Will C3 crops enhanced with the C4 CO2-concentrating ...

Journal of Experimental Botany, Vol. 64, No. 13, pp. 3925–3935, 2013doi:10.1093/jxb/ert103 Advance Access publication 12 April, 2013

© The Author [2013]. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Review papeR

Will C3 crops enhanced with the C4 CO2-concentrating mechanism live up to their full potential (yield)?

Steven M. Driever1,* and Johannes Kromdijk2

1 School of Biological Sciences, University of Essex, Colchester, UK2 Wageningen UR Greenhouse Horticulture, Wageningen, The Netherlands

* To whom correspondence should be addressed. E-mail: [email protected]

Received 8 January 2013; Revised 8 March 2013; Accepted 12 March 2013

Abstract

Sustainably feeding the world’s growing population in future is a great challenge and can be achieved only by increas-ing yield per unit land surface. Efficiency of light interception and biomass partitioning into harvestable parts (harvest index) has been improved substantially via plant breeding in modern crops. The conversion efficiency of intercepted light into biomass still holds promise for yield increase. This conversion efficiency is to a great extent constrained by the metabolic capacity of photosynthesis, defined by the characteristics of its components. Genetic manipulations are increasingly applied to lift these constraints, by improving CO2 or substrate availability for the photosynthetic car-bon reduction cycle. Although these manipulations can lead to improved potential growth rates, this increase might be offset by a decrease in performance under stress conditions. In this review, we assess possible positive or negative effects of the introduction of a CO2-concentrating mechanism in C3 crop species on crop potential productivity and yield robustness.

Key words: C4 photosynthesis, CO2-concentrating mechanism, crop productivity, nitrogen metabolism, photorespiration, redox signalling, yield improvement.

Introduction

As the world population keeps increasing, it is vital for the global production of food to match this growth. The most resource-use-efficient way of producing food that is currently known is the intensive cultivation of plants with edible parts, which is why the production of arable crops has been the main focus area to meet future global food demands. Whereas global resources for new arable land are becoming increasingly scarce, the approach to simply con-vert more land to grow crops seems to be a dead end. It appears, therefore, that future increases in food production should be obtained by increasing crop productivity per unit land surface. During the green revolution, various strategies have already been employed to improve this productivity. As a result, efficiency of light interception and biomass par-titioning into harvestable parts (harvest index) have been improved substantially via plant breeding in modern crops.

Although these areas still deserve further exploration, the scientific consensus appears to be that improving the con-version efficiency of intercepted light into biomass holds most promise for the required substantial increases in crop yield capacity.

The potential of introducing CO2-concentrating mechanisms

Central to plant growth is the required energy from absorbed photons for the acquisition of inorganic carbon and subsequent reduction. Under light-limited conditions, any process that limits the availability of NADPH and ATP for photosynthesis tends to directly reduce the capac-ity for carbon assimilation. The photorespiratory cycle is a

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major example of such a competing process, whereby due to limited discriminating ability of ribulose-bisphosphate carboxylase-oxygenase (Rubisco) between O2 and CO2, the substrate ribulose bisphosphate (RuBP) becomes oxygen-ated instead of carboxylated (for reviews, see, for example, Leegood et al., 1995; Foyer et al., 2009; Sage et al., 2012). One of the products of this reaction is phosphoglycolate, which is toxic when allowed to accumulate. Plants convert phosphoglycolate to phosphoglycerate via photorespira-tion, an intricate chain of metabolic conversions that routes from the chloroplast via the peroxisome to the mitochon-dria and back. Although the end product phosphoglycer-ate feeds back into the Calvin–Benson–Bassham (CBB) cycle, photorespiration reduces the net efficiency of using intercepted light to produce carbohydrates because of the release of CO2 during the mitochondrial conversion of glycine to serine and the overall energetic costs associated with production of phosphoglycerate via photorespiration, which are higher compared to the CBB cycle. Additionally, when light is not limiting, RuBP oxygenation limits Rubisco carboxylation capacity by directly competing with carboxy-lation for Rubisco active sites.

