One hundred million years ago (Mid-Cretaceous), atmospheric CO₂ was between 1500 and 3000 µL L^–1, or four to ten times post-industrial levels. Atmospheric CO₂ declined during the Oligocene (20-30 million years ago) from the high Tertiary levels (>1000 µL L^-1), and oscillated between 180 and 300 µL L^-1 for the last 1-3 million years. The Oligocene was also a time when the Earth was dry and the tropics were relatively hot. The earliest origins of C₄ photosynthesis date back to this period. Curiously, C₄ plants remained in low abundance for a long period of time. According to stable carbon isotopic data, a worldwide expansion of C₄ grasslands and savannas occurred during the Late Miocene and Pliocene (3 to 8 million years ago), most probably through the displacement of C₃ vegetation (Edwards et al. 2010).

Under the early high concentration of CO₂, photorespiration of C₃ plants was inhibited (Section 2.3) so that photosynthetic efficiency was higher than it is now. In addition, maximum photosynthetic rates were double twentieth century values, and the energy cost of photosynthesis would have been around three ATP and two NADPH per molecule of CO₂ fixed. As atmospheric CO₂ concentrations declined to approximately 250–300 µL L^–1, photosynthetic rates were halved, photorespiration increased substantially, photosynthetic efficiency declined and the energetic costs of photosynthesis increased to approximately five ATP and 3.2 NADPH per CO₂ molecule fixed. Such events would have generated a strong selection pressure for genetic variants with increased carboxylation efficiency and increased photosynthetic rates.

Angiosperms have a higher relative specificity of Rubisco for CO₂ than ferns and mosses (see examples of other less evolutionarily advanced species in Figure 2.3). Such differences imply minor evolution in this highly conserved molecule of Rubisco and there is little variation between species of vascular plants. Consequently, alteration of Rubisco in response to a changing atmospheric CO₂ concentration has not been an option.

By contrast, evolution of a new photosynthetic pathway (C₄) has occurred independently and on many occasions in diverse taxa over 25 to 30 million years as CO₂ levels declined. Despite its complexity, C₄ photosynthesis evolved more than 60 independent times in 19 distantly related flowering families. About 50% of C₄ species are grasses (Poaceae) with ~18 distinct origins distributed over 370 genera and ~4600 species (Sage et al. 2011). The oldest identifiable fossils with pronounced bundle sheath layers are seven million years old, although necessary metabolic pathways could have evolved earlier, prior to this adaptation in anatomy. C₄ plants are known to differ from C₃ plants in their discrimination against atmospheric ¹³CO₂, and shifts in the stable carbon isotope signature of soil carbonate layers that reflect emergence of C₄ plants have been dated at 7.5 million years bp. Modern evidence from molecular phylogeny places the origin of the main C₄ taxa at 25-30 million years ago (Christin et al. 2009). By inference, C₄ photosynthesis evolved in response to a significant decline in atmospheric CO₂ concentration, from 1500–3000 µL L^–1 to about 300 µL L^–1. By evolving a CO₂-concentrating mechanism, C₄ plants presented their Rubisco with an elevated partial pressure of CO₂ despite lower atmospheric CO₂. As a consequence, photorespiration was inhibited, maximum photosynthetic rates increased and energetic costs reduced.

The C₄ pathway (Figure 2.3) is ‘a unique blend of modified biochemistry, anatomy and ultra-structure’ (Hatch 1987). The classical C₄ syndrome in most terrestrial plants consists of two photosynthetic cycles (C₃ (or PCR) and C₄) operating across two photosynthetic cell types (mesophyll and bundle sheath), which are arranged in concentric layers around the vascular bundle, also known as the kranz anatomy.

Initial and rapid fixation of CO₂ within mesophyll cells results in the formation of a four-carbon compound which is then pumped to bundle sheath cells for decarboxylation and subsequent incorporation into the PCR cycle in that tissue. This neat division of labour hinges on specialised anatomy and has even resulted in evolution of distinct classes of chloroplasts in mesophyll compared with bundle sheath cells. Three biochemical variants of C₄ photosynthesis (termed subtypes) are known to have evolved from C₃ progenitor and in all cases with a recurring theme where the C₄ cycle of mesophyll cells is complemented by a PCR cycle in bundle sheath cells, where Rubisco is exclusively localised. In effect, a biochemical ‘pump’ concentrates CO₂ at Rubisco sites in bundle sheath cells thereby sustaining faster net rates of CO₂ incorporation and virtually eliminating photorespiration. For this overall mechanism to have evolved, a complex combination of cell specialisation and differential gene expression was necessary. Figure 2.3a shows a low-magnification electron micrograph of a C₄ leaf related to a generalised scheme for the C₄ pathway.

