About 30% of the carbohydrate formed in C3 photosynthesis is lost through photorespiration (Monteith 1977). The loss increases with temperature so that photorespiration is a particularly significant inefficiency for C3 crops in tropical climates and during hot summer weather in temperate climates (Fig. 2). Photorespiration results from the apparently unavoidable oxygenation of RuBP by Rubisco (reviewed, Giordano, Beardall & Raven 2005). Beyond this point, the purpose of photorespiratory metabolism is to recover the carbon diverted into this pathway. Blocking photorespiratory metabolism downstream of Rubisco simply results in this carbon entering a dead-end metabolic pathway. Indeed, mutants that lack any one of the photorespiratory enzymes die unless they are grown at low oxygen or at very high CO2 to inhibit oxygenation of RuBP. The only remaining prospect for decreasing photorespiration then, is decreased oxygenation. Would decreased oxygenation result in higher yields? Photorespiratory metabolism can dissipate excess excitation energy at high PPFD, involves the synthesis of serine and glutamate, and transfers reductive power from the chloroplast to the mitochondrion. This has led some to suggest that photorespiration is essential for normal plant function (e.g. Barber 1998; Evans 1998). However, xanthophylls provide a far more effective means of dissipating excess energy. Unlike photorespiration, this dissipation mechanism is not a significant drain on the ATP and NADPH produced by the light reactions. Further, dissipation of energy as heat through xanthophylls is induced by excess light and is reversed when light is no longer in excess. So unlike photorespiration, it does not continue to divert energy from photosynthesis when light is no longer in excess. In addition, the photosynthetic cell has pathways besides photorespiration for amino acid synthesis and transfer of reductive energy to the cytosol (reviewed, Long 1998), which suggests that the supposed ‘beneficial’ functions of photorespiration are redundant within the cell. Further, photorespiration can be eliminated without detriment to the plant by growing plants in a very high concentration of CO2, a competitive inhibitor of the oxygenase activity of Rubisco. For example, wheat can grow normally and can complete its life cycle under these unusual conditions (Wheeler et al. 1995). Commercial growers of some greenhouse crops increase [CO2] to three or four times the normal atmospheric concentration (Chalabi et al. 2002). This inhibits the oxygenation reaction of Rubisco, increasing photosynthetic efficiency and final yield. At present, the global [CO2] is rising and this, too, is diminishing photorespiration, but atmospheric change also includes many potentially negative effects for crops, including increased temperature, decreased soil moisture and an associated rise in phytotoxic tropospheric ozone (reviewed, Ort & Long 2003; Long et al. 2004, 2005). Healthy C4 plants avoid photorespiration by concentrating CO2 at the site of Rubisco. Despite earlier contradictory arguments, it is now clear that photorespiration is not an essential metabolic pathway in crops. Can it be eliminated? Two possibilities are conversion of C3 crops to C4 or improved specificity of Rubisco for CO2.
C4photosynthesis a means to eliminate photorespiration?
Terrestrial C4 plants differ from C3 plants in containing two distinct layers of photosynthetic tissue, one external to the other, each containing morphologically and functionally distinct chloroplasts. This cellular differentiation within the photosynthetic tissue is termed ‘Kranz’ leaf anatomy. In this arrangement, the mesophyll surrounds the inner photosynthetic bundle sheath where Rubisco is localized. Only the mesophyll has intercellular air spaces and contact with the atmosphere. CO2 is first assimilated into a C4 dicarboxylate through phosphoenolpyruvate (PEP) carboxylase (c) in the mesophyll. The dicarboxylate is transferred to the bundle sheath where it is decarboxylated, releasing CO2 at the site of Rubisco. The resulting pyruvate is transferred back to the mesophyll where it is phosphorylated to provide PEP, completing the C4 cycle. The photosynthetic C4 cycle is in effect a CO2 pump that concentrates CO2 around Rubisco to ca. 10 × current atmospheric concentrations (Hatch 1987; von Caemmerer 2003). It effectively eliminates photorespiration, but requires additional energy to operate the C4 cycle (Table 1). C4 photosynthesis in seed plants has evolved independently at least 45 times (Kellogg 1999; Sage 2003). The first clear evidence of C4 plants in the fossil record coincides with the what appears to be the lowest atmospheric [CO2] in Earth's history, a concentration that was maintained with only minor fluctuations until the Industrial Revolution (Cerling 1999). The repeated evolution of C4 plants, despite the complexity of the process, is strong evidence that there may be no other adaptive variability to use among land plants for decreasing photorespiration. If there were forms of Rubisco with improved ability to discriminate against oxygenation, then it would surely have been a simpler route for evolution than selecting the complex syndrome of changes needed to provide functional C4 photosynthesis. Table 1 shows that from theory, C4 plants will on average have a higher maximum ɛc than C3. This difference increases with temperature because of the increase in photorespiration as a proportion of photosynthesis (Fig. 2), such that this advantage would be most pronounced in the tropics. Indeed the highest known productivity in natural vegetation is for a C4 perennial grass in the central Amazon, which achieves a net production of 100 t (dry matter) ha−1 year−1 (Piedade et al. 1991; Long 1999; Morison et al. 2000). Of our major food crops, only maize and sorghum are C4 (Long 1998). Is there a theoretical advantage in the C4 process and can it be transferred to our major C3 crops?