Because the rate of RuBP oxygenation relative to car-boxylation is linearly dependent on the ratio of [CO2] over [O2] at the site of Rubisco expression, photorespiration can be drastically suppressed by increasing [CO2] in the chlo-roplast stroma. A number of specific selection pressures (Raven et al., 2008; Sage et al., 2012) has spurred evolution to give rise to a large variety of CO2-concentrating mecha-nisms (CCMs), all succeeding in reducing photorespiration by increasing [CO2]/[O2] around Rubisco. Rather than rely-ing on Rubisco catalytic action for both primary fixation of inorganic carbon, as well as reduction following the CBB cycle, CCMs organize the initial capture of inorganic car-bon in a different way. Many CCMs in cyanobacteria, algae, and some higher plants employ active transport and subcel-lular compartmented conversions of CO2/bicarbonate (cata-lysed by specifically localized carbonic anhydrase activity) to concentrate CO2 in subcellular carboxysomes, pyrenoids, or chloroplast stroma (Raven et al., 2008). Fig. 1A shows an example of a proposed single-cell CO2-concentrating mech-anism (CCM) to be engineered into C3 crops (Price et al., 2013) based on these principles. In C4 (Hatch and Slack, 1966) and crassulacean acid metabolism (CAM) metabolism (Nuernbergk, 1961), different types of CCMs which are pre-sent primarily in higher plants, organic C4 acids are used as a temporary carbon store, releasing CO2 around Rubisco by tissue- or organelle-specific decarboxylase activity (Fig 1B). Additional to the primary fixation of carbon, refixation of (photo-)respired CO2 can also be a major source of inor-ganic carbon. In some species, specific chloroplast arrange-ments force the diffusion path of respired CO2 through the chloroplast stroma, to promote high rates of refixation (see, for example, von Caemmerer and Furbank, 2003). Exclusive expression of glycine decarboxylase in mitochondria of bundle sheath cells can aid recapture of photorespired CO2 as well as help raising the stromal [CO2] in bundle sheath

chloroplasts (also referred to as C2 photosynthesis). This phenomenon is often mentioned as one of the evolution-ary events preceding full expression of the C4 syndrome, as extensively reviewed by Sage et al. (2012).

The presence of a CCM can be beneficial from an agro-nomical point of view. First, suppressing photorespiration allows for increases in the maximal rate of CO2 assimilation. Furthermore, since Rubisco is the major sink for nitrogen in plant leaves, nitrogen-use efficiency of primary productivity tends to be improved by the presence of a CCM in carbon acquisition and reduction: namely, when [CO2] surround-ing Rubisco is (close to) saturation, Rubisco’s operating effi-ciency is higher, so less protein is needed to obtain the same turnover. When the primary fixation of inorganic carbon is performed by enzymes with much lower Km for CO2 (or bicar-bonate) than Rubisco, intercellular [CO2] can also be kept at lower levels than C3 photosynthesis, promoting higher instan-taneous water-use efficiency (WUE). These beneficial effects have led to a number of highly productive CCM crop species, in particular from the C4 grasses (including maize, sorghum, and millet), which provide a significant proportion of the global food supply.

It follows naturally from the previous paragraph to investigate the possible introduction of a CCM into exist-ing C3 crop species as a strategy to improve the conversion efficiency of absorbed light into harvestable biomass. It is, therefore, no surprise to find this idea central to a number of research initiatives with the ultimate aim of improving crop productivity per unit land surface (Matsuoka et al., 2001; Hibberd et al., 2008; McCormick et al., 2012; von Caemmerer et al., 2012). Such an introduction, when suc-cessful, would seem to involve a potentially considerable increase in productivity.

What could be the side-effects of these yield improvement strategies?

Whereas the potential productivity increases of introducing a CCM into C3 crop species might be evident, these increases should be accompanied by (in worst case) neutral effects on yield robustness, in order to avoid greater uncertainty in global food production. Whereas under optimal conditions, increasing photosynthetic CO2 assimilation might lead to a yield bonus, the side-effects may determine the robustness of yield under conditions less advantageous. The growth of plants and harvestable organs leading to crop productivity is a very complex trait, influenced by a plethora of heavily entangled processes. It seems unlikely that photosynthesis can be altered without simultaneously affecting many other processes. These alterations can positively, but also nega-tively, affect the applicability of the altered genotypes in the pursuit of increasing global food supply. In the following paragraphs, we aim to address these points by highlighting a number of key processes involved in yield stability that may be affected by the successful introduction of the C4 CCM into a C3 crop species.