By analogy with Calvin’s biochemical definition of the C₃ pathway at Berkeley in the 1950s, the C₄ pathway was also delineated with radioactively labelled CO₂ (see Feature Essay 2.1). Significantly, and unlike C₃ plants, 3-PGA is not the first compound to be labelled after a ¹⁴C pulse (Figure 2.3b). Specialised mesophyll cells carry out the initial steps of CO₂ fixation utilising the enzyme phosphoenolpyruvate (PEP) carboxylase. The product of CO₂ fixation, oxaloacetate, is a four-carbon organic acid, hence the designation ‘C₄’ photosynthesis (or colloquially, C₄ plant). A form of this four-carbon acid, either malate or aspartate depending on the C₄ subtype, migrates to the bundle sheath cells which contain Rubisco and the PCR cycle. In the bundle sheath cells, CO₂ is removed from the four-carbon acid by a specific decarboxylase and a three-carbon product returns to the mesophyll to be recycled to PEP for the carboxylation reaction. Thus, label first appears in the four-carbon acid after ¹⁴C feeding, followed by 3-PGA and, finally, in sucrose and starch (Figure 2.1b).

A physical barrier to CO₂ diffusion exists in the thickened walls of the bundle sheath cells (lined with suberised in some C₄ species), preventing CO₂ diffusion back to the mesophyll and allowing CO₂ build up to levels at least 10 times those of ambient air. Build up of CO₂ in the bundle sheath is also facilitated by the higher activity ratio (2-4 times) of PEP carboxylase relative to Rubisco in C₄ plants. Rubisco is thus exposed to a saturating concentration of CO₂ which both enhances carboxylation due to increased substrate supply, and forestalls oxygenation of RuBP (hence photorespiration) by outcompeting O₂ for CO₂ binding sites on Rubisco (Figure 2.5).

In leaves of C₃ plants, the PCR cycle operates in all mesophyll chloroplasts, but in C₄ plants the PCR cycle is restricted to bundle sheath cells (Figure 2.3). Rubisco is pivotal in this cycle, and can be used as a marker for sites of photosynthetic carbon reduction. Rubisco was visualised by localising this photosynthetic enzyme with antibodies via indirect immunofluorescent labelling (Hattersley et al. 1977; Figure 2.6). In this pioneering method, ‘primary’ rabbit anti-Rubisco serum (from rabbits injected with purified Rubisco) is first applied to fixed transverse sections of leaves. Rabbit antibodies to Rubisco bind to the enzyme in situ. Then ‘secondary’ sheep anti-rabbit immunoglobulin tagged with a fluorochrome (fluorescein isothiocyanate) is applied to the preparation. This fluorochrome binds specifically to the rabbit antibodies and fluoresces bright yellow wherever Rubisco is located (blue light excitation using an epifluorescence light microscope).

In the C₃ grass Microlaena stipoides (Figure 2.6a), all chloroplasts are fluorescing bright yellow and this indicates wide distribution of Rubisco throughout mesophyll tissue. By contrast, only bundle sheath cells are equipped with Rubisco in the C₄ grass Digitaria brownii (Figure 2.6b). These two native Australian grasses co-occur in the ACT but contrast in relative abundance. M. stipoides (weeping grass) is common in dry sclerophyll woodlands throughout southeast temperate Australia, whereas D. brownii (cotton panic grass) in the ACT is at the southern end of its distribution, being far more abundant in subtropical Australia and, in keeping with its C₄ physiology, especially prevalent in semi-arid regions.

One disadvantage of the C₄ pathway is that an energy cost is incurred by C₄ plants to run the CO₂ ‘pump’. This is due to the ATP required for recycling PEP from pyruvate by the chloroplastic enzyme pyruvate, Pi dikinase in the mesophyll cells (Figure 2.4 and Hatch 1987). Under ideal conditions, five ATP and two NADPH are required for every CO₂ fixed in C₄ photosynthesis (two ATP are required to run the CO₂ pump, i.e., regenerate PEP). In addition, a proportion (20-30%) of CO₂ fixed by PEP carboxylase in the mesophyll is not fixed by Rubisco in the bundle sheath, and subsequently leaks back to the mesophyll. This leaked (or overcycled) CO₂ represents an additional, inherent energetic cost of the C₄ pathway.

From the previous section, the C₄ pathway is obviously energetically more expensive than the C₃ pathway in the absence of photorespiration. However, at higher temperatures the ratio of RuBP oxygenation to carboxylation is increased and the energy requirements of C₃ photosynthesis can rise to more than five ATP and three NADPH per CO₂ fixed in air (for these calculations see Hatch 1987).

