Can the Cyanobacterial Carbon-Concentrating Mechanism Increase Photosynthesis in Crop Species? A Theoretical Analysis

Experimental elevation of [CO₂] around C₃ crops in the field has been shown to increase yields by suppressing the Rubisco oxygenase reaction and, in turn, photorespiration. Bioengineering a cyanobacterial carbon-concentrating mechanism (CCM) into C₃ crop species provides a potential means of elevating [CO₂] at Rubisco, thereby decreasing photorespiration and increasing photosynthetic efficiency and yield. The cyanobacterial CCM is an attractive alternative relative to other CCMs, because its features do not require anatomical changes to leaf tissue. However, the potential benefits of engineering the entire CCM into a C₃ leaf are unexamined. Here, a CO₂ and HCO₃⁻ diffusion-reaction model is developed to examine how components of the cyanobacterial CCM affect leaf light-saturated CO₂ uptake (A_sat) and to determine whether a different Rubisco isoform would perform better in a leaf with a cyanobacterial CCM. The results show that the addition of carboxysomes without other CCM components substantially decreases A_sat and that the best first step is the addition of HCO₃⁻ transporters, as a single HCO₃⁻ transporter increased modeled A_sat by 9%. Addition of all major CCM components increased A_sat from 24 to 38 µmol m⁻² s⁻¹. Several Rubisco isoforms were compared in the model, and increasing ribulose bisphosphate regeneration rate will allow for further improvements by using a Rubisco isoform adapted to high [CO₂]. Results from field studies that artificially raise [CO₂] suggest that this 60% increase in A_sat could result in a 36% to 60% increase in yield.C₃ species include the major grain crops rice (Oryza sativa) and wheat (Triticum aestivum) and overall accounted for approximately 75% of all primary foodstuff production in 2012 (FAOSTAT, 2013). The yield of many crop species has been substantially improved through breeding and agronomy, but advancement in yield has substantially slowed in many of the major C₃ crops in the last decade, suggesting that limits on yield improvement using these techniques are being reached and that other approaches are needed (Long and Ort, 2010; Ray et al., 2012).One largely unexploited approach would be to improve the efficiency of photosynthesis in these species (Zhu et al., 2010). The photosynthetic enzyme Rubisco catalyzes the reaction of CO₂ with ribulose bisphosphate (RuBP), which eventually forms carbohydrates. However, Rubisco will also react with oxygen as the first step in photorespiration. This reaction is considered wasteful, since energy is consumed to recover RuBP and CO₂ is lost in the process. In C₃ plants at 25°C and current atmospheric [CO₂], photorespiration results in an approximately 30% decrease in net carbon assimilation (Zhu et al., 2010). Thus, it is a large inefficiency in carbon uptake and a target for improvement.Since CO₂ and oxygen act competitively at Rubisco, photorespiration can be decreased by increasing [CO₂] around Rubisco. That this will increase yield is demonstrated by the many studies that have artificially increased atmospheric [CO₂] around C₃ crops growing in the field (Kimball et al., 2002; Long et al., 2006b). Other photosynthetic organisms have evolved mechanisms to internally elevate [CO₂] at Rubisco to decrease or eliminate photorespiration. Such carbon-concentrating mechanisms (CCMs) include C₄ photosynthesis, as in maize (Zea mays), and the carboxysome and pyrenoid CCMs of single-celled cyanobacteria and algae. The C₄ pathway requires the addition of the photosynthetic C₄ dicarboxylate cycle and inner photosynthetic cells (i.e. bundle sheath), where Rubisco is localized. Converting a C₃ crop to a C₄ crop will require a coordination of changes in photosynthetic tissue differentiation and enzyme and transporter localization. In contrast, cyanobacteria achieve the same effect in a single cell by localizing Rubisco to specialized subcellular compartments called carboxysomes, so in theory they would require fewer changes. Carboxysomes are polyhedral bodies with a protein shell that encloses carbonic anhydrase (CA) and Rubisco packed in an ordered or semiordered array (Long et al., 2007; Yeates et al., 2011). Bicarbonate is actively transported from the environment into the cytosol of the cyanobacteria, and CO₂ in the cytosol is actively hydrated to HCO₃⁻ using NADH. CO₂ is also hydrated to HCO₃⁻, which serves to increase the [CO₂] gradient between the medium and cytosol, increasing CO₂ flux, and also serves to refix CO₂ that leaks from the carboxysome. Since the cytosol lacks CA and the plasma membrane has low permeability to HCO₃⁻, a high cytosolic [HCO₃⁻] far from equilibrium with [CO₂] is achieved. Bicarbonate diffuses through the protein shell of the carboxysome, and since CA is localized to the inner side of the shell, it is rapidly converted to CO₂, given the disequilibrium. The resulting high [CO₂] around Rubisco inside the carboxysome accelerates carboxylation and competitively inhibits the oxygenation reaction (for review, see Price et al., 2008, 2011, 2013; Espie and Kimber, 2011).The C₄ pathway has been well characterized, and several projects are attempting to engineer it into C₃ plants with some success (Slewinski, 2013). However, a primary obstacle has been achieving the necessary two tissue types with the correct localization of the key enzymes (Covshoff and Hibberd, 2012). In this regard, cyanobacterial CCM may provide an attractive parallel approach to eliminating photorespiration in C₃ plants for two reasons. First, the biochemical, structural, and genetic components of the CCM are well understood, in that all of the necessary proteins and their genes have been identified. Second, chloroplasts of higher plants evolved from a common ancestor with modern cyanobacteria (Raven and Allen, 2003) and, therefore, are structurally similar. Thus, engineering carboxysomes into the chloroplast could involve introducing an operon containing the genes associated with the cyanobacterial CCM into the chloroplast genome.There are, however, multiple proteins in the cyanobacterial CCM, and simultaneously transforming all of the required genes into an organism may be particularly challenging. Furthermore, some of the proteins appear to have a similar function, and others may be deleterious if introduced without the full apparatus. A random sequence of gene stacking would likely be inefficient, while the addition of some genes may simply be unnecessary. However, the biophysical reactions involved are well understood, and there are some measurements of the kinetics of the enzymatic reactions and transporters. With this information, a mathematical model can be created to simulate the CCM. Such a model will allow deduction of the minimal component set necessary for an improvement of leaf photosynthetic efficiency and identify a logical sequence of gene additions to deliver progressive improvements and avoid any lethal effects. This model will also be used to determine which aspects of the CCM exert the most control over the overall process of photosynthetic CO₂ assimilation, information that can then be used to optimize the CCM, help identify the ideal set of components, or identify parameters that need to be more accurately measured in order to improve the effectiveness of the CCM model. Similar kinetic models have been applied to propose systems optimization of the Calvin cycle when plants are grown in elevated [CO₂] (Zhu et al., 2007), of the whole C₃ photosynthetic system (Zhu et al., 2013), C₄ photosynthesis (Yu et al., 2014), and to determine the mechanistic basis of mesophyll conductance (Tholen and Zhu, 2011).This paper develops a kinetic model to determine the potential of the cyanobacterial CCM engineered into C₃ crops for improving photosynthesis by determining the necessary components and estimating the potential improvement of CO₂ assimilation rate and efficiency. There are four distinct features to the cyanobacterial CCM: (1) a carboxysome with internally localized Rubisco and CA; (2) active transport of HCO₃⁻ from the medium into the cyanobacterial cytosol (the equivalent compartment in higher plants is the stroma of the chloroplast); (3) active hydration of CO₂ to HCO₃⁻ within the cytosol; and (4) the absence of CA in the cyanobacterial cytosol, in contrast to the higher plant stroma, which contains high activities of CA. Cyanobacteria have three HCO₃⁻ transporters and two CO₂ hydration enzymes that have different kinetics and are induced under different conditions (Price et al., 2011). All of these enzymes were examined, giving a total of seven individual components (listed in Fig. 1A) to the full cyanobacterial CCM model. Because of the common ancestry between cyanobacteria and higher plant chloroplasts, the two have homologous membranes and compartments. Proteins were modeled within a C₃ leaf using locations equivalent to those in cyanobacteria. That is, bicarbonate transporters were localized to the inner chloroplast membrane, and carboxysomes were localized to the stroma. The CO₂ hydration enzymes are bound to the thylakoid and plasma membranes in cyanobacteria, but the reaction for both occurs within the cyanobacterial cytosol; therefore, in this model, hydration of CO₂ by these enzymes was localized to the stroma. This model was used to determine the necessity of each of the features noted above, potential improvements in light-saturated CO₂ uptake (A_sat; irradiance of 1,800 µmol photons m⁻² s⁻¹) that would be achieved on incorporation of each feature in turn, a possible sequence of gene additions that would give an incremental improvement, and the key components of the CCM. The model was also used to test the value of using higher plant isoforms of Rubisco versus prokaryotic isoforms adapted to incorporation within the carboxysome. Sources of uncertainty in the model are also defined.Open in a separate windowFigure 1.A_sat of leaf models with sequential addition of components of the cyanobacterial CCM. Each point represents a leaf that contains the component listed for that column and all of the components in the columns to the left of it. The columns represent the sequence of transformations that produce the fastest increase in A_sat assuming that it is not possible to simultaneously add carboxysomes and remove stromal CA and that it is not desirable to add CO₂ hydration enzymes before removal of stromal CA (A) and assuming that it is possible to simultaneously add carboxysomes and remove stromal CA (B).     

