The first sections of this chapter dealt primarily with the characteristics of photosynthesis of C3 species. There are also species with photosynthetic characteristics quite different from these C3 plants. These so-called C4 species belong to widely different taxo-nomic groups (Table 7); the C4 syndrome is very rare among tree species; Chamaesyce olowaluana (Euphorbiaceae) is a canopy-forming C4 tree from Hawaii (Sage 2004). Although their different anatomy has been well documented for over a century, the biochemistry and physiology of C4 species has been elucidated more recently. It is hard to say who first "discovered" the C4 pathway of photosynthesis
Table 8. Main differences between the three subtypes of C4 species.*
Major substrate moving from
Major decarboxylase Decarboxylation Photosystems
Subtype in BSC occurs in MC to BSC BSC to MC in BSC
NADP-ME NADP-malic enzyme Chloroplast Malate Pyruvate I and IIa
NAD-ME NAD-malic enzyme Mitochondria Aspartate Alanine I and II
PCK PEP carboxykinase Cytosol Aspartate + malate Alanine + PEP I and II
*MC is mesophyll cells; BSC is vascular bundle sheath cells.
aSome NADP-ME monocots, including Zea mays (corn) have only PS I in BSC chloroplasts.
the gas diffusion between the bundle sheath and the mesophyll. In some C4 species (NADP-ME types), the cells of the bundle sheath contain large chloro-plasts with mainly stroma thylakoids and very little grana. The bundle sheath cells are connected via plasmodesmata with the adjacent thin-walled mesophyll cells, with large intercellular spaces.
CO2 is first assimilated in the mesophyll cells, catalyzed by PEP carboxylase, a light-activated enzyme, located in the cytosol. PEP carboxylase uses phosphoenolpyruvate (PEP) and HCO3~ as substrates. HCO3~ is formed by hydratation of CO2, catalyzed by carbonic anhydrase. The high affinity of PEP carboxylase for HCO3~ reduces Ci to about 100 mmol mol—1, less than half the Ci of C3 plants (Sect. 2.2.2). PEP is produced in the light from pyruvate and ATP, catalyzed by pyruvate Pi-diki-nase, a light-activated enzyme located in the chloroplast. The product of the reaction catalyzed by PEP carboxylase is oxaloacetate, which is reduced to malate. Alternatively, oxaloacetate may be transaminated in a reaction with alanine, forming aspartate. Whether malate or aspartate, or a mixture of the two, are formed, depends on the subtype of the C4 species (Table 8). Malate (or aspartate) diffuses via plasmodesmata to the vascular bundle sheath cells, where it is decarboxylated, producing CO2 and pyruvate (or alanine). CO2 is then fixed by Rubisco in the chloroplasts of the bundle sheath cells, which have a normal Calvin cycle, as in C3 plants. Rubisco is not present in the mesophyll cells, which do not have a complete Calvin cycle and only store starch when the bundle sheath chloroplasts reach their maximum starch concentrations.
Fixation of CO2 by PEP carboxylase and the subsequent decarboxylation occur relatively fast,
Figure 37. (Facing page) Schematic representation photosynthetic metabolism in the three C4 types distinguished according to the decarboxylating enzyme. NADP-ME, NADP-requiring malic enzyme; PCK, PEP carboxykinase; NAD-ME, NAD-requiring malic enzyme. Numbers refer to enzymes. (1) PEP carboxylase, (2) NADP-malate dehydrogenase, (3) NADP-malic enzyme, (4) pyruvate Pj-dikinase, (5) Rubisco, (6) PEP carboxykinase, (7) alanine aminotransferase, (8) aspartate amino transferase, (9) NAD-malate dehydrogenase, (10) NAD-malic enzyme (after Lawlor 1993). (Above) Cross-sections of leaves of monocotyledonous C4 grasses (Ghannoum et al. 2005). Chlorophyll a autofluorescence of a leaf cross-section of (Left) Pani-cum miliaceum (French millet, NAD-ME), and (Right) Sorgum bicolor (millet, NAD-ME). The images were obtained using confocal microscopy. Cell walls are shown in green and chlorophyll a autofluorescence in red. Most of the autofluorescence emanates from bundle sheath cells in the NAD-ME species (Left) and from the mesophyll cells in the NADP-ME species (Right), showing the difference in chlorophyll distribution between the two subtypes (courtesy O. Ghannoum, Centre for Horticulture and Plant Sciences, University of Western Sydney, Australia). Copyright American Society of Plant Biologists.