C4 plants have the advantage of eliminating energy loss in photorespiration, but at the expense of additional energy, typically 2 ATPs per CO2 assimilated. Because the specificity of Rubisco for CO2 and the solubility of CO2 relative to O2 decline with increases in temperature, photorespiration as a proportion of photosynthesis increases with temperature. In dim light, when photosynthesis is linearly dependent on the radiative flux, the rate of CO2 assimilation depends entirely on the energy requirements of carbon assimilation (Long, Postl & Bohlàr-Nordenkampf 1993; Long 1999). The additional ATP required for assimilation of one CO2 in C4 photosynthesis, compared with C3 photosynthesis, increases the energy requirement in C4 plants (Hatch 1987). However, when the temperature of a C3 leaf exceeds ca. 25 °C, the amount of light energy diverted into photorespiratory metabolism in C3 photosynthesis exceeds the additional energy required for CO2 assimilation in C4 photosynthesis (Hatch 1992; Long 1999). This means that below ca. 25–28 °C, C4 photosynthesis is less efficient than C3 photosynthesis under light-limiting conditions [i.e. it has a lower quantum yield (ΦCO2)]. This is demonstrated in Fig. 3a, in which values of ΦCO2 were calculated from theory (Long 1999). This is very similar to actual measurements of the temperature response of ΦCO2 in C3 and C4 species (Ehleringer & Björkman 1977; Ehleringer & Pearcy 1983).
Figure 3. (a) The predicted maximum quantum yield of photosynthetic CO2 uptake for leaves of C3 and C4 species under different temperatures following. (b) The predicted rates of gross canopy CO2 uptake integrated over a diurnal course for a range of canopy temperatures. The simulation is for a leaf area index of 3 assuming a spherical distribution of foliar elements, on 30 June and with clear sky conditions (atmospheric transmittance = 0.75) at a latitude of 52 °N. Redrawn from Long (1999).
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Total photosynthesis by a crop canopy, however, reflects a combination of light-limited and light-saturated CO2 assimilation. At light saturation, the efficiency of photosynthesis is independent of the maximum quantum yield of CO2 uptake (ΦCO2) and depends on the maximum rate of photosynthesis (Asat). Here, the C4 plant has an advantage, even below 25 °C, because its maximum rate is greater than that of an equivalent C3 leaf because of the absence of photorespiration, as shown in Fig. 2. Does a higher rate of light-saturated photosynthesis offset the lower rate of light-limited photosynthesis at the crop canopy level at temperatures below 25 °C? Note the dynamic nature of the balance between light-limited and light-saturated photosynthesis within a canopy over the course of a day. By combining established steady-state biochemical models of C3 and C4 leaf photosynthesis (Farquhar et al. 1980; Collatz, Ribascarbo & Berry 1992) with canopy radiation transfer models, the integrals of the diurnal course of photosynthesis can be calculated (Humphries & Long 1995). Using this approach, Fig. 3b shows that while the advantage of C4 photosynthesis diminishes with temperature, there is still an advantage to the simulated daily integral of canopy CO2 uptake even at 5 °C. Thus, even at the cool early growing season temperatures typical of temperate climates, some advantage can theoretically be gained from C4 photosynthesis. That this can occur in practice is supported by the observation that the highest known dry matter productivity for the UK is for the cold-adapted C4 perennial grass Miscanthus × giganteus that produces 29 t (dry matter) ha−1 in southern England with a measured ɛc of 0.039 (Beale & Long 1995; Beale, Morison & Long 1999). At the least, this suggests that with continued improvement in cold tolerance, maize may outyield C3 crops even in cool climates, such as NW Europe.