Crystal Structure and Functional Characterization of Photosystem II-Associated Carbonic Anhydrase CAH3 in Chlamydomonas reinhardtii

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO₂ and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO₂ fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO₃^– increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO₃^– from CO₂. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO₂/HCO₃^– on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO₂ concentration.Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyze the interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃⁻), a reaction that otherwise proceeds slowly at physiological pH. CAs belong to three evolutionary distinct classes, α, β, and γ, which share no significant amino acid sequence identity and are thought to be the result of convergent evolution (Hewett-Emmett and Tashian, 1996; Supuran, 2008; Ferry, 2010; Rowlett, 2010). Animals have only the α-CA type, but as multiple isoforms. By contrast, higher plants, algae, and cyanobacteria may contain members of all three CA families. In algae, CAs has been found in mitochondria and chloroplasts and in the cytoplasm and apoplasm.Many fresh-water and soil-living microalgae face limiting concentrations of inorganic carbon (Ci) in their environments. To overcome this, the green microalga Chlamydomonas reinhardtii, as well as most other unicellular algae and cyanobacteria, actively accumulate Ci inside the cells. This mechanism is known as the carbon-concentrating mechanism (CCM; Raven, 1997; Wang et al., 2011; Meyer and Griffiths, 2013). CCM allows the algae to maintain a high concentration of CO₂ around the carboxylating enzyme, Rubisco, even under limiting external Ci. The increased concentration of CO₂ in the chloroplast increases the CO₂/O₂ specificity for Rubisco that leads to a decreased oxygenation reaction, and hence carboxylation becomes more efficient.CCM can be induced in C. reinhardtii cultures by bubbling air containing CO₂ at ambient or concentrations (≤0.04%; Vance and Spalding, 2005). Full metabolic adaptation is usually reached within 10 to 12 h after transfer to air CO₂ conditions (Renberg et al., 2010). Already within the first few hours after induction, several genes are either up- or down-regulated (Miura et al., 2004; Yamano et al., 2008; Fang et al., 2012). Surprisingly, the global changes in protein expression do not correspond to those in the gene expression; only few proteins are either up- or down-regulated during CCM induction (Manuel and Moroney, 1988; Spalding and Jeffrey, 1989). CAs are important components of the CCM. In C. reinhardtii, 12 genes are expressed that encode for CA isoforms (Moroney et al., 2011). Among the many genes that are significantly up-regulated during CCM induction, there is one encoding for an apoplastic CA (CrCAH1) and two encoding for mitochondrial CAs (CrCAH4 and CrCAH5; Fujiwara et al., 1990; Eriksson et al., 1996).An α-type CA (CrCAH3) located in the thylakoid lumen in C. reinhardtii has also been identified as important at low CO₂ levels (Karlsson et al., 1998). The sequence indicates that it is transported through the thylakoid membrane via the Twin Arg Translocation pathway (Albiniak et al., 2012). A mutant not expressing CrCAH3 (knockout of the cah3 gene) shows no or poor growth under air CO₂ levels (Spalding et al., 1983; Moroney et al., 1986) and has a severely impaired photosynthetic capacity under low Ci conditions. This mutant, called CrCIA3, has been a valuable tool for resolving the CrCAH3 function.It is also established that CrCAH3 is associated with PSII (Stemler, 1997; Villarejo et al., 2002; Blanco-Rivero et al., 2012). Using isolated PSII membranes from C. reinhardtii, Shutova et al. (2008) presented data suggesting that CrCAH3 is important for efficient water oxidation by facilitating the removal of protons that are produced when water is oxidized by PSII. This is in line with recent studies (Zaharieva et al., 2011; Klauss et al., 2012) showing that it is crucial to have alternating electron and proton removals from the oxygen-evolving complex (OEC) during the five-state catalytic cycle, i.e. the Kok cycle (Kok et al., 1970), of photosynthetic water oxidation. If proton removal is slow, this leads to less efficient O₂ production and consequently may lead to donor side photoinhibition (Minagawa et al., 1996). That HCO₃^– acts as a mobile proton carrier has been recently demonstrated for spinach (Spinacia oleracea) PSII membrane fragments using membrane inlet mass spectrometry (MIMS; Koroidov et al., 2014). These results show that PSII possesses a light- and HCO₃^–-dependent CO₂ production for up to 50% of the O₂ produced.Taken together, these data suggest that CrCAH3 plays an important role in regulating PSII reactions. In this work, we present further evidence for its function in PSII primary reactions, in particular at low Ci concentrations. We determined crystal structures of CrCAH3 at 2.6 to 2.7 Å resolution in complex with acetazolamide (AZM) or phosphate ions. Our results support a zinc-hydroxide catalytic mechanism of CrCAH3 similar to that of other α-CAs. CrCAH3 has, however, an activity optimum at lower pH values than CAs of the same type, which normally operate at pH 7.0 and higher (Demir et al., 2000). The activity optimum of CrCAH3 makes it more suitable for CO₂/HCO₃^– interconversion at the pH levels present in the thylakoid lumen under light exposure.     

Responses of Arabidopsis and Wheat to Rising CO2 Depend on Nitrogen Source and Nighttime CO2 Levels

A major contributor to the global carbon cycle is plant respiration. Elevated atmospheric CO₂ concentrations may either accelerate or decelerate plant respiration for reasons that have been uncertain. We recently established that elevated CO₂ during the daytime decreases plant mitochondrial respiration in the light and protein concentration because CO₂ slows the daytime conversion of nitrate (NO₃−) into protein. This derives in part from the inhibitory effect of CO₂ on photorespiration and the dependence of shoot NO₃− assimilation on photorespiration. Elevated CO₂ also inhibits the translocation of nitrite into the chloroplast, a response that influences shoot NO₃− assimilation during both day and night. Here, we exposed Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) plants to daytime or nighttime elevated CO₂ and supplied them with NO₃− or ammonium as a sole nitrogen (N) source. Six independent measures (plant biomass, shoot NO₃−, shoot organic N, ¹⁵N isotope fractionation, ¹⁵NO₃⁻ assimilation, and the ratio of shoot CO₂ evolution to O₂ consumption) indicated that elevated CO₂ at night slowed NO₃− assimilation and thus decreased dark respiration in the plants reliant on NO₃−. These results provide a straightforward explanation for the diverse responses of plants to elevated CO₂ at night and suggest that soil N source will have an increasing influence on the capacity of plants to mitigate human greenhouse gas emissions.The CO₂ concentration in Earth’s atmosphere has increased from about 270 to 400 µmol mol^–1 since 1800, and may double before the end of the century (Intergovernmental Panel on Climate Change, 2013). Plant responses to such increases are highly variable, but plant nitrogen (N) concentrations generally decline under elevated CO₂ (Cotrufo et al., 1998; Long et al., 2004). One explanation for this decline is that CO₂ inhibits nitrate (NO₃−) assimilation into protein in the shoots of C₃ plants during the daytime (Bloom et al., 2002, 2010, 2012, 2014; Cheng et al., 2012; Pleijel and Uddling, 2012; Myers et al., 2014; Easlon et al., 2015; Pleijel and Högy, 2015). This derives in part from the inhibitory effect of CO₂ on photorespiration (Foyer et al., 2009) and the dependence of shoot NO₃− assimilation on photorespiration (Rachmilevitch et al., 2004; Bloom, 2015).A key factor in global carbon budgets is plant respiration at night (Amthor, 1991; Farrar and Williams, 1991; Drake et al., 1999; Leakey et al., 2009). Nighttime elevated CO₂ may inhibit, have a negligible effect on, or stimulate dark respiration, depending on the plant species (Bunce, 2001, 2003; Wang and Curtis, 2002), plant development stage (Wang et al., 2001; Li et al., 2013), experimental approach (Griffin et al., 1999; Baker et al., 2000; Hamilton et al., 2001; Bruhn et al., 2002; Jahnke and Krewitt, 2002; Bunce, 2004), and total N supply (Markelz et al., 2014). The current study is, to our knowledge, the first to examine the influence of N source, NO₃− versus ammonium (NH₄+), on plant dark respiration at elevated CO₂ during the night.Plant organic N compounds account for less than 5% of the total dry weight of a plant, but conversion of NO₃− into organic N expends about 25% of the total energy in shoots (Bloom et al., 1989) and roots (Bloom et al., 1992). During the day, photorespiration supplies a portion of the energy (Rachmilevitch et al., 2004; Foyer et al., 2009), but at night, this energetic cost is borne entirely by the respiration of C substrates (Amthor, 1995) and may divert a substantial amount of reductant from the mitochondrial electron transport chain (Cousins and Bloom, 2004). The relative importance of NO₃− assimilation at night versus the day, however, is still a matter of intense debate (Nunes-Nesi et al., 2010). Here, we estimated NO₃− assimilation using several independent methods and show in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum), two diverse C₃ plants, that NO₃− assimilation at night can be substantial, and that elevated CO₂ at night inhibits this process.     

Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

An antisense construct targeting the C₄ isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO₂ to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO₂ assimilation rates at high intercellular CO₂ were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO₂ (the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C₄ cycle rate, bundle sheath leak rate, and bundle sheath CO₂ concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C₃ and C₄ cycles that exist during environmental perturbations by light and CO₂ can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C₄ pathway could potentially be improved through a reduction in C₄ cycle activity or increased C₃ cycle activity.In the leaves of a range of plants including maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and millet (Pennisetum americanum), a biochemical pathway known as C₄ photosynthesis has evolved to concentrate CO₂ at the site of Rubisco such that Rubisco can operate at close to its maximal activity and photorespiration is reduced, enhancing the rate of photosynthesis in air (Hatch, 1987; Sage, 2004). In most C₄ plants, CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells into four-carbon acids, which diffuse to an inner ring of bundle sheath cells, where they are decarboxylated and the CO₂ is refixed by Rubisco. Plants using the C₄ photosynthetic mechanism have been subdivided into three primary subtypes, the NADP-malic enzyme (ME), NAD-ME, and phosphoenolpyruvate carboxykinase types, according to the decarboxylating enzyme used to generate CO₂ from C₄ acids in the bundle sheath cells (Hatch, 1987). Flaveria bidentis is a typical NADP-ME dicot in which malate and Asp contribute equally in the transfer of CO₂ to bundle sheath cells (Meister et al., 1996). Presumably, in most C₄ plants, the reactions that facilitate the appropriation, transformation, transport, and eventual concentration of CO₂ in the bundle sheath cell chloroplasts (C₄ cycle) are balanced with the reactions that incorporate CO₂ into usable carbon compounds for energy (C₃/Calvin cycle) such that energy is not lost or wasted as environmental conditions fluctuate. This process is important in maintaining the efficiency of the CO₂-concentrating mechanism and of C₄ photosynthesis overall. The nature of the controlling mechanisms for balance and coordination between the C₃ and C₄ cycles is still unclear, however, and concrete evidence for the coordinated regulation of primary carboxylation in the mesophyll and decarboxylation of C₄ acids in the bundle sheath has not been forthcoming. A key approach to revealing these mechanisms has been the use of antisense RNA in the C₄ dicot F. bidentis to reduce levels of key photosynthetic enzymes, including Rubisco (Furbank et al., 1996), NADP-malate dehydrogenase and pyruvate phosphate dikinase (Furbank et al., 1997), Rubisco activase (von Caemmerer et al., 2005), carbonic anhydrase (Cousins et al., 2006), and PEPC protein kinase (Furumoto et al., 2007). This has proven to be a valuable method to help gain insight into enzyme function and regulation during C₄ photosynthesis and to potentially alter the balance between the C₃ and C₄ cycles.In this study, we targeted the gene encoding the chloroplastic C₄ isozyme of NADP-ME in F. bidentis (Marshall et al., 1996) with an antisense construct designed to reduce its activity in vivo. This isoform is thought to catalyze the decarboxylation of l-malate to pyruvate and CO₂ and of NADP to NADPH in bundle sheath chloroplasts during C₄ photosynthesis (Ashton, 1997; Drincovich et al., 2001), allowing the CO₂ to be fixed into the C₃ cycle by Rubisco and pyruvate to return back to mesophyll cells to be recycled into PEP. These antisense lines were generated for two purposes. First, these plants could be used to confirm the identity of the gene encoding the NADP-ME isozyme involved in C₄ photosynthesis. Several other functioning isoforms of NADP-ME have also been identified within Flaveria spp.: a chloroplastic but potentially nonphotosynthetic NADP-ME form and a cytosolic NADP-ME (Marshall et al., 1996; Drincovich et al., 1998; Lai et al., 2002). The specific role and regulation of a C₄ NADP-ME isozyme in F. bidentis is of interest in relation to the “transfer” or “generation” of a functioning C₄ cycle to C₃ plants (Sheehy et al., 2007; Furbank et al., 2009). A greater understanding of the balance and interactions between this enzyme and others in the C₄ and C₃ cycles will aid in deciding the expression locations and levels needed for C₃ plants to gain a functional CO₂-concentrating mechanism.The second use of these antisense plants was to investigate the degree of coordination between the C₄/C₃ cycles in F. bidentis and the possibility of manipulation to improve photosynthetic efficiency. As mentioned above, the mechanisms of regulation (if any) of the C₃ pathway enzymes such as Rubisco in response to the activity and CO₂ supply rate of the C₄ cycle are unknown. It is similarly unclear how much the reactions of the C₃ cycle affect the rates of the initial CO₂-fixing reactions (carbonic anhydrase and PEPC). Leakiness (ϕ), defined as the ratio of CO₂ leak rate from the bundle sheath to the rate of CO₂ supply, reflects the coordination of the C₄ and C₃ cycles by describing the amount of overcycling of the C₄ cycle that has to occur to support a given rate of net CO₂ assimilation (Furbank et al., 1990; von Caemmerer and Furbank, 1999). As a major C₄ enzyme functioning within the bundle sheath, a reduction in NADP-ME should affect both the C₄ cycle rate and the bundle sheath CO₂ concentration (C_s), possibly disrupting the enzymatic balance and coordination in F. bidentis. Here, we have designed experiments to simultaneously look at in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ), facilitating estimates of ϕ, C₄ cycle rate, and the possible range of C_s within transgenic α-NADP-ME and antisense Rubisco small subunit (α-SSu) F. bidentis plants (Furbank et al., 1996). These measurements aim to show the impact of our perturbations of the C₃/C₄ balance, highlighting possible communication pathways between the cycles and also other possible targets for future genetic manipulation to improve the rate and/or efficiency of photosynthesis in C₄ plants.     

The Minimal CO2-Concentrating Mechanism of Prochlorococcus spp. MED4 Is Effective and Efficient