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Will there be enough reducing power left for N metabolism?

Plants take nitrogen from the root environment, in the shape of nitrate or ammonium ions. Sufficient supply of nitrogen is required for the establishment of a productive photo-synthetic apparatus. Chlorophyll (Evans, 1989), as well as

various CBB-cycle enzymes, in particular Rubisco (Makino et al., 1994), comprise major investments of leaf nitrogen. Leaf nitrogen metabolism can be an important determinant of rate-limiting processes in C3 photosynthesis (Yamori et al., 2011), due to the negative correlation between leaf N and the ratio Jmax/Vcmax (Sage et al., 1990; Makino et al., 1994).

Fig. 1. The two types of CO2-concentrating mechanism. (A.) An example of a proposed single-cell CO2-concentrating mechanism (CCM), to be engineered into higher plants. This type, after Price et al. (2013), depends on the presence of a carboxysome in the chloroplast, as well as inorganic carbon transporters that transport bicarbonate (HCO3 –) from outside the cell into the chloroplast and the carboxysome. Carbonic anhydrase (CA) is only present inside the carboxysome, where CO2 is released and Rubisco is located and the initial step of the Calvin–Benson–Bassham (CBB) cycle takes place. (B.) An example of a two-cell CO2-concentrating mechanism (CCM), the NADP-malic enzyme (NADP-ME) type of C4 photosynthesis. This mechanism depends on the fixation of CO2 in one cell type (mesophyll) and the transport to and release in another cell type (bundle sheath). In the mesophyll cytosol, HCO3 – is equilibrated with CO2 by CA, and oxaloacetate (OAA) is produced from the carboxylation of phosphoenolpyruvate (PEP) mediated by PEP carboxylase (PEPC). Inside the mesophyll chloroplast, OAA is converted into malate by malate dehydrogenase (MDH), which is transported into the chloroplast of the bundle sheath cell. Here, malate is decarboxylated into pyruvate and CO2 by NADP-ME and the CO2 is used in the CBB cycle. Pyruvate is transported back to the mesophyll chloroplast where it is phosphorylated to regenerate PEP in a reaction mediated by pyruvate orthophosphate dikinase (PPDK).

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The photorespiratory pathway is strongly intertwined with the pathway of nitrate reduction in C3 species (Fig. 2). Various studies report impaired nitrate metabolism as a result of increased stromal [CO2] (Bloom et al., 1989, 2002, 2010, 2012; Smart et al., 1998; Searles and Bloom, 2003; Rachmilevitch et al., 2004). Interactive effects between N-supply and atmospheric [CO2] were also found for a range of C3 crop species grown in the free-air CO2 enrich-ment (FACE) experiments (Ainsworth and Long, 2005); however, specific effects of nitrate as the source of N were not evaluated. Interestingly, Bloom et al. (2012) suggest that C3 capacity for nitrate metabolism could be acclimated to historical CO2 concentrations (i.e. lower than current) and might already be reduced under current atmospheric conditions.

The reasons for the apparent correlation between pho-torespiration and nitrate metabolism are not completely clear. A number of underlying mechanisms can be distin-guished, although the supporting evidence for their relative importance remains inconclusive (for a complete discussion, see Foyer et al., 2009). First, a decrease in the photorespira-tory production of malate may lead to diminished reductive power (NADH) for the cytosolic reduction of NO3

– to NO2–

by nitrate reductase, although this effect might be counter-acted by the increased export of triose phosphate from the chloroplast at high CO2 (Kaiser et al., 2000). Additionally, transport of nitrite from cytosol to chloroplast stroma, requires co-transport of protons across the chloroplast enve-lope. As diffusion is considered the main mode of transport, this can be hampered by acidification of the stroma relative