Figure 3b shows that for a tropical C3 crop such as rice, substantial gains in ɛc may be made by engineering the addition of the photosynthetic C4 cycle into the crop. Genes coding for the enzymes of the photosynthetic C4 cycle have been isolated from maize and other C4 plants, and have been used, both singly and in combination, to transform rice and other C3 crop species (reviewed in detail by Raines 2006). While high activity of the introduced C4 enzymes is achieved in many cases, there is little evidence that over-expression of C4 genes in C3 species alters photosynthetic characteristics or increases yield (Häusler et al. 2002; Miyao 2003), with only a few exceptions (e.g. Sheriff et al. 1998; Ku et al. 2001). Furthermore, while it is now possible to transform C3 plants to express the C4 pathway enzymes in a single cell, C4 plants differ not only in their use of the photosynthetic C4 cycle, but also in spatial separation of PEPc and Rubisco. In C4 plants, there is a semi-impermeable barrier between the mesophyll and bundle sheath cells, which limits the diffusion of CO2 released in the bundle sheath back into the mesophyll. Any CO2 that diffuses back must be reassimilated, increasing the requirement of ATP and energy requirement per CO2 molecule assimilated. Figure 3b assumes a leakage rate of 10% (i.e. 1 in 10 CO2 molecules diffuses back into the mesophyll). If the entire mechanism is engineered within a single cell as is being attempted in rice (i.e. PEPc in the cytoplasm and Rubisco in the chloroplast), then leakage of CO2 would be very much higher. As such, the additional ATP required in recycling CO2 would drive the maximum ɛc well below that of C3 photosynthesis. von Caemmerer (2003) shows from theory that such a single cell system would be very inefficient because of the leakage of a large proportion of the CO2 released at Rubisco. As such, a single-cell C4 system would allow a plant to maintain a positive carbon balance under high light and drought conditions, but would be very inefficient at low light or in dense canopies. Two naturally occurring C4 plants have been identified in which the process occurs within a single cell. However, these are elongated cells in which PEPc and Rubisco are spatially separated by distance (Voznesenskaya et al. 2001, 2002; Edwards, Franceschi & Voznesenskaya 2004). Both are slow-growing species of hot semiarid environments consistent with the theoretical analysis of von Caemmerer (2003). Although higher photosynthetic rates have been suggested to occur in rice transformed with pyruvate orthophosphate dikinase (PPDK) and PEPc, this appears a result of increased stomatal aperture rather than of increased capacity within the mesophyll (Ku et al. 2001). The analysis of von Caemmerer (2003) shows that simple expression of the C4 enzymes in the mesophyll of C3 crops is not adequate in obtaining the ɛc advantages of C4 photosynthesis. This requires understanding of the integrated development of Kranz anatomy, localization of C4 and C3 enzymes, and necessary membrane transporters. Understanding of the development of C4 photosynthesis is still too incomplete to determine the necessary transformations (Monson 1999), although an alternative route may involve the search for a simple ‘genetic switch’ that, when triggered, would induce the formation of Kranz anatomy (Surridge 2002). At present, a more viable approach to concentrating CO2 within a single cell may be to use some of the successful concentrating mechanisms found in algae (reviewed, Giordano et al. 2005). Equally, it should be noted that there are likely opportunities to improve the Yp of C4 crops in cool climates. Although maize and sorghum show a low Yp north of ca. 50 °N, the related C4 grass, M. × giganteus has been shown to be highly productive. Understanding how this is achieved may be critical to increasing the Yp of our existing C4 crops (Beale & Long 1995; Naidu et al. 2003).
An alternative means of decreasing photorespiration is to decrease the oxygenation capacity of Rubisco, but as subsequently explained, this may come with the penalty of decreased carboxylation capacity.
Increasing the efficiency of Rubisco
In considering how to redesign plant canopies, it was noted that photosynthesis at the leaf level is saturated by a PPFD well below full sunlight (Fig. 1c). Referring back to Fig. 1c, it can be seen that the solar radiation exceeds the PPFD needed to saturate photosynthesis for much of a sunny day. Are there other approaches to using this excess energy? The response of photosynthesis to solar energy describes a non-rectangular hyperbola, rising rapidly with increasing solar radiation at low PPFD, but saturating at about 25% of full sunlight. Why does this saturation occur?
Several analyses suggest a colimitation by Rubisco and by capacity for regeneration of RuBP, the primary substrate for CO2 assimilation in C3 leaves. So why not just increase the amount of Rubisco per unit of leaf area? Rubisco is already the most abundant protein in crop leaves, constituting about 50% of the soluble protein of the leaf. Calculations of volumes suggest there may not be physical capacity to add more (Pyke & Leech 1987).
Rubisco appears to carry a double penalty. Firstly, it catalyses oxygenation of RuBP leading to photorespiration. Secondly, the maximum catalytic rate of Rubisco (kcc) is remarkably slow compared with most plant enzymes, such that large amounts of the protein are required to achieve the photosynthetic rates necessary to support high productivities in C3 crops. This inefficiency explains why Rubisco is so much more abundant than any other protein in leaves.
It has long been recognized that genetic modification of Rubisco to increase the specificity for CO2 relative to O2 (τ) would decrease photorespiration and would potentially increase C3 crop productivity. However, when the kinetic properties of Rubisco forms from different photosynthetic organisms are compared, it appears that forms with high τ have low maximum catalytic rates of carboxylation per active site (kcc) (Bainbridge et al. 1995). Theoretical considerations also suggest that increased τ may only be achieved at the expense of kcc. If a fixed inverse relationship between kcc and τ implied from measurements is assumed, and if increased concentration of Rubisco per unit leaf area is not an option, will increased τ result in increased leaf and canopy photosynthesis?