As an oligotrophic specialist, Prochlorococcus spp. has streamlined its genome and metabolism including the CO₂-concentrating mechanism (CCM), which serves to elevate the CO₂ concentration around Rubisco. The genomes of Prochlorococcus spp. indicate that they have a simple CCM composed of one or two HCO₃⁻ pumps and a carboxysome, but its functionality has not been examined. Here, we show that the CCM of Prochlorococcus spp. is effective and efficient, transporting only two molecules of HCO₃⁻ per molecule of CO₂ fixed. A mechanistic, numerical model with a structure based on the CCM components present in the genome is able to match data on photosynthesis, CO₂ efflux, and the intracellular inorganic carbon pool. The model requires the carboxysome shell to be a major barrier to CO₂ efflux and shows that excess Rubisco capacity is critical to attaining a high-affinity CCM without CO₂ recovery mechanisms or high-affinity HCO₃⁻ transporters. No differences in CCM physiology or gene expression were observed when Prochlorococcus spp. was fully acclimated to high-CO₂ (1,000 µL L⁻¹) or low-CO₂ (150 µL L⁻¹) conditions. Prochlorococcus spp. CCM components in the Global Ocean Survey metagenomes were very similar to those in the genomes of cultivated strains, indicating that the CCM in environmental populations is similar to that of cultured representatives.The marine picocyanobacteria genus Prochlorococcus along with its sister group the marine genus Synechococcus dominate primary production in oligotrophic marine environments (Partensky et al., 1999). Prochlorococcus spp. is an oligotrophic specialist with several key adaptations allowing it to outcompete other phytoplankton in the stable, low-nutrient regions where it thrives. These adaptations include small cell size (less than 1 μm), allowing it to effectively capture nutrients and light, and genome streamlining, which minimizes nutrient requirements (Partensky and Garczarek, 2010). At approximately 1,900 genes, the genomes of high-light-adapted Prochlorococcus spp. are the smallest known among photoautotrophs, suggesting that this is about the minimum number of genes needed to make a cell from inorganic constituents and light (Rocap et al., 2003). Genome reduction has been accomplished by both the loss of entire pathways and complexes, such as the phycobilisomes and many regulatory capabilities, and the paring down of systems to their minimal components, as is the case for the circadian clock and the photosynthetic complexes (Rocap et al., 2003; Kettler et al., 2007; Partensky and Garczarek, 2010).As part of this genome streamlining, the CO₂-concentrating mechanism (CCM), which enhances the efficiency of photosynthesis by elevating the concentration of CO₂ around Rubisco, has been reduced to what appears to be the minimal number of components necessary for a functional CCM (Badger and Price, 2003; Badger et al., 2006). In typical cyanobacteria, the CCM is composed of HCO₃⁻ transporters, CO₂ uptake systems, and the carboxysome, a protein microcompartment in which Rubisco and carbonic anhydrase (CA) are enclosed. HCO₃⁻ is accumulated in the cytoplasm by direct import from the environment and by the active conversion of CO₂ to HCO₃⁻ via an NADH-dependent process, which constitutes the CO₂ uptake mechanism (Shibata et al., 2001). The accumulated HCO₃⁻ then diffuses into the carboxysome, where CA converts it to CO₂, elevating the concentration of CO₂ around Rubisco (Reinhold et al., 1987; Price and Badger, 1989).Whereas some cyanobacteria have up to three different families of HCO₃⁻ transporters with differing affinities for use under different environmental conditions, Prochlorococcus spp. has only one or two families (Badger et al., 2006)_. Most cyanobacteria have low-affinity and high-affinity CO₂ uptake systems, but no CO₂ uptake systems are apparent in Prochlorococcus spp. genomes. The carboxysome of Prochlorococcus spp. and other α-cyanobacteria has apparently been laterally transferred from chemoautotrophs, but all of the required components of the carboxysome are present and it is functional (Badger et al., 2002; Roberts et al., 2012). Despite its simplicity, this CCM is likely functional. HCO₃⁻ can be accumulated in the cytoplasm by the HCO₃⁻ transporters and then diffuse into the carboxysome for conversion to CO₂ and subsequent fixation by Rubisco. However, the functionality of the CCM in Prochlorococcus spp. has not yet been tested. Prochlorococcus spp. is a representative of the α-cyanobacteria, a group with distinct CCMs, which have been much less well studied than the CCMs of β-cyanobacteria (Rae et al., 2011, 2013; Whitehead et al., 2014).We characterized inorganic carbon (C_i) acquisition and processing in Prochlorococcus spp. MED4, examined the effect of long-term acclimation to different CO₂ concentrations on CCM physiology and gene expression, and searched metagenomes for Prochlorococcus spp. CCM genes to determine if CCMs in the natural populations are similar to cultured strains.     

The Rubisco Small Subunit Is Involved in Tobamovirus Movement and Tm-22-Mediated Extreme Resistance

The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.Plant viruses use at least one movement protein (MP) to facilitate viral spread between plant cells via plasmodesmata (PD; Lucas and Gilbertson, 1994; Ghoshroy et al., 1997). Among viral MPs, the MP of tobamoviruses, such as Tobacco mosaic virus (TMV) and its close relative Tomato mosaic virus (ToMV), is the best characterized. TMV MP specifically accumulates in PD and modifies the plasmodesmatal size exclusion limit in mature source leaves or tissues (Wolf et al., 1989; Deom et al., 1990; Ding et al., 1992). TMV MP and viral genomic RNA form a mobile ribonucleoprotein complex that is essential for cell-to-cell movement of viral infection (Watanabe et al., 1984; Deom et al., 1987; Citovsky et al., 1990, 1992; Kiselyova et al., 2001; Kawakami et al., 2004; Waigmann et al., 2007). TMV MP also enhances intercellular RNA silencing (Vogler et al., 2008) and affects viral symptom development, host range, and host susceptibility to virus (Dardick et al., 2000; Bazzini et al., 2007). Furthermore, ToMV MP is identified as an avirulence factor that is recognized by tomato (Solanum lycopersicum) resistance proteins Tobacco mosaic virus resistance-2 (Tm-2) and Tm-2² (Meshi et al., 1989; Lanfermeijer et al., 2004). Indeed, tomato Tm-2² confers extreme resistance against TMV and ToMV in tomato plants and even in heterologous tobacco (Nicotiana tabacum) plants (Lanfermeijer et al., 2003, 2004).To date, several host factors that interact with TMV MP have been identified. These TMV MP-binding host factors include cell wall-associated proteins such as pectin methylesterase (Chen et al., 2000), calreticulin (Meshi et al., 1989), ANK1 (Ueki et al., 2010), and the cellular DnaJ-like protein MPIP1 (Shimizu et al., 2009). Many cytoskeletal components such as actin filaments (McLean et al., 1995), microtubules (Heinlein et al., 1995), and the microtubule-associated proteins MPB2C (Kragler et al., 2003) and EB1a (Brandner et al., 2008) also interact with TMV MP. Most of these factors are involved in TMV cell-to-cell movement.Rubisco catalyzes the first step of CO₂ assimilation in photosynthesis and photorespiration. The Rubisco holoenzyme is a heteropolymer consisting of eight large subunits (RbCLs) and eight small subunits (RbCSs). RbCL was reported to interact with the coat protein of Potato virus Y (Feki et al., 2005). Both RbCS and RbCL were reported to interact with the P3 proteins encoded by several potyviruses, including Shallot yellow stripe virus, Onion yellow dwarf virus, Soybean mosaic virus, and Turnip mosaic virus (Lin et al., 2011). Proteomic analysis of the plant-virus interactome revealed that RbCS participates in the formation of virus complexes of Rice yellow mottle virus (Brizard et al., 2006). However, the biological function of Rubisco in viral infection remains unknown.In this study, we show that RbCS plays an essential role in virus movement, host susceptibility, and Tm-2²-mediated extreme resistance in the ToMV-host plant interaction.     

The Benefits of Photorespiratory Bypasses: How Can They Work?