Representative light response curves for photosynthesis in C₃ cf. C₄ plants (Figure 2.7) can be used to demonstrate some of these inherent differences in photosynthetic attributes. At low temperature (10°C in Figure 2.7) a C₃ leaf shows a steeper initial slope as well as a higher value for light-saturated photosynthesis. By implication, quantum yield is higher and photosynthetic capacity is greater under cool conditions. In terms of carbon gain and hence competitive ability, C₃ plants will thus have an advantage over C₄ plants at low temperature and especially under low light.

By contrast, under warm conditions (35°C, upper curves in Figure 2.7) C₄ photosynthesis in full sun greatly exceeds that of C₃, while quantum yield (inferred from initial slopes) remains unaffected by temperature. Significantly, C₃ plants show a reduction in quantum yield under warm conditions (compare 10°C and 35°C curves; right side of Figure 2.7). At 35°C C₃ plants also show lower rates of light-saturated assimilation compared with C₄ plants. Increased photorespiratory losses from C₃ leaves at high temperature are responsible (Section 2.3). C₄ plants will thus have a competitive advantage over C₃ plants under warm conditions at both high and low irradiance.

C₄ photosynthesis calls for metabolic compartmentation which is in turn linked to specialised anatomy (Figure 2.4). Three biochemical subtypes of C₄ photosynthesis have evolved which probably derive from subtle differences in the original physiology and leaf anatomy of their C₃ progenitors.

CO₂ assimilation by all three C₄ subtypes (Figure 2.8) involves five stages:

1. carboxylation of PEP in mesophyll cells, thereby generating four-carbon acids (malate and/or aspartate);
2. transport of four-carbon acids to bundle sheath cells;
3. decarboxylation of four-carbon acids to liberate CO₂;
4. re-fixation of this CO₂ via Rubisco within the bundle sheath, using the C₃ pathway;
5. transport of three-carbon acid products following decarboxylation back to mesophyll cells to enable synthesis of more PEP.

Recognising some systematic distinctions in whether malate or aspartate was transported to bundle sheath cells, C₄ plants were further subdivided into three subtypes according to their four-carbon acid decarboxylating systems and ultrastructural features (Hatch et al. 1975). Members of each subtype contain high levels of either NADP-malic enzyme (NADP-ME), phosphoenolpyruvate carboxykinase (PCK) or NAD-malic enzyme (NAD-ME) (so designated in Figure 2.8). High NADP-malic enzyme activity is always associated with higher NADP-malate dehydrogenase activity, while those species featuring high activities of either of the other two decarboxylases always contain high levels of aminotransferase and alanine aminotransferase activities. As a further distinction, each of the decarboxylating enzymes is located in bundle sheath cells; NAD-malic enzyme is located in mitochondria but PEP carboxykinase is not.

In all three subtypes, the primary carboxylation event occurs in mesophyll cytoplasm with PEP carboxylase acting on HCO₃^– to form oxaloacetate. However, the fate of this oxaloacetate varies according to subtype (Table 2.1; Figure 2.8). In NADP-ME species, oxaloacetate is quickly reduced to malate in mesophyll chloroplasts using NADPH. By contrast, in NAD-ME and PCK species, oxaloacetate is transaminated in the cytoplasm, with glutamate donating the amino group, to generate aspartate. Thus, malate is transferred to bundle sheath cells in NADP-ME species and aspartate is transferred in NAD-ME and PCK species. The chemical identity of three-carbon acids returned to mesophyll cells varies accordingly.

In NADP-ME species, only chloroplasts are involved in decarboxylation and subsequent carboxylation via the PCR cycle (Figure 2.8). By contrast, in NAD-ME and PCK species, chloroplasts, cytoplasm and mitochondria are all involved in moving carbon to the PCR cycle of bundle sheath chloroplasts. In NAD-ME and PCK species, aspartate arriving in bundle sheath cells is reconverted to oxaloacetate in either mitochondria (NAD-ME) or cytoplasm (PCK) (Table 2.2). Reduction and decarboxylation of oxaloacetate occurs in mitochondria of NAD-ME species and CO₂ is thereby released for fixation by chloroplasts of bundle sheath cells. In PCK species, oxaloacetate in the cytoplasm is decarboxylated by PCK, thereby releasing CO₂ for fixation in bundle sheath chloroplasts (Figure 2.8).

Transport of metabolites to bundle sheath cells

A rapid transfer of malate and aspartate to bundle sheath cells from mesophyll cells is required if the CO₂ concentration in bundle sheath cells is to stay high. A very high density of plasmodesmata linking bundle sheath cells to mesophyll cells facilitates this traffic. Consequently, the permeability coefficient of C₄ bundle sheath cells to small metabolites such as four-carbon acids is about 10 times larger than that of C₃ mesophyll cells (Table 2.2). However, coupled with this need for a high permeability to metabolites moving into bundle sheath cells is a low permeability to CO₂ molecules so that CO₂ released through decarboxylation in the bundle sheath does not diffuse rapidly into mesophyll air spaces. For some species, a layer of suberin in the cell wall of bundle sheath–mesophyll junctions (suberin lamella) significantly reduces CO₂ efflux (Table 2.2).

