C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves



    1. Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, PO Box 475, Canberra City, ACT 2601, Australia
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Susanne von Caemmerer. Fax: + 61 26125 5075; e-mail: Susanne.caemmerer.@anu.edu.au


Attempts are being made to introduce C4 photosynthetic characteristics into C3 crop plants by genetic manipulation. This research has focused on engineering single-celled C4-type CO2 concentrating mechanisms into C3 plants such as rice. Herein the pros and cons of such approaches are discussed with a focus on CO2 diffusion, utilizing a mathematical model of single-cell C4 photosynthesis. It is shown that a high bundle sheath resistance to CO2 diffusion is an essential feature of energy-efficient C4 photosynthesis. The large chloroplast surface area appressed to the intercellular airspace in C3 leaves generates low internal resistance to CO2 diffusion, thereby limiting the energy efficiency of a single-cell C4 concentrating mechanism, which relies on concentrating CO2 within chloroplasts of C3 leaves. Nevertheless the model demonstrates that the drop in CO2 partial pressure, pCO2, that exists between intercellular airspace and chloroplasts in C3 leaves at high photosynthetic rates, can be reversed under high irradiance when energy is not limiting. The model shows that this is particularly effective at lower intercellular pCO2. Such a system may therefore be of benefit in water-limited conditions when stomata are closed and low intercellular pCO2 increases photorespiration.


A number of biotechnological approaches are being taken to increase photosynthetic capacity of leaves (Surridge 2002). One aim of this work has been to introduce C4 characteristics into C3 plants such as tobacco, potato and rice by genetic manipulation (for review see Matsuoka et al. 2001; Häusler et al. 2002; Leegood 2002). The research has focused on engineering single-celled C4-type CO2 concentrating mechanisms as found in the aquatic plant Hydrilla verticillata (Bowes et al. 2002). Leegood (2002) outlined the biochemical necessities of enzyme activities and metabolite transporters that need to be engineered into a C3 cell for a functional C4 pathway in single-cell systems. Alternative approaches include the introduction of CO2 concentrating mechanisms from aquatic algae and cyanobacterial systems (Badger & Spalding 2000; Lieman-Hurwitz et al. 2003). In this paper a mathematical model of single-cell C4 photosynthesis adapted to C3 leaves is used to discuss the CO2 diffusional constraints inherent in such approaches (von Caemmerer & Furbank 2003).


The inefficiencies of C3 photosynthesis are largely due to the inefficiencies of the bifunctional enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC. The carboxylation of ribulose bisphosphate (RuBP) is the first step of the photosynthetic carbon reduction (PCR) cycle, which leads to CO2 uptake of leaves, whereas the oxygenation of RuBP necessitates the recycling of phosphoglycolate in the photorespiratory carbon-oxidation (PCO) cycle with a concomitant CO2 loss (Andrews & Lorimer 1987). Rubisco carboxylation has a low catalytic turnover rate of approximately 3.5 s−1 per catalytic site at CO2 saturation (von Caemmerer et al. 1994). At ambient pCO2 and O2 partial pressure (pO2) the turnover rate of carboxylation is reduced to approximately 1 s−1 depending on temperature. Thus to achieve high photosynthetic rates C3 leaves invest as much as 30% of soluble protein and 25% of leaf nitrogen into Rubisco (von Caemmerer & Quick 2000). Energy is required in the form of ATP and NADPH not only in the PCR cycle to regenerate RuBP but also in the PCO recycle to regenerate phosphoglycolate. This reduces the quantum yield of C3 photosynthesis at atmospheric pO2 and limits the potential CO2 fixation at low light particularly at higher temperatures. The quantum efficiency is particularly pertinent in a crop situation in which CO2 fixation of most leaves is light limited (Long 1991). Furthermore for CO2 assimilation to occur, CO2 needs to diffuse from the air surrounding leaves to the chloroplasts. A low conductance to CO2 diffusion reduces the efficiency of Rubisco further. The conductance of CO2 diffusion into leaves is regulated by stomatal movements that are governed by the compromise of increasing CO2 fixation over excessive water loss and which vary with environmental conditions. The diffusion of CO2 from intercellular airspace to the chloroplast is determined by leaf anatomy, liquid path lengths and membrane properties (Evans et al. 1994; Syvertsen et al. 1995; Evans & von Caemmerer 1996; Bernacchi et al. 2002). C3 leaves have large intercellular airspaces and chloroplasts are appressed to cell walls exposed to intercellular airspace, thus greatly increasing internal CO2 diffusion. As a result the chloroplast surface area appressed to intercellular airspace is 15–20 times the projected leaf area (Lloyd et al. 1992; Evans et al. 1994).