Bypassing the photorespiratory pathway is regarded as a way to increase carbon assimilation and, correspondingly, biomass production in C₃ crops. Here, the benefits of three published photorespiratory bypass strategies are systemically explored using a systems-modeling approach. Our analysis shows that full decarboxylation of glycolate during photorespiration would decrease photosynthesis, because a large amount of the released CO₂ escapes back to the atmosphere. Furthermore, we show that photosynthesis can be enhanced by lowering the energy demands of photorespiration and by relocating photorespiratory CO₂ release into the chloroplasts. The conductance of the chloroplast membranes to CO₂ is a key feature determining the benefit of the relocation of photorespiratory CO₂ release. Although our results indicate that the benefit of photorespiratory bypasses can be improved by increasing sedoheptulose bisphosphatase activity and/or increasing the flux through the bypass, the effectiveness of such approaches depends on the complex regulation between photorespiration and other metabolic pathways.In C₃ plants, the first step of photosynthesis is the fixation of CO₂ by ribulose bisphosphate (RuBP). For every molecule of CO₂ fixed, this reaction produces two molecules of a three-carbon acid, i.e., 3-phosphoglycerate (PGA), and is catalyzed by the Rubisco enzyme. A small portion of the carbon in PGA is used for the production of Suc and starch, whereas the remainder (i.e. five-sixths) is used for the regeneration of RuBP (Fig. 1). The regeneration of the Rubisco substrate RuBP in the Calvin-Benson-Bassham (CBB) cycle ensures that ample RuBP is available for carbon fixation (Bassham, 1964; Wood, 1966; Beck and Hopf, 1982). Rubisco is a bifunctional enzyme that catalyzes not only RuBP carboxylation but also RuBP oxygenation (Spreitzer and Salvucci, 2002). RuBP oxygenation generates only one molecule of PGA and one molecule of 2-phosphoglycolate (P-Gly; Ogren, 1984). The photorespiratory pathway converts this P-Gly back to RuBP in order to maintain the CBB cycle.Open in a separate windowFigure 1.Schematic representation of the C3 photosynthesis kinetic model with three different photorespiratory bypass pathways. The bypass described by Kebeish et al. (2007) is indicated in blue, the bypass described by Maier et al. (2012) in pink, and the bypass described by Carvalho et al. (2011) in green. The original photorespiratory pathway is marked in orange, and CO₂ released from photorespiration (including the original pathway and bypass pathways) is indicated in red. 2PGA, 2-Phosphoglyceric acid; ASP, Asp; CIT, citrate; ICIT, isocitrate; PGA, 3-phosphoglycerate; DPGA, glycerate-1,3-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; SBP, sedoheptulose-1,7-bisphosphate; S7P, sedoheptulose-7-phosphate; Ri5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; FBP, fructose-1,6-bisphosphatase; F6P, fructose 6-phosphate; Xu5P, xylulose-5-phosphate; G6P, glucose-6-phosphate; G1P, glucose-1-phosphate; ADPG, ADP-glucose; F26BP, fructose-2,6-bisphosphate; UDPG, uridine diphosphate glucose; SUCP, sucrose-6F-phosphate; SUC, Suc; PEP, phosphoenolpyruvate; OAA, oxaloacetate; PGCA, phosphoglycolate; GCA, glycolate; GOA, glyoxylate; GCEA, glycerate; MAL, malate; PYR, pyruvate; GLU, glutamate; KG, alfa-ketoglutarate; GLN, Gln; HPR, hydroxypyruvate; RuBP, ribulose bisphosphate; SER, Ser; GLY, Gly; TS, tartronic semialdehyde.In higher plants, P-Gly is dephosphorylated to glycolate, which is transferred into the peroxisomes, where it is oxidized to hydrogen peroxide and glyoxylate. Then, glyoxylate is aminated to produce Gly, which is subsequently transferred to the mitochondria. There, two molecules of Gly are converted into one Ser plus one CO₂ and one NH₃ (Ogren, 1984; Peterhansel et al., 2010). The Ser is ultimately converted back to PGA (Tolbert, 1997). CO₂ and NH₃ are gasses that can escape to the atmosphere (Sharkey, 1988; Kumagai et al., 2011), and the loss of carbon and nitrogen essential for biomass accumulation will decrease the efficiency of photosynthesis and plant growth (Zhu et al., 2010). Fortunately, both substances are partially reassimilated in the chloroplast, but this results in decreased photosynthetic energy efficiency. At 25°C and current atmospheric CO₂ concentrations, approximately 30% of the carbon fixed in C₃ photosynthesis may be lost via photorespiration and the size of this loss increases with temperature (Sharkey, 1988; Zhu et al., 2010). As a result, photorespiration has been regarded as a pathway that could be altered to improve photosynthetic efficiency (Zelitch and Day, 1973; Oliver, 1978; Ogren, 1984; Zhu et al., 2008, 2010).There are several approaches that may be used to alter photorespiration to improve photosynthetic efficiency. First, it might be possible to increase the specificity of Rubisco to CO₂ versus oxygen (S_c/o; Dhingra et al., 2004; Spreitzer et al., 2005; Whitney and Sharwood, 2007). However, previous studies have shown that there is an inverse correlation between S_c/o and the maximum carboxylation rate of Rubisco (Jordan and Ogren, 1983; Zhu et al., 2004), and there are some indications that the S_c/o of different organisms may be close to optimal for their respective environments (Tcherkez et al., 2006; Savir et al., 2010). Second, a CO₂-concentrating mechanism could be engineered into C₃ plants. For example, introducing cyanobacterial bicarbonate transporters (Price et al., 2011) or introducing C₄ metabolism could be used to concentrate CO₂ in the vicinity of Rubisco and, thereby, suppress the oxygenation reaction of Rubisco (Furbank and Hatch, 1987; Mitchell and Sheehy, 2006). Past efforts to introduce a C₄ pathway into C₃ plants have focused on biochemical reactions related to C₄ photosynthesis without taking into account the anatomical differences between C₃ and C₄ plants, which may have been responsible for the limited success of such endeavors (Fukayama et al., 2003). Recently, there has been renewed interest in engineering C₄ photosynthetic pathways into C₃ plants, with efforts focusing on understanding and engineering the genetic regulatory network related to the control of both the anatomical and biochemical properties related to C₄ photosynthesis (Mitchell and Sheehy, 2006; Langdale, 2011).Transgenic approaches have been used to knock down or knock out enzymes in the photorespiratory pathway. Unfortunately, the inhibition of photorespiration by the deletion or down-regulation of enzymes in the photorespiratory pathway resulted in a conditional lethal phenotype (i.e. such plants cannot survive under ambient oxygen and CO₂ concentrations but may be rescued by growing them under low-oxygen or high-CO₂ conditions; for review, see Somerville and Ogren, 1982; Somerville, 2001). Another approach to reduce photorespiration is to block (or inhibit) enzymes in this pathway using chemical inhibitors. Zelitch (1966, 1974, 1979) reported that net photosynthesis increased by inhibiting glycolate oxidase or glycolate synthesis. However, other groups showed that the inhibition of glycolate oxidase or Gly decarboxylation led to the inhibition of photosynthesis (Chollet, 1976; Kumarasinghe et al., 1977; Servaites and Ogren, 1977; Baumann et al., 1981). It turns out that plants cannot efficiently metabolize photorespiratory intermediates without a photorespiratory pathway, and suppression of this pathway inhibits the recycling of carbon back toward RuBP, which is necessary for maintaining the CBB cycle (Peterhansel et al., 2010; Peterhansel and Maurino, 2011). Moreover, the accumulation of toxic metabolic intermediates (e.g. P-Gly) can strongly inhibit photosynthesis (Anderson, 1971; Kelly and Latzko, 1976; Chastain and Ogren, 1989; Campbell and Ogren, 1990). This may explain why earlier attempts to block or reduce photorespiration have failed to improve carbon gain.Instead of reducing photorespiration directly, a promising idea is to engineer a photorespiratory bypass pathway. Such a pathway would metabolize P-Gly produced by RuBP oxygenation but minimize carbon, nitrogen, and energy losses and avoid the accumulation of photorespiratory intermediates. Kebeish et al. (2007) introduced the glycolate catabolic pathway from Escherichia coli into Arabidopsis (Arabidopsis thaliana); we will subsequently call this type of bypass the Kebeish bypass. In such transgenic plants, glycolate is converted to glycerate in the chloroplasts without ammonia release (Fig. 1). Previous studies suggested that this pathway theoretically requires less energy and shifts CO₂ release from mitochondria to chloroplasts (Peterhansel and Maurino, 2011; Peterhansel et al., 2013); experimental results indicated that the bypass allowed for increased net photosynthesis and biomass production in Arabidopsis (Kebeish et al., 2007). There are reports of two other photorespiratory bypass pathways in the literature (Carvalho, 2005; Carvalho et al., 2011; Maier et al., 2012). In the Carvalho bypass (Carvalho, 2005; Carvalho et al., 2011), glyoxylate is converted to hydroxypyruvate in the peroxisome. Similar to the Kebeish bypass, the ammonia release is abolished, one-quarter of the carbon from glycolate is released as CO₂ in the peroxisomes, and three-quarters of the carbon from glycolate is converted back to PGA. However, this pathway has only been partially realized in tobacco (Nicotiana tabacum); that is, the enzyme of the second reaction of this pathway was not detectable in the transgenic plants, and plants expressing this pathway showed stunted growth when grown in ambient air (Carvalho et al., 2011). The Maier bypass (Maier et al., 2012) is characterized by complete oxidation of glycolate in the chloroplasts. Initial results suggested that the photosynthesis and biomass of transgenic Arabidopsis with this pathway were enhanced (Maier et al., 2012).Recently, the design and benefits of the three bypass pathways were reviewed (Peterhansel et al., 2013), and it was suggested that a photorespiratory bypass can contribute to an enhanced photosynthetic CO₂ uptake rate by lowering energy costs and minimizing carbon and nitrogen losses. However, a systematic and quantitative analysis of the potential contributions of these different factors to photosynthesis improvement has not yet been conducted. Systems modeling can help to design new metabolic pathways and improve our understanding of biochemical mechanisms (McNeil et al., 2000; Wendisch, 2005; Zhu et al., 2007; Bar-Even et al., 2010; Basler et al., 2012). Such models have been used successfully to gain insight into the photosynthetic metabolism (Laisk et al., 1989, 2006; Laisk and Edwards, 2000; Zhu et al., 2007, 2013; Wang et al., 2014). In this study, we use an extended kinetic model of C₃ photosynthesis based on earlier work by Zhu et al. (2007) to systematically analyze the potential of three photorespiratory bypass pathways for improving photosynthetic efficiency (Supplemental Model S1). In addition, we determined under what conditions such bypass pathways may lead to increased photosynthesis and biomass production in C₃ plants and how to further improve the photosynthesis of plants with such a bypass. Our analysis suggests that the benefit of a photorespiratory bypass varies dramatically if it is engineered into different crops.     