Fig. 2. Schematic diagram of photorespiration and nitrogen metabolism, illustrating interactions between these processes. In the process of photorespiration, the oxygenation reaction by Rubisco uses O2 and produces phosphoglycolate (P-Glyco) and glycerate-3-phospate (Gly-3-P). The latter is used in the Calvin–Benson–Bassham (CBB) cycle. P-Glyco is hydrolysed into glycolate (Glyco) and transported into the peroxisome. Glyco is turned into glyoxylate (Glyox), which consumes O2 and produces H2O2. Glyox is then turned into glycine (Gly) and transported to the mitochondrion, where it is decarboxylated. In the mitochondrion, CO2 and NH3 are released, as well as serine (Ser). Ser is transported back into the peroxisome, where it is converted into glycerate via hydroxypyruvate (Hpyr). Glycerate is subsequently returned to the chloroplast where it feeds back into the CCB cycle. Nitrogen metabolism is closely interlinked with photorespiration. The 2-oxoglutarate (2-OG) from the peroxisome is recycled to glutamate (Glu) and returned back to the peroxisome, involving malate. However, in the chloroplast this cycle is shared with the conversion of glutamine (Gln) to Glu, which reassimilates the NH3 released in the mitochondrion. In this reaction, NH3 from other sources can also be used, such as the photosynthetic electron transport-driven, ferredoxin (Fd)-dependent conversion of nitrite from nitrate.

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to the cytosol by higher [CO2] (Shingles et al., 1996; Bloom et al., 2002). Finally, chloroplastic reduction of nitrite and the incorporation of ammonium into amino acids compete with NADP reduction for reduced ferredoxin supplied by photosynthetic electron transport. However, the Km of ferre-doxin-NADP reductase is much lower compared to nitrite reductase and glutamate synthase (Knaff, 1996; Bloom et al., 2010), strongly suppressing nitrate metabolism in the pres-ence of high [NADP+]/[NADPH] in the chloroplast stroma. Reducing photorespiration decreases the proportional demand for ATP relative to NADPH by the photosynthetic dark reactions. As a result, increases in stromal CO2 can lead to higher [NADP+]/[NADPH], thereby limiting the available reducing power for nitrate metabolism.

Higher atmospheric CO2 leads to less capacity for nitrate reduction in C3 plants, but not in crops with a naturally occur-ring CCM, such as the NADP-ME C4 species Zea mays and Flaveria bidentis, the C4 NAD-ME species Amaranthus ret-roflexus, and the CAM species Crassula ovata (Cousins and Bloom, 2003; Bloom et al., 2012). Bloom et al. (2012) also surveyed two Flaveria C3–C4 intermediates, which showed intermediate sensitivity. Although the reasons for these dif-ferences are not clear, a hypothesis could be that nitrate metabolism is tuned to the prevailing chloroplastic stromal CO2 concentration or availability of reductive power in the cytosol. If true, this potentially seriously limits the yields of CCM-enhanced C3 crops when grown with nitrate as the prime nitrogen source. As nitrate is the predominant form of the total N-availability to plants (Epstein and Bloom, 2005), this side-effect could harm the applicability of a CCM-enhanced C3 crop (also noted by Rachmilevitch et al., 2004). Therefore, retuning of the nitrate assimilatory machinery might be required along with the introduction of a CCM in current C3 crop species.

A trade-off between water-use efficiency and drought tolerance?

The exchange of water vapour and CO2 between plants and the surrounding atmosphere share to a large extent the same path of diffusion. As a result, water exchange is usu-ally strongly connected to photosynthetic CO2 assimilation. Loss of leaf water tends to occur via transition from liq-uid to gaseous phase into intercellular airspaces, followed by diffusion through the stomata, whereas intake of CO2 moves in the opposite direction. Although the internal pathway of water movement is not identical to that of CO2, crop water relations can also significantly affect the inter-nal resistance to CO2 (for review, see Flexas et al., 2012). Additionally, leaf internal conductance and stomatal con-ductance can be tightly connected to maintain leaf water status (Savvides et al., 2012) and, as a result, feedback regu-lation from leaf water status can also impact photosynthetic carbon acquisition.