The C4 photosynthetic pathway is a biochemical CO2 concentrating mechanism that in most terrestrial C4 species relies on the co-ordinated functioning of mesophyll and bundle sheath cells. Exceptions have been discovered in the chenopods Borszczowia aralocaspia and Bientertia cycloptera in which the C4 photosynthetic mechanism operates in single cells and these are discussed separately below (Freitag & Stichler 2000; Voznesenskaya et al. 2001, 2002). In all C4 species a complex combination of both biochemical and morphological specialization provides elevated pCO2 at the site of Rubisco carboxylation. This suppresses photorespiration and allows Rubisco to operate close to its maximal rate, such that CO2 assimilation in C4 plants is effectively CO2 saturated in air (Hatch 1987). It is thought that the appearance of C4 plants in the fossil record correlates with a marked and sustained declines in atmospheric pCO2 during the Tertiary period (Ehleringer et al. 1991; Sage 2001). This suggests that low pCO2 may have been an important driving force for evolution of the pathway. Nevertheless C4 species are also characterized by greater nitrogen and water use efficiency relative to C3 plants. The increased nitrogen use efficiency is largely accounted for by the saving of nitrogen in Rubisco protein (Evans & von Caemmerer 2000).

In C4 photosynthesis CO2 is initially converted to bicarbonate by carbonic anhydrase, which is then fixed by phosphoenolpyruvate (PEP) carboxylase into C4 acids. These diffuse to and are decarboxylated in the bundle sheath to supply CO2 for Rubisco. The efficiency of the C4 pathway and the pCO2 attained in the bundle sheath is related to both the bundle sheath resistance to CO2 diffusion and the relative biochemical capacities of the C3 and C4 cycle. The efficiency of the C4 concentrating mechanisms is intimately linked to the leakiness of the bundle sheath. Leakiness is defined as that fraction of CO2 generated by C4 acid decarboxylation in the bundle sheath that subsequently leaks out (Farquhar 1983). Since the C4 cycle consumes energy in the form of ATP during the regeneration of PEP, leakage of CO2 out of the bundle sheath is an energy cost to the leaf. The higher energetic demands of C4 photosynthesis in comparison with C3 photosynthesis were elegantly demonstrated with quantum yield measurements under varying pCO2, pO2 and temperature by Ehleringer & Bjorkman (1977). These data showed that C4 species have lower quantum yields than C3 species at low temperatures, but superior quantum yields at high temperature where high photorespiratory rates decrease the quantum yields of C3 species.