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (MLs), p-coumaryl alcohol, coniferyl alcohol (CA), and sinapyl alcohol (SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (Sarkanen and Ludwig, 1971; Boerjan et al., 2003; Ralph et al., 2004). The contribution of various MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three MLs, the lignification process has a high degree of metabolic plasticity (Boerjan et al., 2003; Ralph et al., 2004; Ralph, 2007; Vanholme et al., 2012). Of particular interest are ML conjugates in which the ester group can be acetate (Ac; Sarkanen et al., 1967; Ralph, 1996; Ralph and Lu, 1998; Del Río et al., 2007; del Río et al., 2008; Martínez et al., 2008), p-hydroxybenzoate (pBz; Venverloo, 1971; Monties and Lapierre, 1981; Landucci et al., 1992; Tomimura, 1992a, 1992b; Hibino et al., 1994; Sun et al., 1999; Kuroda et al., 2001; Lu et al., 2004, 2015; Morreel et al., 2004; Rencoret et al., 2013), p-coumarate (pCA; Monties and Lapierre, 1981; Ralph et al., 1994; Crestini and Argyropoulos, 1997; del Río et al., 2008, 2012a, 2012b; Withers et al., 2012; Rencoret et al., 2013; Petrik et al., 2014), or ferulate (FA; Grabber et al., 2008; Ralph, 2010; Wilkerson et al., 2014). In all cases, the MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (Lu and Ralph, 2002, 2008; Del Río et al., 2007; Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in Angelica sinensis and transformed into poplar (Populus alba × grandidentata), which naturally incorporates other acylated MLs, namely ML-pBz conjugates, into its lignin (Wilkerson et al., 2014). Plants that incorporate ML-FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which ML-FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (DFRC), that can release diagnostic chemical marker compounds from lignins containing ML-FAs (Lu and Ralph, 2014; Wilkerson et al., 2014). The DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that ML-FA conjugates are fully incorporated into the lignin of the FMT poplar (Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of pCA in lignin has been highly speculative. It is hypothesized that the pCA moieties may function as a radical sensitizer (Takahama and Oniki, 1996, 1997; Takahama et al., 1996; Ralph et al., 2004; Hatfield et al., 2008; Ralph, 2010). Peroxidases and/or laccases readily oxidize pCA to a radical but are poor oxidizers for SA. Free radicals of pCA readily undergo radical transfer to SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating pCA to an ML, like SA, to form SA-pCA, the most prevalent ML-pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating MLs in monocots (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014).In rice (Oryza sativa), enzymes have been characterized that function specifically in the addition of pCA onto hemicelluloses (Bartley et al., 2013) or lignin (Withers et al., 2012; Petrik et al., 2014). The p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many ML biosynthetic enzymes (Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward p-coumaroyl-CoA and an ML, and has kinetic efficiency with p-coumaryl alcohol > SA > CA. In most grasses, the PMT enzyme predominantly produces SA-pCA conjugates that are then incorporated into the lignin polymer (Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of PMT genes has been performed in Brachypodium distachyon and maize (Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of MLs before polymerization and not in the acylation of hemicelluloses (Petrik et al., 2014). RNA interference-mediated suppression of BdPMT resulted in decreased incorporation of ML-pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the BdPMT gene, which resulted in a complete absence of pCA-acylating B. distachyon lignins, did not affect plant growth or development (Petrik et al., 2014). The arabinose-bound FA and pCA levels remained virtually unchanged in the PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the p-coumaroyl-CoA substrate and its ML acylation. The PMT enzyme identified in maize (pCAT = ZmPMT) also displayed the highest catalytic efficiency with p-coumaroyl-CoA and SA as substrates (Marita et al., 2014). RNAi-mediated suppression of ZmPMT also resulted in decreased production of the ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (Ralph et al., 1994; Ralph, 2010). As a result, the pCA itself can be completely quantified by simple saponification. The units to which the pCA is attached are, like their normal ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the pCA’s terminal location makes p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (Arabidopsis thaliana) and poplar, neither of which naturally produces ML-pCA conjugates, to express a PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of PMT has on the native levels of ML-pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.     

Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins

Spatial segregation of metabolism, such as cellular-localized CO₂ fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are self-assembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.A key enzyme in photosynthesis is the CO₂ fixation enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco not only fixes CO₂, resulting in carbon assimilation, but it can also fix O₂, leading to photorespiration. Suppressing the unwanted oxygenase activity of Rubisco by sequestering Rubisco with a source of CO₂ is Nature’s solution to this substrate discrimination problem. While C4 plants compartmentalize CO₂ fixation in specific cells (Hibberd et al., 2008; Parry et al., 2011), cyanobacteria have evolved a specialized organelle composed entirely of protein to encapsulate Rubisco—the carboxysome.The carboxysome is just one type of bacterial microcompartment (BMC), widespread, functionally diverse bacterial organelles (Axen et al., 2014). All BMCs consist of an enzymatic core surrounded by a selectively permeable protein shell (Kerfeld et al., 2005; Tanaka et al., 2008; Chowdhury et al., 2014; Kerfeld and Erbilgin, 2015). While the encapsulated enzymes differ among functionally distinct BMCs, they share an architecturally similar shell composed of three types of proteins: BMC-H, BMC-T, and BMC-P forming hexamers, pseudohexamers, and pentamers, respectively (Kerfeld and Erbilgin, 2015). These constitute the building blocks of a self-assembling, apparently icosahedral shell with a diameter ranging from 40 to 400 nm (Shively et al., 1973a,b, 1998; Price and Badger, 1991; Bobik et al., 1999; Iancu et al., 2007, 2010; Petit et al., 2013; Erbilgin et al., 2014). Recent studies have also shown that in the biogenesis of BMCs an encapsulation peptide (EP) (Fan and Bobik, 2011; Kinney et al., 2012; Aussignargues et al., 2015; Jakobson et al., 2015), a short (approximately 18 residues) amphipathic α-helix mediates interactions between a subset of core protein and the shell (Fan and Bobik, 2011; Choudhary et al., 2012; Kinney et al., 2012; Lawrence et al., 2014; Lin et al., 2014; Aussignargues et al., 2015). Indeed, because they are self-assembling organelles composed entirely of protein, BMCs hold great promise for diverse applications in bioengineering and development of bionanomaterials (Frank et al., 2013; Chowdhury et al., 2014; Chessher et al., 2015; Kerfeld and Erbilgin, 2015); the key features of BMCs include selective permeability, spatial colocalization of enzymes, the establishment of private cofactor pools, and the potentially beneficial effects of confinement on protein stability. For example, introducing carboxysomes into plants could provide a saltational enhancement of crop photosynthesis (Price et al., 2013; Zarzycki et al., 2013; Lin et al., 2014; McGrath and Long, 2014).The β-carboxysome, which sequesters form 1B Rubisco, has been an important model system for the study of the structural basis of carboxysome function, assembly, and engineering (Kerfeld et al., 2005; Tanaka et al., 2008; Cameron et al., 2013; Aussignargues et al., 2015; Cai et al., 2015). Beta-carboxysomes assemble from the inside out (Cameron et al., 2013; Gonzalez-Esquer et al., 2015). Two proteins that are absolutely conserved and unique to β-carboxysomes, CcmM and CcmN, play essential roles in this process: CcmM crosslinks Rubisco through its C-terminal Rubisco small subunit-like domains (SSLDs; pfam00101); CcmM and CcmN interact through their N-terminal domains; and C-terminal EP of CcmN interacts with the carboxysome shell.Here we describe a system for producing synthetic β-carboxysome shells and encapsulating nonnative cargo. We constructed a synthetic operon composed of ccmK1, ccmK2, ccmL, and ccmO, genes encoding, respectively, two BMC-H proteins, a BMC-P protein, and a BMC-T protein of the carboxysome shell of the halotolerant cyanobacterium, Halothece sp. PCC 7418 (Halo hereafter). Recombinant shells composed of all four proteins were produced and purified. We also demonstrated that the terminal α-helices of CcmK1 and CcmK2 are not, as had been proposed (Samborska and Kimber, 2012), required for the shell formation, and that the synthetic shell is a single-layered protein membrane. Cargo could be targeted to the interior of the synthetic shells using either the EP of CcmN or the N-terminal domain of CcmM; the latter observation provides new insight into the organization of the β-carboxysome. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly but also provide a platform to study carboxysome shell permeability. These results will be useful in guiding the design and optimization of carboxysomes and other BMCs for introduction into plants.     