Centrifugal versus centripetal chloroplasts

Not all species contain a suberin layer, but all C₄ plants have a need to prevent CO₂ from diffusing quickly out of bundle sheath cells, so that the location of chloroplasts of bundle sheath cells becomes critical in those species lacking a suberin layer (Figure 2.9). Where species have a suberin layer, chloroplasts are located in a centrifugal position, that is, on the wall furtherest away from the centre of the vascular bundle lying in the middle of the bundle sheath (Figure 2.9E, F). In those C₄ species lacking a suberin layer, chloroplasts are located centripetally, that is, on the wall closest to the centre of the vascular bundle lying within the bundle sheath (Figure 2.9A, B). Such a location would help restrict CO₂ diffusion from bundle sheath to mesophyll cells.

Regulation of C₄ photosynthesis

Fixation of CO₂ by C₄ plants involves the coordinated activity of two cycles in separate anatomical compartments (Figure 2.8). The first cycle is C₄ (carboxylation by PEP carboxylase), the second is C₃ (carboxylation by Rubisco). Given this biochemical and anatomical complexity, close regulation of enzyme activities is a prerequisite for efficient coordination.

PEP carboxylase, NADP-malate dehydrogenase and pyruvate orthophosphate dikinase are all light-regulated and their activities vary according to irradiance. NADP-malate dehydrogenase is regulated indirectly by light via the thioredoxin system.

PEP carboxylase in C₄ plants exists in the same homo-tetramer in light- and dark-acclimated leaves. This is in marked contrast to CAM species where different forms exist in light- and dark-acclimated leaves. In C₄ plants, PEP carboxylase has extremely low activity at night, thus preventing uncontrolled consumption of PEP. Such complete loss of activity in darkness is mediated via divalent metal ions, pH plus allosteric activators and inhibitors. As a consequence, and over a period of days, C₄ plants can increase or decrease PEP carboxylase in response to light regime.

Rubisco is characterised by its low affinity for its productive substrate, CO₂ and slow catalytic turnover rate (i.e., 1-3 cycles per sec). Importantly, Rubisco reacts with O₂ (photorespiration), and this culminates in loss of CO₂ and energy. In C₃ plants, photorespiration can drain more than 25% of fixed CO₂ under non-stressful conditions. The ratio of photorespiration to photosynthesis increases with increasing temperature and decreasing intercellular CO₂ such as occurs when stomatal conductance is reduced under water stress. C₃ plants compensate for Rubisco’s inefficiencies by (i) opening their stomata to increase CO₂ diffusion into chloroplasts, which increases water loss and lowers leaf-level water use efficiency, WUE; and (ii) investing up to 50% of leaf nitrogen in Rubisco, which lowers their leaf-level nitrogen use efficiency, NUE.

The C₄ pathway supercharges photosynthesis and suppresses photorespiration by operating a CO₂ concentrating mechanism which elevates CO₂ around Rubisco. Although C₄ photosynthesis incurs additional energy, the energy cost of photorespiration exceeds that of the CO₂ concentrating mechanism above 25^oC. Hence, higher radiation use efficiencies (i.e., efficiency of converting absorbed radiation into biomass) have been recorded for C₄ than C₃ crops. High bundle sheath CO₂ concentration saturates C₄ photosynthesis at relatively low intercellular CO₂, allowing C₄ plants to operate with lower stomatal conductance. Thus, leaf-level WUE is usually higher in C₄ than C₃ plants. Relative to C₃ plants, Rubisco of C₄ plants is faster (higher turnover rate) and operates under saturating CO₂. Thus, C₄ plants typically achieve higher photosynthetic rates with about 50% less Rubisco and less leaf nitrogen. Hence, photosynthetic NUE is higher in C₄ than C₃ plants. Accordingly, C₄ plants are advantaged relative to C₃ plants in hot and nitrogen-poor environments with short growing seasons, hence their great abundance in wet/dry tropics such as Northern Territory savannas.

As mentioned earlier, more than 50% of C₄ plants are grasses. C₄ grasses are confined to low latitudes and altitudes, whereas C₃ species dominate at higher latitudes and altitudes. Generally, C₄ species frequently occur in regions of strong irradiance. Ehleringer and colleagues (Ehleringer et al. 1997) proposed that these distribution patterns are best explained by the different responses of photosynthetic quantum yields to temperature between C₃ and C₄ plants.