It is vital to keep in mind that many of these important parameters of the C4 concentrating mechanism, such as bundle sheath resistance to CO2 diffusion, bundle sheath pCO2, and leakiness of the bundle sheath cannot be measured directly and that estimates vary widely. For example, estimates of bundle sheath resistance vary between 100 and 1000 m2 s mol−1 and estimates of leakiness range between 8 and 55% (for review see von Caemmerer & Furbank 2003). Models of C4 photosynthesis can quantitatively relate these parameters (Berry & Farquhar 1978; von Caemmerer & Furbank 1999), but uncertainties in values of the kinetic constants of Rubiscos of C4 species (von Caemmerer & Quick 2000) make it impossible to calculate accurately the bundle sheath pCO2 required to saturate Rubisco of C4 species. Nevertheless, these models have been able to demonstrate that a low bundle sheath conductance is one of the most essential features of the C4 photosynthetic pathway (Berry & Farquhar 1978; von Caemmerer & Furbank 1999). Bundle sheath conductance expressed on a leaf area basis is dependent on the conductance across the mesophyll/bundle sheath interface and the bundle sheath surface area to leaf area ratio (Sb). Estimates of Sb range between 0.6 and 3.1 m2 m−2 (Apel & Peisker 1978; Brown & Byrd 1993). This contrasts with the large chloroplast surface area to leaf area ratios of 15–20 cited above for C3 leaves. However, the conductance to CO2 diffusion across the mesophyll bundle sheath interface is also several-fold less in comparison with the equivalent conductance across the cell wall and chloroplast interface in C3 species (Evans & von Caemmerer 1996; von Caemmerer & Furbank 2003).


Recently, research has focused on engineering single-celled C4-type CO2 concentrating mechanisms. Examples of such CO2 concentrating mechanism have been found in some aquatic plants such as Hydrilla verticillata (Bowes et al. 2002). This freshwater angiosperm induces C4-like characteristics when it encounters low dissolved inorganic carbon, high temperatures, light and pO2 in summer when it can form dense mats of vegetation below the water surface (Reiskind et al. 1997). Hydrilla has PEP carboxylase in the cytosol and presumably carbonic anhydrase and produces malate, which is decarboxylated in the chloroplast by NADP-ME malic enzyme. Rubisco fixes the CO2 released and PEP is regenerated from pyruvate by pyruvate Pi dikinase (Bowes et al. 2002). The discovery of a single-cell C4 photosynthetic mechanism in the chenopods Borszczowia aralocaspica and Bienertia cycloptera have shown that Kranz antatomy may also not be essential for terrestrial C4 plant photosynthesis (Freitag & Stichler 2000; Voznesenskaya et al. 2001, 2002). However these single-cell terrestrial C4 systems (unlike Hydrilla) rely on a dimorphic cell structure with functionally distinct chloroplasts at either end of elongated single cells. They are therefore more like the conventional C4 photosynthetic system. The photosynthetic rates of these naturally occurring single-cell C4 systems are low when compared with some of the common C3 and C4 crop plants (Table 1). However when compared to their C3 counterparts they show the important characteristic of having increased their photosynthetic rates per Rubisco catalytic sites (Salvucci & Bowes 1981; Voznesenskaya et al. 2001).

Table 1.  Comparison of literature values of CO2 assimilation rates (A) between species with a single-cell C4 photosynthetic mechanism, several C3 and C4 species. A is expressed on a chlorophyll basis
SpeciesPhotosynthetic typeA (µmol g chl−1 s−1)ConditionsReference
Hydrilla verticillataAquatic single cell C4  4320 µL L−1 CO2, 30 °C, 1000 µmol quanta m−2 s−1Salvucci & Bowes (1981)
Bienertia cyclopteraSingle cell C4  8320 µL L−1 CO2, 25 °C, 1300 µmol quanta m−2 s−1Voznesenskaya et al. (2002)
Borszczowia aralocaspicaSingle cell C4 17320 µL L−1 CO2, 25 °C, 1200 µmol quanta m−2 s−1Voznesenskaya et al. (2001)
Nicotiana tabacumC3 47–49320 µL L−1 CO2, 25 °C, 1000 µmol quanta m−2 s−1Evans et al. (1994)
Oryza sativaC3 36300 µL L−1 CO2, 25 °C, 1400 µmol quanta m−2 s−1Makino et al. (1983)
Triticum aestivumC3 54350 µL L−1 CO2, 25 °C, 2000 µmol quanta m−2 s−1Watanabe et al. (1994)
Gossypium hirsutumC3 77320 µL L−1 CO2, 30 °C, 2000 µmol quanta m−2 s−1Wong (1979)
Zea maysC4118320 µL L−1 CO2, 30 °C, 2000 µmol quanta m−2 s−1Wong (1979)
Amaranthus edulisC4134320 µL L−1 CO2, 30 °C, 1600 µmol quanta m−2 s−1Leegood & von Caemmerer (1988)