The majority of studies focus on the importance of supe-rior CO2 acquisition under photorespiratory conditions to explain the evolutionary rise of C4 photosynthesis (Edwards

et al., 2001; Sage, 2004; Sage et al., 2012). However, more recently, the beneficial effects of the C4 syndrome from the perspective of plant water relations were highlighted (Osborne and Sack, 2012), as well as the structural benefits of a bundle sheath to prevent xylem cavitation in areas with high evaporative demand (Griffiths et al., 2013). When pri-mary fixation of CO2 is performed by an enzyme with higher affinity for CO2 than Rubisco, introduction of a CCM into a C3 crop species could result in higher instantaneous WUE through reduced stomatal opening, which is commonly found in most C4 species (Long, 1999). This could allow for more conservative water supply during irrigation, although this is also heavily influenced by crop total leaf area and climatic conditions during the growing period.

As stomata are the main gateways for water as well as CO2 exchange, it is not surprising to find adjustments in plant spe-cies with a naturally occurring CCM. For example, in faculta-tive CAM plants, the diurnal rhythmicity in the opening and closing of stomata during CAM metabolism almost perfectly opposes the stomatal behaviour observed for the same plant in C3 mode (Borland et al., 2011). In C4 photosynthesis, devel-opment and regulation of stomatal conductance has also been modified compared to the C3 ancestral form, although the changes are more subtle. Whereas C3 stomata tend to respond more directly to a change in light intensity, Huxman and Monson (2003) found the main mode of response by C4 stomata to be indirect as a result of the corresponding change in intercellular [CO2] (ci). Taylor et al. (2010) also showed lower operating stomatal conductance in C4 compared to C3 in a comparison of phylogenetically closely related spe-cies. Taylor et al. (2012) further elaborated on this difference, demonstrating C4 grasses as having lower maximum stoma-tal conductance than C3 grasses, due to smaller stomata at a given stomatal density. They also provided evidence for this difference to be evolutionarily coupled specifically to the pho-tosynthetic pathway.

Experiments studying the difference in drought-response of C4 compared to C3 species yield remarkable results, as reviewed by (Ghannoum, 2009). Under well-watered con-ditions, C4 species generally tend to have higher instanta-neous WUE (per CO2 fixed), as well as WUE per biomass produced. However, these comparative differences become substantially reduced, and sometimes even disappear alto-gether (Ripley et al., 2007), when drought is applied exper-imentally (Taylor et al., 2011) or when monitored in arid habitats (Ibrahim et al., 2008; Ripley et al., 2010). Whereas the decline of C3 photosynthesis under drought primar-ily reflects a (reversible) reduction in CO2 supply, in C4 photosynthesis the observed reduction is mostly due to a decrease in biochemical capacity. As a result, after an ini-tial decline in ci at mild drought (e.g. Williams et al., 2001; Leakey et al., 2004), ci often actually increases in C4 species when drought is sustained and photosynthetic reductions become insensitive to high atmospheric [CO2] (Ghannoum et al., 2003; Ghannoum, 2009). Limited potential for alter-native electron sinks has been suggested to explain greater sensitivity to water stress in C4 (Ghannoum, 2009). Energy-use efficiency of the C4 CCM also tends to be negatively

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influenced by drought stress, due to increased overcycling (Buchmann et al., 1996; Williams et al., 2001). This could be explained by the biochemical activity of the CCM being less affected by drought than the activity of the CBB cycle. However, increased overcycling may also reflect readjust-ment to balance ATP consumption between photorespira-tory losses and excessive phosphoenolpyruvate regeneration due to leakiness (leak rate of CO2 away from bundle sheath cells to neighbouring mesophyll cells, divided by rate of pri-mary bicarbonate fixation). At high light intensity, such a futile cycle may presumably also act as a protective mecha-nism, when it helps to keep the plastoquinone pool in bun-dle sheath chloroplasts less reduced. At lower light intensity, when CO2 released via leakage primarily originates from mitochondrial respiration, overcycling may actually be ener-getically less costly than a relative increase in photorespira-tion (Kromdijk et al., 2010).