allowing the build-up of a high concentration of CO2 in the vascular bundle sheath. When the outside CO2 concentration is 380 mmol mol-1, that at the site of Rubisco in the chloroplasts of the vascular bundle is 1000-2000 mmol mol-1. The Ci, that is the CO2 concentration in the intercellular spaces in the mesophyll, is only about 100 mmol mol-1. With such a steep gradient in the CO2 concentration it is inevitable that some CO2 diffuses back from the bundle sheath to the mesophyll, but this is only about 20%. In other words, C4 plants have a mechanism to enhance the CO2 concentration at the site of Rubisco to an extent that its oxygenation reaction is virtually fully inhibited. Consequently, C4 plants have negligible rates of photorespiration.
Based on the enzyme involved in the decarbox-ylation of the C4 compounds transported to the vascular bundle sheath, three groups of C4 species are discerned: NADP-malic enzyme-, NAD-malic enzyme- and PEP carboxykinase-types (Table 8, Fig. 37). The difference in biochemistry is closely correlated with anatomical features of the bundle sheath and mesophyll of the leaf blade as viewed in transverse sections with the light microscope (Ellis 1977). In NAD-ME-subtypes, which decarbox-ylate malate (produced from imported aspartate) in the bundle sheath mitochondria, the mitochondrial frequency is several-fold higher than that in NADP-ME-subtypes. The specific activity of the mitochon-drial enzymes involved in C4 photosynthesis is also greatly enhanced (Hatch & Carnal 1992). The NAD-ME group of C4 species tends to occupy the driest habitats, although the reason for this is unclear (Ellis et al. 1980, Ehleringer & Monson 1993).
Decarboxylation of malate occurs only during assimilation of CO2, and vice versa. The explanation for this is that the NADP needed to decarboxylate malate is produced in the Calvin cycle, during the assimilation of CO2. At least in the more ''sophisticated'' NADP-ME C4 plants such as Zea mays (corn) and Saccharum officinale (sugar cane), the NADPH required for the photosynthetic reduction of CO2 originates from the activity of NADP malic enzyme. Since two molecules of NADPH are required per molecule of CO2 fixed by Rubisco, this amount of NADPH is not sufficient for the assimilation of all CO2. Additional NADPH is required to an even larger extent if aspartate, or a combination of malate and aspartate, diffuses to the bundle sheath. It is assumed that this additional NADPH can be imported via a ''shuttle'', involving PGA and dihy-droxyacetone phosphate (DHAP). Part of the PGA that originates in the bundle-sheath chloroplasts returns to the mesophyll. Here it is reduced, producing DHAP, which diffuses to the bundle sheath.
Alternatively, NADPH required in the bundle sheath cells might originate from the removal of electrons from water. This reaction requires the activity of PS II, next to PS I. PS II is only poorly developed in the bundle sheath cells, at least in the ''more sophisticated'' C4 species. The poor development of PS II activity in the bundle sheath indicates that very little O2 is evolved in these cells that contain Rubisco, which greatly favors the carboxylation reaction over the oxygenation.
The formation of PEP from pyruvate in the mesophyll cells catalyzed by pyruvate Pi-dikinase, requires one molecule of ATP and produces AMP, instead of ADP; this corresponds to the equivalent of two molecules of ATP per molecule of PEP. This represents the metabolic costs of the CO2 pump of the C4 pathway. It reduces photosynthetic efficiency of C4 plants, when compared with that of C3 plants under nonphotorespiratory conditions. In summary, C4 photosynthesis concentrates CO2 at the site of carboxylation by Rubisco in the bundle sheath, but this is accomplished at a metabolic cost.
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