Genome-Wide Association of Carbon and Nitrogen Metabolism in the Maize Nested Association Mapping Population

Carbon (C) and nitrogen (N) metabolism are critical to plant growth and development and are at the basis of crop yield and adaptation. We performed high-throughput metabolite analyses on over 12,000 samples from the nested association mapping population to identify genetic variation in C and N metabolism in maize (Zea mays ssp. mays). All samples were grown in the same field and used to identify natural variation controlling the levels of 12 key C and N metabolites, namely chlorophyll a, chlorophyll b, fructose, fumarate, glucose, glutamate, malate, nitrate, starch, sucrose, total amino acids, and total protein, along with the first two principal components derived from them. Our genome-wide association results frequently identified hits with single-gene resolution. In addition to expected genes such as invertases, natural variation was identified in key C₄ metabolism genes, including carbonic anhydrases and a malate transporter. Unlike several prior maize studies, extensive pleiotropy was found for C and N metabolites. This integration of field-derived metabolite data with powerful mapping and genomics resources allows for the dissection of key metabolic pathways, providing avenues for future genetic improvement.Carbon (C) and nitrogen (N) metabolism are the basis for life on Earth. The production, balance, and tradeoffs of C and N metabolism are critical to all plant growth, yield, and local adaptation (Coruzzi and Bush, 2001; Coruzzi et al., 2007). In plants, there is a critical balance between the tissues that are producing energy (sources) and those using it (sinks), as the identities and locations of these vary through time and developmental stage (Smith et al., 2004). While a great deal of research has focused on the key genes and proteins involved in these processes (Wang et al., 1993; Kim et al., 2000; Takahashi et al., 2009), relatively little is known about the natural variation within a species that fine-tunes these processes in individual plants.In addition, a key aspect of core C metabolism involves the nature of plant photosynthesis. While the majority of plants use standard C₃ photosynthetic pathways, some, including maize (Zea mays) and many other grasses, use C₄ photosynthesis to concentrate CO₂ in bundle sheath cells to avoid wasteful photorespiration (Sage, 2004). Under some conditions (such as drought or high temperatures), C₄ photosynthesis is much more efficient than C₃ photosynthesis. Since these conditions are expected to become more prevalent in the near future due to climate change, various research groups are working to convert C₃ crop species to C₄ metabolism in order to boost crop production and food security (Sage and Zhu, 2011). Beyond this, better understanding of both C₃ and C₄ metabolic pathways will aid efforts to breed crops for superior yield, N-use efficiency, and other traits important for global food production.In the last two decades, quantitative trait locus (QTL) mapping, first with linkage analysis and later with association mapping, has been used to dissect C and N metabolism in several species, including Arabidopsis (Arabidopsis thaliana; Mitchell-Olds and Pedersen, 1998; Keurentjes et al., 2008; Lisec et al., 2008; Sulpice et al., 2009), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Hirel et al., 2001; Limami et al., 2002; Zhang et al., 2006, 2010a, 2010b). These studies identified key genetic regions underlying variation in core C and N metabolism, many of which include candidate genes known to be involved in these processes.Previous studies of genetic variation for C and N metabolism are limited by the fact that they identified trait loci only through linkage mapping in artificial families or through association mapping across populations of unrelated individuals. Linkage mapping benefits from high statistical power due to many individuals sharing the same genotype at any given location, but it suffers from low resolution due to the limited number of generations (and hence recombination events) since the initial founders. Association mapping, in turn, enjoys high resolution due to the long recombination histories of natural populations but suffers from low power, since most genotypes occur in only a few individuals. In addition, many of these studies focused on C and N in artificial settings (e.g. greenhouses or growth chambers) instead of field conditions, running the risk that important genetic loci could be missed if the conditions do not include important (and potentially unknown) natural environmental variables.To address these issues and improve our understanding of C and N metabolism in maize, we used a massive and diverse germplasm resource, the maize nested association mapping (NAM) population (Buckler et al., 2009; McMullen et al., 2009), to evaluate genetic variation underlying the accumulation of 12 targeted metabolites in maize leaf tissue under field conditions. This population was formed by mating 25 diverse maize lines to the reference line, B73, and creating a 200-member biparental family from each of these crosses. The entire 5,000-member NAM population thus combines the strengths of both linkage and association mapping (McMullen et al., 2009), and it has been used to identify QTLs for important traits such as flowering time (Buckler et al., 2009), disease resistance (Kump et al., 2011; Poland et al., 2011), and plant architecture (Tian et al., 2011; Peiffer et al., 2013). Most importantly, this combination of power and resolution frequently resolves associations down to the single-gene level, even when using field-based data.The metabolites we profiled are key indicators of photosynthesis, respiration, glycolysis, and protein and sugar metabolism in the plant (Sulpice et al., 2009). By taking advantage of a robotized metabolic phenotyping platform (Gibon et al., 2004), we performed more than 100,000 assays across 12,000 samples, with two independent samples per experimental plot. Raw data and the best linear unbiased predictors (BLUPs) of these data were included as part of a study of general functional variation in maize (Wallace et al., 2014), but, to our knowledge, this is the first in-depth analysis of these metabolic data. We find strong correlations among several of the metabolites, and we also find extensive pleiotropy among the different traits. Many of the top QTLs are also near or within candidate genes relating to C and N metabolism, thus identifying targets for future breeding and selection. These results provide a powerful resource for those working with core C and N metabolism in plants and for improving maize performance in particular.     

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO₂ limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO₂-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO₂ are not available.In photosynthesis, photon energy absorbed by PSI and PSII in thylakoid membranes oxidizes the reaction center chlorophylls (Chls), P700 in PSI and P680 in PSII, and drives the photosynthetic electron transport (PET) system. In PSII, water is oxidized to oxygen as the oxidized P680 accepts electrons from water. These electrons then reduce the cytochrome b₆/f complex through plastoquinone (PQ) in the thylakoid membranes. Photooxidized P700 in PSI accepts electrons from the reduced cytochrome b₆/f complex through plastocyanin or cytochrome c₆. Electrons released in the photooxidation of P700 are used to produce NADPH through ferredoxin and ferredoxin NADP⁺ reductase. Thus, electrons flow from water to NADPH in the so-called photosynthetic linear electron flow (LEF). Importantly, LEF induces a proton gradient across the thylakoid membranes, which provides the driving force for ATP production by ATP synthases in the thylakoid membranes. NADPH and ATP serve as chemical energy donors in the photosynthetic carbon reduction cycle (Calvin cycle).It recently has been proposed that, in cyanobacteria, the photorespiratory carbon oxidation cycle (photorespiration) functions simultaneously with the Calvin cycle to recover carbon for the regeneration of ribulose-1,5-bisphosphate, one of the substrates of Rubisco (Hagemann et al., 2013). Rubisco catalyzes the primary reactions of carbon reduction as well as oxidation cycles. However, the presence of a specific carbon concentration mechanism (CCM) in cyanobacteria had been thought to prevent the operation of photorespiration. CCM maintains a high concentration of CO₂ around Rubisco so that the oxygenase activity of Rubisco is suppressed (Badger and Price, 1992). However, recent studies on mutants deficient in photorespiration enzymes have shown that photorespiration functions, particularly under CO₂-limited conditions, in cyanobacteria as it does in higher plants (Eisenhut et al., 2006, 2008).Decreased consumption of NADPH under CO₂-limited or high-light conditions causes electrons to accumulate in the PET system. As a result, the photooxidation and photoreduction cycles of the reaction center Chls in PSI and PSII become uncoupled from the production of NADPH, inducing alternative electron flow (AEF) pathways (Mullineaux, 2014). In cyanobacteria, several AEFs that differ from those in higher plants are proposed to function as electron sinks (Mullineaux, 2014). Electrons accumulated in the PET system flow to oxygen through FLAVODIIRON1 (FLV1) and FLV3 proteins in PSI and the terminal oxidase, cytochrome c oxidase complex, and cytochrome bd-quinol oxidase (Pils and Schmetterer, 2001; Berry et al., 2002; Helman et al., 2003; Nomura et al., 2006; Lea-Smith et al., 2013). Cyanobacterial FLV comprises a diiron center, a flavodoxin domain with an FMN-binding site, and a flavin reductase domain (Vicente et al., 2002). In Synechocystis sp. PCC 6803, Helman et al. (2003) identified four genes encoding FLV1 to FLV4 and showed that FLV1 and FLV3 were essential for the photoreduction of oxygen by PSI. FLV1 and FLV3 were proposed to function as a heterodimer (Allahverdiyeva et al., 2013). FLV2/4 have been proposed to function in energy dissipation associated with PSII (Zhang et al., 2012). In addition, hydrogenases convert H⁺ to H₂ with NADPH as an electron donor (Appel et al., 2000). Furthermore, Flores et al. (2005) suggested that the nitrate assimilation pathway functions in AEF when the cells live in medium containing nitrate.To elucidate the physiological functions of these AEFs, evaluation of the presence and capacity of each AEF pathway is required. Therefore, in vivo analyses of electron fluxes are essential. We had found that an electron flow uncoupled from photosynthetic oxygen evolution functioned under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803 but not in Synechococcus elongatus PCC 7942 (Hayashi et al., 2014), indicating that an AEF operated in Synechocystis sp. PCC 6803. This AEF was induced in high-[CO₂]-grown Synechocystis sp. PCC 6803 during the transition from CO₂-saturated photosynthesis to CO₂-limited photosynthesis (Hayashi et al., 2014). In contrast, in Synechocystis sp. PCC 6803 grown at ambient CO₂ concentration, AEF was detected immediately following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014), suggesting that AEF was already induced under ambient atmospheric conditions.The expression of the AEF activity observed under CO₂-limited photosynthesis required the presence of oxygen in Synechocystis sp. PCC 6803 (Hayashi et al., 2014). In Synechocystis sp. PCC 6803, FLV1/3 were proposed to catalyze the photoreduction of oxygen (Helman et al., 2003). However, Hayashi et al. (2014) found no evidence that FLV1/3 operated under CO₂-limited photosynthesis: a mutant Synechocystis sp. PCC 6803 deficient in FLV1/3 maintained almost constant electron flux under CO₂-limited photosynthesis after the transition from CO₂-saturated conditions. Thus, the postulated photoreduction of oxygen by FLV1/3 was not responsible for the electron flux observed under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803.In this study, we aimed to elucidate the molecular mechanism of the oxygen-dependent AEF functioning under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. The possibility that FLV2 and FLV4 catalyze the photoreduction of oxygen under CO₂-limited photosynthesis could not be excluded, given that AEF in high-[CO₂]-grown Synechocystis sp. PCC 6803 was induced following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014). Both FLV2 and FLV4 are predicted to possess oxidoreductase motifs, similar to FLV1 and FLV3 (Helman et al., 2003; Zhang et al., 2012). Furthermore, the expression of two FLV genes (flv2 and flv4) was enhanced under low-[CO₂] conditions (Zhang et al., 2009). Zhang et al. (2012) proposed that FLV2 and FLV4 did not donate electrons to oxygen on the basis of the finding that the Synechocystis sp. PCC 6803 mutants deficient in FLV1/3 showed no light-dependent oxygen uptake (Helman et al., 2003). However, Helman et al. (2003) cultivated Synechocystis sp. PCC 6803 strains deficient in FLV1 and FLV3 proteins under high-[CO₂] conditions, and we cannot exclude the possibility that the FLV2 and FLV4 proteins were not produced in the studied cells. Taken together, it seems plausible that FLV2 and FLV4 mediate oxygen-dependent AEF following the transition to CO₂-limited photosynthesis. To evaluate this possibility, we constructed Synechocystis sp. PCC 6803 mutants deficient in flv2 and flv4 and measured their oxygen evolution and Chl fluorescence simultaneously. The mutants showed suppressed LEF after transition to CO₂-limited photosynthesis, similar to S. elongatus PCC 7942. We also tested the possibility that photorespiration functions as an electron sink under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. A recent study revealed photorespiratory oxygen uptake in a flv1/3 mutant under CO₂-depleted conditions (Allahverdiyeva et al., 2011). In this study, we found that the quantum yield of photosystem II [Y(II)] of mutants deficient in genes encoding proteins that function in photorespiration was similar to that of wild-type Synechocystis sp. PCC 6803. Thus, FLV2 and FLV4 appear to function in the oxygen-dependent AEF under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. This inference is further supported by the lack of FLV2 and FLV4 homologs in the genome of S. elongatus PCC 7942 (Bersanini et al., 2014). In addition, we found oxygen-reducing activities of recombinant glutathione S-transferase (GST)-FLV4 fusion protein, similar to those of recombinant FLV3 protein (Vicente et al., 2002). In light of these results, we discuss the molecular mechanism of the oxygen-dependent AEF under CO₂-limited photosynthesis and the physiological function of FLV proteins in Synechocystis sp. PCC 6803.     