Considering the differences between C4 and C3 photosyn-thesis mentioned in the preceding paragraphs, introduction of a CO2-concentrating mechanism into C3 crop species may increase biomass production, as well as WUE, provided that stomatal regulation can be modified to adjust to the pres-ence of the CO2-concentrating mechanism. However, the higher sensitivity of the performance of C4 photosynthesis to drought highlights a potential decrease in yield robustness, when drought events occur. The quickly reversible nature of the drought-induced reduction in C3 photosynthesis also pro-motes higher yield robustness, compared to the slower recov-ery after rewatering in C4 (Ripley et al., 2010).

An advantage at high temperature, but a disadvantage at low temperature?

The comparative advantage of a CCM changes markedly with temperature. The catalytic action of RuBP oxygenation is favoured over carboxylation when temperature increases. CO2 solubility in water also decreases relative to O2 at higher temperatures, leading to potentially higher rates of photores-piration at higher temperatures (Sage and Kubien, 2007). Since the presence of a CCM suppresses photorespiration, the comparative advantage increases at higher (physiologi-cally relevant) temperatures (Raven, 1991; Long, 1999; Raven et al., 2002). However, in CCMs that occur in zooxanthel-lae, high temperature may actually impair the concentrating mechanism (Jones et al., 1998).

Early work (Long, 1983; Long et al., 1983) on C4 chill-ing sensitivity led to the hypothesis that C4 photosynthesis, although theoretically advantageous even at low tempera-tures (Long, 1983, 1999), might be inherently incompetent at sustaining photosynthetic capacity at low temperatures (below approximately 15 °C). This incompetence was pos-tulated to stem from the lack of cold stability of key C4 proteins, phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase. However, later studies have confirmed the presence of more stable isoforms of these proteins in C4 species adapted to cooler climates, such as Miscanthus × giganteus (Naidu et al., 2003; Wang et al.,

2008). The common chilling sensitivity of these enzymes in C4 species is therefore more likely to be associated with ecological adaptation to warmer climates (Sage and Kubien, 2007).

Since the kinetic properties of Rubisco respond strongly to temperature (Bernacchi et al., 2001), at low temperatures the maximum turnover rate of Rubisco can become severely reduced and be the main factor limiting CO2 assimilation. Because the presence of a CCM commonly results in lower leaf nitrogen investment in Rubisco protein (e.g. Makino et al., 2003), limitation by Rubisco activity in C4 photo-synthesis tends to occur at relatively less cool temperatures compared to C3 photosynthesis (Pittermann and Sage, 2000; Kubien et al., 2003). Together with limited phospho-enolpyruvate regeneration capacity due to declining pyru-vate orthophosphate dikinase activity at low temperature (Naidu et al., 2003; Wang et al., 2008), this results in a severe reduction of the plateau for CO2 assimilation rate in C4 spe-cies below 15 °C. The significance of this reduction is most obvious at both the start and end of the growing season, when the combination of high light intensity and low day-time temperatures (early in the day) occurs frequently, creat-ing considerable risk for photoinhibition in C4 crops (Long, 1983; Long et al., 1994).

Sage and McKown (2006) also suggested that acclima-tion of C4 photosynthesis to low temperatures by increasing Rubisco content may be constrained by space limitations due to the relatively small fraction of leaf tissue where Rubisco is expressed, as well as by generally less phenotypic plasticity associated with the greater structural and biochemical com-plexity to maintain functional C4 compared to C3 photosyn-thesis. However, if any spatial limitations to increase Rubisco content are caused by specific expression of Rubisco in bun-dle sheath chloroplasts, these could presumably be overcome when the introduced CCM can operate within a single cell (such as the example in Fig. 1A).

It appears that the natural cold tolerance found in a minor-ity fraction of C4 species is associated with greater potential for cold acclimation. Work by Liu and Osborne (2008) showed that 20 days chilling pretreatment (15/5 °C day/night) induced cold acclimation in C3 and C4 grass species (naturally co-occurring on a site with regular low temperatures). Although maximal CO2 assimilation rates were more reduced in C4 than C3 species, acclimation to the chilling pretreatment provided significant protection to subsequent high-light freezing treat-ments in both photosynthetic types. The extent of protection was similar in both photosynthetic types and associated with an upregulation of osmotic potential. Although this mode of acclimation does only occur in specific cold-tolerant C4 spe-cies, it provides evidence for a lack of inherent barriers to cold acclimation and subsequent freezing injury in C4 grasses (Liu and Osborne, 2008).