C₄ photosynthesis suppresses photorespiration by operating a CO₂ concentrating mechanism that comes at additional energetic cost. This cost is independent of ambient CO₂ and temperatures. In contrast, photorespiration (and its associated energy cost) increases steeply with temperature in C₃ plants and is highly dependent on CO₂ concentrations. Under saturating irradiance and current ambient atmospheric CO₂ concentration, the threshold temperature where the cost of photorespiration in C₃ plants exceeds that of the CO₂ concentrating mechanism in C₄ plants is estimated around 25^oC. This model provides a physiological basis for understanding today’s contrasting geographic distribution between C₃ and C₄ grasses.

As an example, the C₄ grasses of the northern Australian savannas are relatively un-shaded because of the low tree density and sparse canopy. Light is abundant and since the CO₂ concentration inside C₄ leaves is high, a potentially high rate of light-saturated assimilation can be exploited. Most C₃ species reach light saturation in the range of one-eight to one-half full sunlight (Figure 2.7). In C₄ species, canopy assimilation might not become light saturated even in full sunlight. C₄ plants thus maintain a competitive advantage over C₃ plants in tropical locations, where average daily light receipt is much larger than in temperate zones, and associated with warmer conditions that also favour C₄ photosynthesis (Figure 2.6). Given strong sunlight, warmth and seasonally abundant water, biomass production by C₄ plants is commonly double the rate for C₃ plants. Typically, C₃ plants produce 15–25 t ha^–1 but C₄ plants easily produce 35–45 t ha^–1.

Physiological characteristics of the C₄ subtypes

As outlined in previous sections, characteristic biochemical, anatomical and physiological traits are associated with each of the three “classical” C₄ subtypes (Table 2.3). However, it should be noted that many C₄ plants have leaf structures that fall outside the “classical” subtype division (eg, NADP-ME tribes, Arundinelleae and Neurachneae). As many as 11 anatomical-biochemical suites have been identified in C₄ grasses. A curious aspect about the subtypes of C₄ grasses is their biogeography. In Australia and elsewhere, NADP-ME grasses are more frequent at higher rainfall, NAD-ME grasses predominate at lower rainfall, while the distribution of PCK grasses is even across rainfall gradients (Hattersley 1992).

The fundamental paradigm underpinning the efficiency of C₄ photosynthesis in terrestrial plants is the ‘division of labour’ between the initial fixation of CO₂ into C₄ acids, and their subsequent utilisation to generate high concentrations of CO₂ for ultimate fixation by Rubisco. The basic model for C₄ plants with classical kranz anatomy consists of two photosynthetic cycles (C₃ and C₄) operating across two photosynthetic cell types (mesophyll and bundle sheath), with strict cell- and organelle-specific localisation of key enzymes and with sufficient resistance to CO₂ back-diffusion. Indeed, the discovery of the kranz anatomy by Haberlandt preceded that of C₄ biochemistry by a century. The prevailing consensus has been that efficient C₄ photosynthesis necessitates the collaboration of two cell types.

Recently, this notion has been challenged by the discovery of non-kranz or single-cell C₄ photosynthesis in shrubs (Borszczowia aralocaspica and Bienertia cycloptera; Chenopodiaceae family) found in the salt deserts of Central Asia (Voznesenskaya et al. 2002, 2003). These plants show CO₂ and O₂ responses typical of C₄ photosynthesis but lack the kranz anatomy. They perform C₄ photosynthesis through the spatial localisation of dimorphic chloroplasts (as well as other organelles and photosynthetic enzymes) in distinct positions within a single chlorenchyma cell. Yet, the details of the partitioning differ between the two species (Edwards et al. 2004).

In Bienertia, the central cytoplasmic compartment of the chlorenchyma cell plays the role of bundle sheath cells in kranz-type C₄ (NAD-ME) plants; it is filled with mitochondria surrounded by chloroplasts. The peripheral cytoplasm lacks mitochondria and plays the role of the mesophyll cell in kranz-type C₄ plants. Accordingly, chloroplastic Rubisco and mitochondrial NAD-ME and glycine decarboxylase are restricted to the central compartment; chloroplastic pyruvate, Pi dikinase is restricted to the peripheral compartment, which is highly enriched with cytosolic PEP carboxylase. In Borszczowia, the compartmentation occurs at the distal (mesophyll equivalent) and proximal (bundle sheath equivalent) ends of the elongated, cylindrical chlorenchyma cell. The inter-connecting cytoplasm between the two intra-cellular compartments provides a liquid diffusion path, thus replacing the role of the bundle sheath cell wall in kranz-type C₄ plants (Edwards et al. 2004).