These examples of single-cell C4 photosynthesis raise intriguing and important questions. How efficiently does the C4 photosynthetic pathway function in leaves with low photosynthetic capacities? What is the diffusive resistance required between the sites of initial CO2 fixation by PEP carboxylase and decarboxylation of CO2 in these systems with low photosynthetic capacity? Could these systems function at higher photosynthetic capacities? Von Caemmerer & Furbank (2003) demonstrated with the use of a C4 model that, particularly at low photosynthetic capacities, a high diffusive resistance is essential for energy-efficient C4 photosynthesis. An example is reproduced here focusing on low bundle sheath resistance values and a comparison of high and low photosynthetic capacities. The simulations use the model described by von Caemmerer & Furbank (1999) with the kinetic constants taken from their Table 2. Figure 1 shows modelled rates of CO2 assimilation, bundle sheath pCO2 and leakiness (the fraction of CO2 generated by C4 acid decarboxylation in the bundle sheath that leaks back out) as a function of bundle sheath resistance. Leakiness is a good measure for the efficiency of the CO2 concentrating mechanism. Three examples of photosynthetic capacity are shown with the same ratios of maximal Rubisco activity (Vcmax) and PEP carboxylase activity (Vpmax) (measures of C3 and C4 cycle capacities). The lowest photosynthetic capacity was chosen to mimic photosynthetic rates of species such as B. aralocaspica (Voznesenskaya et al. 2001). The simulation shows that leakiness is highest at low photosynthetic capacity (low values of Vcmax and Vpmax) when comparison is made at the same bundle sheath resistance. High values of leakiness imply an energy inefficient CO2 concentrating mechanism. Therefore leaves with low photosynthetic capacities require higher bundle sheath resistances than leaves with higher photosynthetic capacities to achieve a similar efficiency of the CO2 concentrating mechanism. The model outputs in Fig. 1 thus suggest that low CO2 diffusion resistances are not necessarily what limits the photosynthetic capacity of these single cell organisms.

Figure 1.

Modelled (a) C4-CO2 assimilation rate (b) bundle sheath pCO2, and (c) leakiness as a function of bundle sheath resistance. Three different photosynthetic capacities are shown which have the same ratio of maximal PEP carboxylase to Rubisco activity (Vpmax/Vcmax), where Vpmax was 120 (solid line), 60 (dashed lines) or 30 µmol m−2 s−1 (dotted lines). Note bundle sheath pCO2 is less and leakiness (φ) is greater at the lower photosynthetic capacities. Leakiness is the ratio of CO2 leak rate from the bundle sheath cells to the rate of PEP carboxylation. The simulations use the model described by von Caemmerer & Furbank (1999) with kinetic constants taken from their Table 2. Mesophyll pCO2 and pO2 for this simulation were 10 Pa and 20 kPa, respectively.

The bundle sheath resistance in this model does not have to be a bundle sheath cell wall but can be viewed more generally as the resistance to CO2 diffusion between the site of initial CO2 fixation and the site of C4 acid decarboxylation. Anatomical observations of B. aralocaspica by Freitag & Stichler (2000) suggest that this species may well have the high diffusion resistance required for efficient C4 photosynthesis, since the length of the liquid diffusion path in the cells of B. aralocaspica is approximately 40 µm. CO2 diffusion through water is 10 000 times slower than through air so that this path length provides a considerable barrier to CO2 diffusion. Furthermore the ratio of the surface area of this diffusion barrier to leaf area may also be low. Using values for diffusivity of CO2 in water, and the calculations described by von Caemmerer & Furbank (2003) one obtains a diffusion resistance greater than 500 m2 s mol−1. This is well within the range of values observed for C4 species (von Caemmerer & Furbank 2003). In terms of single-cell C4 photosynthesis it will be of great interest to determine the nature of the diffusion barrier in these terrestrial species and a single-cell system such as Hydrilla.