To summarize temperature effects, the introduction of a CCM into C3 crops is likely to lead to superior maximum assimilation rates at temperatures ranging between 20 and 25 °C to approximately 38 to 45 °C and reduced tempera-ture sensitivity of the quantum yield of CO2 fixation (Sage and Kubien, 2007). However, below this temperature range,

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maximum assimilation rates are likely to be lower than the original C3 crop due to limitations by Rubisco content and the compromised ability to acclimate. As a result, biomass production may be boosted when sufficiently high tempera-tures prevail, but beneficial effects on yield could be counter-acted due to a shortened growing period in strongly seasonal climates. At lower temperatures, presence of a CCM is likely to lead to increased risk of photoinhibition, and thus the introduction of a CCM should be accompanied by upregula-tion of photoprotective mechanisms to avoid problems with yield robustness due to cold spells.

Does perturbation of photorespiration affect redox signalling?

The role of photorespiration as a photoprotective mechanism has been known for some time. The photoprotective role is realized through NADH production in the mitochondria and the use of NADH in the peroxisomes, while reducing power from the chloroplast is exported, which facilitates energy dis-sipation (Wingler et al., 2000). However, photorespiration does not only provide photoprotection: it has been shown to be directly involved in repair of the D1 protein (Takahashi et al., 2007). Furthermore, photorespiration is a source of metabolic H2O2, produced in the peroxisome. Through this, photorespiration is involved in the redox status of the peroxi-some and the cell as a whole.

Under the current atmospheric oxygen concentration, production of reactive oxygen species (ROS) is unavoid-able in many different parts of the cell, such as peroxisomes, mitochondria, and chloroplasts. They are produced by pri-mary processes such as photosynthetic electron transport, photorespiration, and mitochondrial respiration, as well as enzymes like glucose oxidase, xanthine oxidase, and several plant peroxidases (Mittler, 2002). Due to their reactive nature, ROS are potentially dangerous for cellular processes, causing oxidative damage to proteins, DNA, and lipids (Halliwell and Gutteridge, 1999). An extensive array of enzymic and non-enzymic detoxification mechanisms allow for management of ROS within safe levels and are found in almost every cellular compartment (Mittler et al., 2004; Torres et al., 2006).

However, when plants are exposed to severe environmen-tal conditions, changes in oxygen metabolism can perturb the balance between reduction and oxidation reactions in the cell in favour of the latter and induce oxidative stress. Under oxi-dative stress, the production of ROS and the subsequent oxi-dation reactions exceed the rate of scavenging as well as the rate of repair of oxidative damage to cell components. When this situation continues, damage may become irreversible and result in a loss of physiological function of the cell or death.

Production of ROS may be deleterious to the cell under certain conditions, but under conditions that are within the adaptive range of the plant, they can also fulfil several beneficial functions. It has been shown that ROS can be involved in redox regulation (Buchanan and Balmer, 2005; Dietz, 2003) and signalling (e.g. Apel and Hirt, 2004; Baier and Dietz, 2005; Mullineaux and Baker, 2010). It has also

been suggested that ROS production could potentially dis-sipate excess excitation energy through the water–water cycle and act as a photoprotective mechanism (Asada, 1999, 2000; Ort and Baker, 2002). Although it has been shown that there is production of ROS in chloroplasts at high light intensi-ties in the presence of alternative electron transport, the low capacity of the water–water cycle makes it unlikely that this mechanism contributes significantly to photoprotection (Driever and Baker, 2011). Therefore, it is more likely that the water–water cycle operates in conjunction with processes such as photorespiration. Furthermore, ROS produced in response to high light are expected to contribute to stress sig-nalling in response to changes in environmental conditions. Particularly H2O2 has been identified as a trigger for retro-grade signalling events from the chloroplast to the nucleus (Galvez-Valdivieso and Mullineaux, 2010; Maruta et al., 2012). However, not only ROS produced in the chloroplast can act as a trigger for retrograde signalling events but also the redox status of the plastoquinone pool itself can act as a trigger (Pfannschmidt et al., 2009) and both events can even occur at the same time (Galvez-Valdivieso and Mullineaux, 2010). This illustrates that the photosynthetic function of chloroplasts acts as an integrator of cues from environmen-tal changes such as increases in light intensity into molecu-lar signals that are vital for adaptation to these changes. As mentioned already, photorespiration is involved in the over-all redox status of the peroxisome and the whole cell, and as a result is directly linked to the ROS signalling network (Fujita et al., 2006; Foyer and Noctor, 2009). This highlights that the prevailing balance between photosynthesis and pho-torespiration could be vital for redox and ROS signalling network functioning.