A low conductance for CO₂ diffusion out of the bundle sheath cells (or its equivalent cellular compartment) is critical for the efficient operation of C₄ photosynthesis. The total diffusive resistance to CO₂ has multiple components with different levels of contribution. These components include bundle sheath walls, membranes, bundle sheath chloroplast position, the site of C₄ acid decarboxylation, and the liquid-phase diffusion path. For kranz-type C₄ plants, calculated total bundle sheath resistance on a leaf area basis can range from 50 to 150 m² s^-1 mol^-1 (von Caemmerer and Furbank 2003). Evidently, single-cell C₄ plants have sufficient resistance to CO₂ back-diffusion which is essentially made of the cytoplasmic liquid phase and the special localisation of the (Rubisco-containing) chloroplasts surrounding the mitochondria (site of C₄ acid decarboxylation).

Thus, single-cell C₄ plants have efficient photosynthesis which is not inhibited by O₂, and their carbon isotope values are similar to kranz-type C₄ plants. Although, single-cell C₄ photosynthesis breaks away from the classical kranz anatomy, it remains within the general ‘division of labour’ paradigm.

More than 40 eudicot and monocot species distributed over 21 lineages have been reported to possess intermediate C₃ and C₄ photosynthetic characteristics and CO₂ compensation points. These intermediate species are likely remnants of the complex processes that led to the evolution of C₄ plants from C₃ ancestors, although reversions from the C₄ condition have also been suggested. Moreover, a number of identified C₃-C₄ species occur in taxa that are not closely related to any C₄ lineage, raising the possibility that the C₃-C₄ photosynthetic pathway may be a distinct adaptation. This, in addition to the small number of intermediate species found so far cast doubts over their physiological and ecological fitness, and whether they represent living fossils of evolutionary paths or evolutionary dead-ends (Rawsthorne 1992; Sage et al. 2011).

Leaves of all C₃-C₄ intermediates have partial or full kranz anatomy, with prominent bundle sheath cells containing chloroplasts and other organelles, and intermediate interveinal distances. Bundle sheath chloroplasts contain Rubisco and functional PCR cycle in both mesophyll and bundle sheath cells. Intermediate leaves also have CO₂ compensation points that are lower than what is observed for C₃ leaves and can be indistinguishable from C₄ leaves, due to reduced photorespiration. Biochemically, C₃-C₄ intermediates differ in the level of activity of the C₄ cycle and the extent to which CO₂ is concentrated in bundle sheath cells.

C₃-C₄ intermediate plants reduce photorespiration (and hence, CO₂ compensation point) using a ‘photorespiratory pump’ based on modified localisation of the mitochondrial photorespiratory enzyme, glycine decarboxylase (Figure 2.10). In these plants, glycine decarboxylase activity is restricted to bundle sheath cells and excluded from mesophyll cells. Consequently, photorespired CO₂ is released in the bundle sheath where it is largely refixed by Rubisco and the bundle sheath PCR before it diffuses back to the mesophyll. Such a system may weakly elevate CO₂ in bundle sheath cells. Intermediate species that rely on the ‘photorespiratory pump’ are termed C₃-like or Type I intermediates (e.g., Panicum milioides, Flaveria pubescens) and have intermediate CO₂ compensation points and negligible C₄ cycle activity.

In Type II and C₄-like intermediates (e.g., Flaveria brownii), up to 70% of atmospheric CO₂ may be first fixed into C₄ acids. These plants have C₄-like CO₂ compensation points but are not classified as C₄ plants because they lack the strict localisation of photosynthetic enzymes (e.g., Rubisco is present in mesophyll cells) and their bundle sheath cell walls have high CO₂ permeability, resulting in only a partial CO₂ concentrating mechanism (Brown 1980; Ku et al. 1991; Rawthorne 1992; Vogan and Sage 2011).

The physiological advantages of the intermediate photosynthetic pathway in all its naturally occurring forms remain unclear. It may be hypothesised that lowered photorespiration may lead to reduced CO₂ limitation of photosynthesis, and thus allow C₃-C₄ plants to operate with lower stomatal conductance, thus conferring higher water use efficiency relative to C₃ counterparts. Moreover, increased nitrogen cost associated with ‘building’ another set of photosynthetic cells (bundle sheath) may reduce nitrogen use efficiency if the gains in CO₂ uptake are not substantial.

Work conducted with C₃-C₄ species yielded inconclusive evidence on the likely advantages of C₃-C₄ photosynthesis relative to the ancestral C₃ mode. Generally, these studies demonstrated that, short of substantial C₄ cycle activity and advanced cell-specific localisation of C₃ and C₄ cycle enzymes between the mesophyll and bundle sheath cells, C₃-C₄ photosynthesis does not improve photosynthetic efficiency (Bolton and Brown 1980; Pinto et al. 2011, Vogan and Sage 2011). Therefore, partial recycling of photorespired CO₂ or a partial CO₂ concentrating mechanism reduce photorespiratory loss normally associated with C₃ photosynthesis, without leading to significant gains in plant fitness or productivity.