To construct a ‘single-cell C4 model based on the Hydrilla system, von Caemmerer & Furbank (2003) adapted their C4 photosynthetic model (Fig. 2; von Caemmerer & Furbank 1999). The aim of this model was to see how such a single-cell C4 system would operate in current C3 mesophyll cells, in which the chloroplast surface area is large. The model uses C3 kinetic constants for Rubisco since Rubisco isolated from C3 species has been found to have a lower Km CO2 than Rubisco isolated from C4 species (Yeoh, Badger & Watson 1981). The internal diffusive conductances for the cell wall/plasmalemma interface and the chloroplast envelope were estimated from measurements of internal leaf conductance to CO2 diffusion in tobacco using a value 0.8 mol m−2 s−1 for both the cell wall plasmalemma interface and the chloroplast envelope (Evans et al. 1994). This gives a total conductance of 0.4 mol m−2 s−1 for the C3 comparison. Output from this model shows that CO2 assimilation rate increases with increasing C4 cycle activity (quantified in the model by the maximal PEP carboxylase activity, Vpmax) (Fig. 3). Thus the model demonstrates that the chloroplast envelope may enable a limited C4 cycle activity particularly at low pCO2 (see von Caemmerer & Furbank 2003). However, a certain amount of C4 cycle activity is required before chloroplast CO2 partial pressure is greater than cytosolic pCO2 so that leakiness becomes positive (Fig. 3b & c). Also shown are the associated energy costs and it is clear that with the high CO2 conductance values of the chloroplast cytosol/interface the system is energetically inefficient (Fig. 3d).

Figure 2.

A schematic representation of the model of single-cell C4 photosynthesis developed by von Caemmerer & Furbank (2003). CO2 diffuses into the mesophyll cell were it is converted to HCO3 and fixed by PEP carboxylase. It is assumed that C4 acid decarboxylation occurs in the chloroplast at the same rate. In the chloroplast CO2 released by C4 acid decarboxylation is either fixed by Rubisco or leaks back to the cytosol. Photorespiratory CO2 is released in the cytosol. The model incorporates a resistance to CO2 diffusion at the chloroplast envelope and across the cell wall and plasmalemma interface. The resistance at the chloroplast envelope separates the initial CO2 fixation by PEP carboxylase from the decarboxylation step and is analogous to the bundle sheath resistance in the model of C4 photosynthesis. A denotes net CO2 assimilation rate, PCR denotes the photosynthetic carbon reduction cycle and PCO stands for photorespiratory carbon oxidation cycle.

Figure 3.

Modelled single-cell C4 photosynthesis as a function of C4 cycle activity, which is represented by different maximal PEP carboxylation rates at a constant intercellular pCO2 partial pressure of 20 Pa. The CO2 diffusion resistances across the cell wall and plasmalemma and across the chloroplast envelope were both 0.8 mol m−2 s−1. Maximal Rubisco activity was 80 µmol m−2 s−1. Mesophyll pO2 was 20 kPa. Mitochondrial respiration was assumed to be zero. The equations of the model, which uses a modified version of the C4 model described by von Caemmerer & Furbank (1999) with Rubisco kinetic constants suitable for C3 photosynthesis (von Caemmerer & Quick 2000; Table 2.3) are described in von Caemmerer & Furbank (2003). Shown are: (a) CO2 assimilation rate; (b) chloroplast and cytosolic pCO2; (c) leakiness; (d) ATP required per CO2 fixed.

The model also considers a diffusion conductance across the cell wall and plasmalemma interface of 0.8 mol m−2 s−1. Increasing C4 cycle activity therefore lowers the cytosolic pCO2 relative to intercellular pCO2 slightly (Fig. 3b). This is exacerbated if this cell wall/plasmalemma conductance to CO2 diffusion is reduced further (data not shown). It is therefore easily demonstrated that a low conductance to CO2 diffusion across the cell wall and plasmalemma is of no benefit to the system. On the other hand a low conductance at the chloroplast/cytosol interface is important since it separates the initial CO2 fixation from the decarboxylation step. Therefore by decreasing the chloroplast envelope conductance the efficiency of the CO2 concentrating mechanism can be increased.

