Update on C 4 Evolution The Path from C 3 to C 4 Photosynthesis 1

C4 PHOTOSYNTHESIS 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), NADdependent 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. 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 1 This work was supported by the Deutsche Forschungsgemeinschaft through the Collaborative Research Center SFB590 and the Research Group FOR1186, and by the Bill and Melinda Gates Foundation in the framework of the C4 Rice Project. * Corresponding author; e-mail gowik@uni-duesseldorf.de. www.plantphysiol. org/cgi/doi/10.1104/pp.110. 165308