Does Low Stomatal Conductance or Photosynthetic Capacity Enhance Growth at Elevated CO2 in Arabidopsis?

The objective of this study was to determine if low stomatal conductance (g) increases growth, nitrate (NO₃⁻) assimilation, and nitrogen (N) utilization at elevated CO₂ concentration. Four Arabidopsis (Arabidopsis thaliana) near isogenic lines (NILs) differing in g were grown at ambient and elevated CO₂ concentration under low and high NO₃⁻ supply as the sole source of N. Although g varied by 32% among NILs at elevated CO₂, leaf intercellular CO₂ concentration varied by only 4% and genotype had no effect on shoot NO₃^– concentration in any treatment. Low-g NILs showed the greatest CO₂ growth increase under N limitation but had the lowest CO₂ growth enhancement under N-sufficient conditions. NILs with the highest and lowest g had similar rates of shoot NO₃^– assimilation following N deprivation at elevated CO₂ concentration. After 5 d of N deprivation, the lowest g NIL had 27% lower maximum carboxylation rate and 23% lower photosynthetic electron transport compared with the highest g NIL. These results suggest that increased growth of low-g NILs under N limitation most likely resulted from more conservative N investment in photosynthetic biochemistry rather than from low g.The availability of water varies in time and space, and plants in a given environment are expected to evolve a stomatal behavior that optimizes the tradeoff of CO₂ uptake for photosynthesis at the cost of transpirational water loss. The resource of CO₂ also varies over time, and plant fossils indicate that stomatal characteristics have changed in response to periods of high and low atmospheric CO₂ over the past 65 million years (Beerling and Chaloner, 1993; Van Der Burgh et al., 1993; Beerling, 1998; Kürschner, 2001; Royer et al., 2001). Relatively low atmospheric CO₂ concentrations (less than 320 µmol mol⁻¹) over the last 23 million years (Pearson and Palmer, 2000) are associated with increased stomatal conductance (g) to avoid CO₂ starvation (Beerling and Chaloner, 1993). Atmospheric CO₂ concentration has risen rapidly from 280 to 400 µmol mol⁻¹ since 1800 and has resulted in lower stomatal density (Woodward, 1987; Woodward and Bazzaz, 1988; Lammertsma et al., 2011). At the current atmospheric CO₂ concentration (400 µmol mol⁻¹), further decreases in g reduce water loss but also restrict CO₂ assimilation and, thus, limit the effectiveness of low g in water-stressed environments (Comstock and Ehleringer, 1993; Virgona and Farquhar, 1996). Elevated CO₂ concentration enhances the diffusion gradient for CO₂ into leaves, which allows g to decrease without severely restricting photosynthetic carbon gain (Herrick et al., 2004). Most consider such an improvement in water use efficiency in C₃ plants to be the main driving force for decreased g at elevated CO₂ concentration, especially in dry environments (Woodward, 1987; Beerling and Chaloner, 1993; Brodribb et al., 2009; Franks and Beerling, 2009; Katul et al., 2010).Water is the most common factor limiting terrestrial plant productivity, but declining stomatal density has also occurred in wetland environments where water stress is uncommon (Wagner et al., 2005). Improved water use efficiency at elevated CO₂ concentration may be shifting the most common factor limiting plant productivity from water to nitrogen (N). In herbarium specimens of 14 species of trees, shrubs, and herbs, leaf N decreased 31% as atmospheric CO₂ increased from about 270 to 400 μmol mol⁻¹ since 1750 (Penuelas and Matamala, 1990). Indeed, many studies have shown that N availability limits the stimulation of plant growth at elevated CO₂ concentration (Luo et al., 2004; Dukes et al., 2005; Reich et al., 2006). That most plants at elevated CO₂ concentration exhibit both lower g and greater N limitation suggests a relationship between these factors.Plants primarily absorb N as nitrate (NO₃^–) in most temperate soils and assimilate a major portion of this NO₃^– in shoots (Epstein and Bloom, 2005). Elevated CO₂ increases the ratio of CO₂ to oxygen in the chloroplast, decreasing photorespiration and improving photosynthetic efficiency (Sharkey, 1988) but inhibiting photorespiration-dependent NO₃^– assimilation (Rachmilevitch et al., 2004; Bloom et al., 2010, 2012; Bloom, 2014). Greater rhizosphere NO₃^– availability tends to enhance root NO₃^– assimilation and decrease the influence of elevated CO₂ concentration on plant organic N accumulation (Kruse et al., 2002, 2003; Bloom et al., 2010).The most important factor regulating chloroplast CO₂ concentration among natural accessions of Arabidopsis (Arabidopsis thaliana) is g and to a lesser extent mesophyll conductance (Easlon et al., 2014). Low g may decrease the ratio of CO₂ to oxygen in the chloroplast at elevated CO₂ concentration, enhancing photorespiration-dependent NO₃^– assimilation. Alternatively, increasing atmospheric CO₂ may down-regulate the need to synthesize enzymes such as Rubisco to support photosynthesis, which conserves organic N, and g may decline as a by-product of lower photosynthetic capacity (Sage et al., 1989; Moore et al., 1998).Here, we examined the influence of atmospheric CO₂ concentration and NO₃^– supply on photosynthesis, leaf N, and growth in near isogenic lines (NILs) of Arabidopsis differing in g. Arabidopsis accessions differ in many traits (including g) and likewise differ in DNA sequence at a large percentage of genes across the genome (Cao et al., 2011). Use of these NILs greatly reduces the proportion of the genome that varies and minimizes the influence of variation in other traits that are frequently associated with low g and could limit growth (Arp et al., 1998). We tested the extent to which (1) low g was associated with greater CO₂ growth enhancement at low and high NO₃⁻ supply; (2) low leaf intercellular CO₂ concentration (C_i) increased shoot NO₃^– assimilation; and (3) low g at elevated CO₂ concentration was associated with altered N utilization in photosynthetic biochemistry.