When striving to improve yield through increasing photo-synthesis, the role of redox and ROS signalling, the antioxi-dant capacity, and the involvement of both the chloroplasts and mitochondria in these should not be overlooked. Introduction of any CCM and the effects this may have on ROS metabolism and the redox status of the cell as a whole can have great impact on both energy flow and ROS signal-ling networks and may compromise the ability of the plant to cope with environmental changes.

Combining high-throughput screening and physiology: phenomics+

A popular strategy to improve crop productivity is phe-nomics, the high-throughput phenotypic screening of large numbers of plants to identify outstanding genotypes (Furbank, 2009). Recently, a number of phenomics cen-tres have been established to apply this approach for yield improvement. To assess any potential side-effects apart from the trait selected by the screening condition, the iden-tified genotypes from a phenomics approach need either to be evaluated mechanistically to explore the physiological basis for the superior performance or empirically by exten-sive (and expensive) further performance assessments. With regards to the potential side-effects or pitfalls mentioned

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in this review, we suggest that these secondary screens could be informed by similar mechanistic analyses for the crop under evaluation. For this purpose, we expect cur-rent advances in combining various omics fields (genomics, transcriptomics, proteomics, metabolomics, phenomics) and (eco)physiological knowledge to be particularly useful, by allowing evaluation of G × E at various levels of inte-gration (Leakey and Lau, 2012).

Conclusions and recommendations

The strategy of enhancing crop yield by means of introduc-tion of a CCM in C3 crop species undoubtedly holds great promise to increase the maximum energy conversion effi-ciency of intercepted solar irradiance into biomass. However, to what extent this improvement can be used to increase agri-cultural production efficiency depends on a range of addi-tional interacting factors. We have tried to define some of these factors by identifying potential side-effects that could coincide with the implementation of a CCM. It is difficult to assess the potential impact of each side-effect, as their real importance remains to be found, as well as to what extent they can be overcome by further genetic manipulations. In the worst case, unwanted side-effects could (1) narrow the range of climatic conditions under which the implementation of a CO2-concentrating-mechanism-enhanced could lead to an actual yield increase or (2) increase the sensitivity of the CO2-concentrating-mechanism-enhanced crop to abiotic stress conditions, leading to a decrease in yield robustness.

By providing a summary of the main potential side-effects that could be expected based on current knowledge, we hope to provide a list for further targeted testing, whenever the first CCM-enhanced C3 genotypes become available. We have focused specifically on side-effects with regards to the crop response to abiotic stress. To allow any potential yield increase to be translated into actual yield increases while preventing an increase in vulnerability, the crop’s response to biotic stress also needs to remain in good shape. We there-fore suggest that a similar analysis should be done to assess whether any interactive effects may occur with regards to crop defence reactions or disease tolerance or proneness, as well as the ability of the CCM-enhanced crop to compete with weed species.

An important assumption interacting with the issues mentioned in this paper is the strength of the correlation between photosynthetic conversion efficiency and actual biomass production and yield. Although there is a good case to expect a correlation (Long et al., 2006; Zhu et al., 2010), this can often be limited to certain phases in the topology of the crop. The specific conditions during these topological phases are especially important when deter-mining to what extent improvements in potential yield will translate into actual yield. Although climatic conditions are relatively stable for a given geographic region, year-by-year variation in seasonal timing and the occurrence of extreme weather events can be substantial. This variation will be reflected in the extent of the improvement of yield

due to the presence of a CCM as well as on the impact of potential side-effects on yield robustness.

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