Joseph Holtum¹, Klaus Winter² and Barry Osmond³

¹Centre for Tropical Biodiversity and Climate Change, James Cook University, Australia; ²Smithsonian Tropical Research Institute, Balboa, Ancón, Republic of Panama; ³School of Biological Sciences, University of Wollongong, and Research School of Biology, Australian National University, Australia

Crassulacean acid metabolism (CAM) is a water-conserving mode of photosynthesis that, like C₄ photosynthesis, is a modification of the C₃ photosynthetic pathway fitted with a CO₂ concentrating mechanism (CCM) that can increase the [CO₂] around ribulose bisphosphate carboxylase/oxygenase (Rubisco) by more than 10-fold and suppress photorespiration. The overall energy demand of the CAM pathway is only about 10% more than that of C₃ photosynthesis, as costs of the CCM machinery are partially offset by reducing photorespiration.

In C₄ plants, as explained earlier in Section 2.2.2, this CCM is most commonly achieved by an “in-line turbocharger” based on initial CO₂ fixation by phosphoenolpyruvate carboxylase (PEPC) into C₄ acids in the cytoplasm of outer mesophyll cells. These acids diffuse rapidly to adjacent relatively CO₂-tight bundle-sheath cells (Figure 2.31 right) where CO₂ is released again. High [CO₂] builds up in this spatially separated compartment where it is refixed by Rubisco.

In CAM plants enzyme systems analogous to those in C₄ plants achieve the same result through a “battery-like” dark accumulation of CO₂ into the 2^nd carboxyl group of malic acid (acidification phase) in the vacuole of large mesophyll cells (Figure 2.31 left). Malic acid can accumulate to very high concentrations, attaining concentrations of greater than 1 mole acid per litre in mesophyll cells of tropical tree-CAM plants (Clusia spp.). Indeed one can sometimes taste the acid, with acid taste-testing for the presence of CAM being possibly first recorded in Aloe sp. by Nehemiah Grew in 1682 and in field reports from India by Benjamin Heyne in 1815.

In the light, malic acid returns to the cytoplasm where it is rapidly decarboxylated (deacidification phase). The CO₂ released, which accumulates to high internal [CO₂] as stomata close, is refixed by Rubisco in chloroplasts of the same mesophyll cell where it is further assimilated by the photosynthetic carbon reduction (PCR) cycle (Figure 2.32).  

Ultimately three of the four carbons recovered from the malic acid must be stored as starch and/or sugars in order to provide to PEPC the C₃ substrate required for CO₂ uptake during the following night. The fourth carbon, effectively that obtained from the atmosphere, is available for growth. Deacidification may generate high [CO₂] behind closed stomata but photorespiration is not completely abolished (Lüttge 2002) since photosynthesis also generates high internal [O₂]. While exploring Lake Valencia in Venezuela in 1800, Alexander von Humboldt measured elevated [O₂] in bubbles streaming from the cut base of presumably CAM Clusia leaves standing in water in the light.

Of course by closing stomata in the light CAM plants minimize water loss when evaporative demand is highest (von Caemmerer and Griffiths 2009). The biomass production per unit water utilized in CAM was 6 times higher than for C₃ plants and 2 times higher than for C₄ plants when plants exhibiting all three photosynthetic pathways were grown together in a garden outdoors (Winter et al. 2005). The attribute of water-use efficiency undoubtedly contributes significantly to the success of CAM photosynthesis in nature, with CAM species outnumbering C₄ species by about two to one. The paradoxes of CAM, a mode of photosynthesis that involves stomatal opening and CO₂ uptake during the dark, continue to inform many aspects of plant biochemistry, physiology, ecology and evolution. This article draws heavily on two recent reviews (Borland et al. 2011; Winter et al. 2015).

Vascular plants often inhabit regions subject to tidal submergence while others carry out their entire life cycle under water. Examples of common submerged aquatic macrophytes are pond weeds and seagrasses. Once again, an evolutionary selective pressure for these plants has been the availability of CO₂. Low levels of dissolved CO₂ are common in both inland and marine waters, particularly at more alkaline pH. In more productive inland lakes, CO₂ content can vary enormously, requiring considerable flexibility in the actual mode of carbon acquisition. At high pH, HCO₃^– becomes the more abundant form of inorganic carbon, whereas dissolved CO₂ will predominate at low pH (Section 18.2). Consequently, when SAM plants evolved from their C₃ progenitors on land, there was some adaptive advantage in devices for CO₂ accumulation because CO₂ rather than HCO₃^– is substrate for Rubisco. The nature of this ‘CO₂ pump’ and the energetics of carbon assimilation are not fully characterised in SAM plants but considerable CO₂ concentrations do build up within leaves, enhancing assimilation and suppressing photorespiration.