A single-cell C4 system with the current C3 diffusion characteristics is clearly not an efficient CO2 concentrating mechanism with its high leakiness and energy requirements. Nevertheless the model has demonstrated that by introducing a C4 system it may be possible to reduce the drop in pCO2 between intercellular airspace and chloroplast, which in this simulation is 4 Pa (at Vpmax = 0; Fig. 3b) but has been shown to be up to 8 Pa in C3 leaves (Evans & von Caemmerer 1996). Introducing a C4 pathway into C3 cells may therefore be useful in ameliorating internal CO2 diffusion limitations particularly at low intercellular pCO2. In C3 leaves the largest reductions in pCO2 from intercellular airspace to chloroplasts occur at high light, in which the ATP supply is not limiting and considerations of energy efficiency may be of secondary importance. The model shows that this is particularly effective at lower intercellular pCO2 (von Caemmerer & Furbank 2003). Such a system may therefore be of benefit in water-limited conditions when stomata are closed and low intercellular pCO2 increases photorespiration. It is unlikely that this approach will lead to increased leaf CO2 assimilation rates under low irradiance because of the increased ATP requirement, which may be an important consideration in crop situations, where lower canopy leaves are light limited.


Alternative approaches to improve CO2 fixation in C3 plants using genetic manipulations include the introduction of CO2 concentrating mechanisms from aquatic algae and cyanobacterial systems. Most algae and cyanobacteria concentrate inorganic carbon and there appear to be a diversity of mechanisms and possibilities (Badger & Spalding 2000; Badger, Hanson & Price 2002). Recently the first transgenic plants expressing a cyanobacterial gene involved in HCO3 accumulation were generated by Lieman-Hurwitz et al. (2003). They observed small increases in photosynthetic rate and growth. This system has the same requirements for a diffusion barrier at the chloroplast envelope and is thus mathematically similar to the single-cell C4 photosynthetic system discussed above. It will be interesting to investigate the additional energy cost of such a system.

A different approach is being taken by Whitney et al. (2001). They are attempting to substitute the native Rubisco in tobacco with non-green algal Rubiscos, which have improved kinetic properties with higher CO2/O2 specificity and lower Michaelis constants for CO2 accompanied however, by a small reduction in the maximum catalytic turnover rate of the carboxylase function. Nevertheless, if successful, this can increase the carboxylation efficiency of Rubisco and hence may not only improve photosynthesis on a leaf area basis but also improve CO2 fixation on a leaf nitrogen basis. More importantly an increase in the CO2/O2 specificity of Rubisco will not only increase CO2 fixation rate at high irradiance but also improve the quantum yield and therefore CO2 fixation at low irradiance. A negative aspect of this approach is the fact that an increase of Rubisco's catalytic capacity will further increase the drop in pCO2 between intercellular and chloroplasts diminishing the potential benefits.


If biotechnological approaches are to be successful in introducing CO2 concentrating mechanisms into C3 cells, diffusion characteristics within C3 leaves need to be considered. The large chloroplast surface area appressed to intercellular airspace in C3 leaves generates low internal resistance to CO2 diffusion. This makes it difficult to design an efficient CO2 concentrating mechanism within chloroplasts of C3 leaves without also changing anatomical and diffusional aspects. On the other hand, approaches that aim to increase photosynthetic capacity within chloroplasts may exacerbate existing diffusion limitations. Clearly a further challenge will be to develop a better understanding of CO2 diffusion across cell walls and membranes inside leaves.


I would like to thank Dr John Evans and Dr Bob Furbank for many interesting discussions on CO2 diffusions and single-cell C4 photosynthesis. In particular Bob Furbank's suggestions that I model the single-cell systems has led to many of the ideas expressed here. I would also like to thank Christine Raines for helpful comments on the manuscript.