Zhu, Portis & Whitmarsh (2004b) use a mathematical model to explore these questions. From values of τ and kcc reported for Rubisco across diverse photosynthetic organisms, an inverse relationship between kcc on τ was defined. Following the steady-state biochemical model of leaf photosynthesis of Farquhar et al. (1980), the C3 photosynthetic CO2 uptake rate (A) is either limited by the maximum Rubisco activity (Vc,max) or by the rate of regeneration of RuBP, which, in turn, is determined by the rate of whole chain electron transport (J). If J is limiting, increase in τ would increase net CO2 uptake because products of the electron transport chain would be partitioned away from photorespiration into photosynthesis. The effect of an increase in τ on Rubisco-limited photosynthesis depends on both kcc and [CO2]. As in the case of C4 photosynthesis, there are conflicting consequences at the level of the canopy. Increased τ would increase light-limited photosynthesis, while the associated decrease in kcc would lower the light-saturated rate of photosynthesis. Zhu et al. (2004b) simulated the consequences of variation in τ assuming an inverse change in kcc for carbon gain by crop canopies. An increase in τ results in an increase in leaf CO2 uptake at low light, but it decreases CO2 uptake in high light. Over the course of a day, total crop canopy CO2 uptake (Ac′) results from significant amounts of both light-limited and light-saturated photosynthesis. Simulation of Ac′ suggests that the present average τ found in C3 terrestrial plants is supraoptimal for the present atmospheric [CO2] of 370 µmol mol−1, but would be optimal for 200 µmol mol−1, a value remarkably close to the average of the last 400 000 years. This suggests that Rubisco in higher terrestrial plants has adapted to the past atmospheric [CO2], but that further adaptation has been slow and has failed to change in response to the relatively rapid rise in [CO2] that has occurred since the start of the Industrial Revolution.
The thesis that increased [CO2] favours the selection of forms of Rubisco with increased kcc and decreased τ is consistent with the observation that Rubisco from C4 plants, in which the enzyme functions in a high [CO2], typically has a higher kcc and lower τ than in C3 land plants (Seemann, Badger & Berry 1984; Sage 2002;). Similarly, Galmes et al. (2005) suggest that lower [CO2] is found in the mesophyll of plants from saline and arid habitats because of their persistently lower gs, and they provide evidence that this has led to the selection of higher τ forms of Rubisco in some C3 species. Zhu et al. (2004b) show that if Rubisco from the non-green algae Griffithsia monilis can be expressed in place of the present C3 crop Rubisco, then canopy carbon gain can be increased by 27%. These simulations suggest that very substantial increases in crop carbon gain may result if exotic forms of Rubisco can be successfully expressed in C3 plants. Much evidence and theory points towards a strong negative relationship between specificity and catalytic rate of carboxylation in Rubisco. In this case, an indirect result of engineering higher specificity would be lower crop canopy photosynthesis because the detrimental effect of lowered catalytic rate would outweigh the beneficial effect of increased specificity (Zhu et al. 2004b). Ideally, a crop would express a high kcc Rubisco in the upper canopy leaves exposed to full sunlight and a high τ Rubisco in the shaded lower canopy leaves.
Most C3 annual crop canopies form leaves at progressively higher levels so that leaves start life at the top of the canopy and then become progressively shaded as new leaves form above. Shading is sensed in plant leaves by the balance of red/far-red light via the phytochrome system (Gilbert, Jarvis & Smith 2001). One possibility would be for plants to trigger the replacement of a high kcc Rubisco with a high τ form as the leaf acclimates to shade. Table 2 shows that such a system can increase ɛc by 31%, in comparison with an equivalent crop canopy with the current typical Rubisco of C3 crops. This would increase ɛc both by decreasing photorespiratory losses in the lower canopy and by increasing the light-saturated rate of photosynthesis in the upper canopy.
Table 2. Estimates of the daily canopy carbon gain (Ac′) after Zhu et al. (2004b) and assuming the hypothetical replacement of the average form of Rubisco from C3 crop species with Rubiscos from other species
|Species||Ac′ (mmol m−2 d−1)||Ac′ (%)||Asat (µmol m−2 s−1)|
|Current average C3 crop (kcc = 2.5, τ = 92.5)||1040||100||14.9|
|Griffithsia monilis (kcc = 2.6, τ = 167)||1430||127%||21.5|
|Amaranthus edulis (kcc = 7.3, τ = 82)||1250||117%||28.3|
|A. edulis/current (kcc = 2.5, τ = 92.5)||1360||131%||28.3|