In summary:

Regardless of photosynthetic mode, and despite catalytic limitations, Rubisco is ubiquitous and remains pivotal to carbon gain in our biosphere. As a corollary, carbon loss via photorespiration is an equally universal feature of C₃ leaves, and the evolution of devices that overcome such losses have conferred significant adaptive advantages to C₄, CAM and SAM plants.

 

Bolton JK, Brown RH (1980) Photosynthesis of grass species differing in carbon dioxide fixation pathways. V. Response of Panicum maximum, Panicum milioides, and tall fescue (Festuca arundinacea) to nitrogen nutrition. Plant Physiol 66: 97-100

Brown DA (1980). Photosynthesis of grass species differing in carbon dioxide fixation pathways. IV. Analysis of reduced oxygen response in Panicum milioides and Panicum schenckii. Plant Physiol 65: 346-349

Christin PA, Samaritani E, Petitpierre B et al. (2009) Evolutionary insights on C₄ photosynthetic subtypes in grasses from genomics and phylogenetics. Genom Biol Evol 1: 221–230

Edwards GE, Franceschi VR, Voznesenskaya EV (2004) Single-cell C₄ photosynthesis versus the dual-cell (Kranz) paradigm. Annu Rev Plant Biol 55: 173–196

Ehleringer JR, Cerling TE, Helliker BR (1997) C₄ photosynthesis, atmospheric CO₂, and climate. Oecologia 112: 285-299

Ghannoum O, von Caemmerer S, Conroy JP (2002) The effect of drought on plant water use efficiency of nine NAD–ME and nine NADP–ME Australian C₄ grasses. Funct Plant Biol 29: 1337-1348

Ghannoum O, Evans JR, Chow WS et al. (2005) Faster rubisco is the key to superior nitrogen-use efficiency in NADP-malic enzyme relative to NAD-malic enzyme C₄ grasses. Plant Physiology 137: 638-650

Hatch MD (1987) C₄ photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895: 81–106

Hatch MD, Kagawa T, Craig S (1975) Subdivision of C₄-pathway species based on differing C₄ acid decarboxylating systems and ultrastructural features. Aust J Plant Physiol 2: 111-128.

Hattersley PW, Watson L, Osmond CB (1977) In situ immunofluorescent labelling of ribulose-1,5-bisphosphate carboxylase in leaves of C₃ and C₄ plants. Aust J Plant Physiol 4: 523-539

Hattersley PW (1992) In ‘Desertified Grasslands: their Biology and Management’ (ed. Chapman GP) pp 181-212. Academic Press: London

Ku MSB, Wu JR, Dai ZY et al. (1991) Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol 96: 518-528

Pinto H, Tissue DT, Ghannoum O (2011) Panicum milioides (C₃-C₄) does not have improved water or nitrogen economies relative to C₃ and C₄ congeners exposed to industrial-age climate change. J Exp Bot 62: 3223-3234

Portis AR Jr, Salvucci ME (2002). The discovery of Rubisco activase – yet another story of serendipity. Photosyn Res 73: 257–264

Portis AR Jr, Li C, Wang D, Salvucci ME (2008) Regulation of Rubisco activase and its interaction with Rubisco. J Exp Bot 59: 1597-1604

Rawsthorne S (1992) C₃-C₄ intermediate photosynthesis - Linking physiology to gene expression. Plant J 2: 267-274

Sage RF (2004). The evolution of C₄ photosynthesis. New Phytol 161: 341-370

Sage RF, Christin PA, Edwards EJ (2011) The C₄ plant lineages of planet Earth. J Exp Bot 62, 3155-3169

Spreitzer RJ, Salvucci ME (2002) Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. Annu Rev Plant Biol 53: 449-475

Vogan PJ, Sage RF (2011) Water-use efficiency and nitrogen-use efficiency of C₃-C₄ intermediate species of Flaveria. Plant Cell Environ 34: 1415–1430.

von Caemmerer S, Furbank RT (2003) The C₄ pathway: an efficient CO₂ pump. Photosyn Res 77: 191–207

Voznesenskaya EV, Franceschi VR, Kiirats O et al. (2002) Proof of C₄ photosynthesis without Kranz anatomy in Bienertia cycloptera. Plant J 31: 649–662

Voznesenskaya EV, Edwards GE, Kiirats O et al. (2003) Development of biochemical specialization and organelle partitioning in the single celled C₄ system in leaves of Borszczowia aralocaspica. Amer J Bot 90: 1669-1680