4 leaf and the establishment of Kranz anatomy are the of related C3 and C4 species from or et al., will the evolutionary of C4 photosynthesis and the gene that is required for the of a C3 into a C4 The successful of these different the of the of C4 and the of a of how the C3 plant must be altered to introduce the C4 pathway should a in the relatively of are to an to this