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Plant, Cell and Environment (2014) 37, 1981–1984

doi: 10.1111/pce.12386

Commentary

Correlating Rubisco catalytic and sequence diversity within C3 plants with changes in atmospheric CO2 concentrations

CO2 fixation in higher plants is catalysed by Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1 .39) whose catalysis has a pervasive influence on the efficiency of photosynthesis and growth. Surprisingly, Rubisco is endowed with catalytic imperfections, which include confusion of substrate CO2 with O2 and a slow turnover rate. The confusion between CO2 and O2 has become exacerbated through the onset of oxygenic photosynthesis that has significantly reduced the earth’s atmospheric CO2/O2 ratio. To compensate for these imperfections in catalysis, the simpler evolutionary route for C3 plants is to make more Rubisco to maintain viable CO2 assimilation rates – despite the significant nitrogen and energy cost to the plant. In this issue of Plant, Cell & Environment, Galmes et al. (2014) presented Rubisco catalytic data from diverse C3 terrestrial plants from different phylogenetic lineages that have adapted to varying environmental conditions. Their findings help better understand how Rubisco function in C3 plants has adapted to atmospheric CO2 and O2 changes. Understanding such puzzles is a key to identifying solutions for improving Rubisco catalysis that, in coordination with other photosynthesis-enhancing approaches, are aimed at delivering a second ‘green revolution’ to increase productivity in globally important C3 grain crops such as wheat and rice (Evans & von Caemmerer 2011).

RUBISCO CATALYSIS AND ASSEMBLY Constraining Rubisco performance is the complexity of its catalytic chemistry. Activation of each catalytic site requires slow reversible binding of non-substrate CO2 to the ε-amino group of a conserved lysine residue to form a carbamate, which is stabilized by the fast binding of Mg2+ (Cleland et al. 1998). Productive binding and enolization of the substrate ribulose-1,5-bisphosphate (RuBP) can now commence, producing an enediol that is susceptible to both carboxylation and oxygenation (Fig. 1). Addition of CO2 produces a carboxyketone, which is hydrated prior to scission of the C2–C3 bond to produce two molecules of 3-phosphoglycerate (3-PGA) that are cycled through the Calvin cycle to regenerate RuBP or produce triose phosphates which are used to make carbohydrates. In contrast, addition of O2 produces a peroxyketone that is hydrated prior to division into one molecule of 2-phosphoglycolate (2-PG) and one of 3-PGA. As 2-PG is toxic to the chloroplast (Zelitch et al. 2009), Correspondence: R. E. Sharwood. Fax: +61261250758; e-mail: [email protected] © 2014 John Wiley & Sons Ltd

it is recycled back to 3-PGA via the energetically costly photorespiratory pathway that also releases fixed carbon (Fig. 1). Altering Rubisco catalysis in C3 leaves to reduce oxygenase activity, particularly under conditions (such as water stress and high temperatures) where photorespiration is exacerbated, poses a viable pathway towards improving CO2 assimilation rates and plant resource use efficiency (Long et al. 2006). As Rubisco typically comprises 20–50% of the leaf protein in C3 plants, it poses a significant resource cost. Biogenesis of Rubisco in leaves requires a myriad of co-evolved ancillary proteins – many still unknown – to facilitate the highly regulated synthesis and assembly of its eight chloroplast-made large subunits (LSu) and eight cytosol-made small subunits (SSu) (Whitney et al. 2011a). The LSu’s form an L8 core of four L2 dimers with two catalytic sites located at the interface of each paired LSu, with conserved amino acid residues from both LSu contributing to each catalytic site. The SSu’s arrange as tetramers to cap either end of the L8 core to provide structural stability and enable maximal catalysis. The L8S8 enzyme in C3 plants also requires close interaction with Rubisco activase, a protein that maintains Rubisco catalysis by removing inhibitory sugar–phosphate ligands from its catalytic sites in an ATP-dependent process (Andrews 1996).

TESTING RUBISCO STRUCTURE-FUNCTION IN C3-CHLOROPLASTS The complex biogenesis requirements of plant Rubisco render the LSu intolerant to certain amino acid changes and also prevent their functional assembly in hosts such as Escherichia coli. Mutagenic testing of Rubisco plant is however feasible in chloroplasts via replacement of the LSu gene (rbcL) by plastome transformation (Whitney & Sharwood 2008; Whitney et al. 2011b). By this approach, hybrid L8S8 Rubisco comprising tobacco SSu and various sources of plant LSu have been made with the enzyme’s catalytic phenotype matching the Rubisco from which the LSu was sourced (Sharwood et al. 2008; Whitney et al. 2011b). An expansion of this work tested bioinformatic predictions from LSu sequence comparisons to show that only one amino acid was responsible for interchanging Rubisco catalysis in Flaveria species between C3 catalysis (Met-309) and C4 catalysis (Ile-309), which increased carboxylation rate c ) but reduced CO affinity. This demonstrated the fea( kcat 2 sibility, and importance, of experimentally validating bioinformatic amino acid predictions to distinguish those that are catalytic switches from those likely facilitating 1981

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Figure 1. RuBP carboxylation or oxygenation by Rubisco and the requirement for photorespiration. During Rubisco catalysis, CO2 reacts with the enediol of RuBP, followed by a series of complex partial reactions to yield two molecules of 3-phosphoglycerate (3-PGA) that feed into the Calvin cycle to either regenerate substrate RuBP or produce precursors for carbohydrate (CHO) synthesis. Rubisco can also fix O2 to the enediol, leading to the production of one molecule of 3-PGA and one of 2-phosphoglycolate (2-PG); the latter is recycled via photorespiration back into 3-PGA, resulting in CO2 loss and energy consumption.

compatibility with co-evolving ancillary proteins involved in the biogenesis (e.g. generic or Rubisco-specific chaperones) and/or regulation of Rubisco activity (e.g. Rubisco activase).

plants across diverse evolutionary lineages and ecological c , K and K (the apparent K for origins. By measuring kcat c O m O2) for Rubisco from previously under-sampled phylogenetic (mosses, ferns, basal angiosperms) and ecological groups (carnivorous plants, CAM-Crassulacean acid metabolism species and aquatic plants using HCO3−), variations in Rubisco catalysis are uncovered that show correlations in response to evolutionary changes in atmospheric CO2 and O2 levels. They show that reductions in atmospheric CO2/O2 ratios correlate with an increase in Rubisco CO2 affinity, c – consistent with previous albeit at the expense of kcat reports (Tcherkez et al. 2006). In addition, this adaptation was generally accompanied by an increased investment in Rubisco, thereby reducing N-use efficiency. This strategy contrasts with C4 plants that invest energy in operating a CO2 concentrating mechanism (CCM) around Rubisco to impede the resource costs of photorespiration. This has allowed C4 c with limited concern as to plants to select for increases in kcat the trade-off to CO2 affinity. This adaptation has proven particularly favourable for improving resource use efficiency in C4 grasses, where genotypes producing faster Rubisco produce less of the enzyme and thereby show better N-use efficiency (Ghannoum et al. 2005).

IS SCREENING C3 PLANTS THE ANSWER TO FINDING NATURAL IMPROVEMENTS IN RUBISCO CATALYSIS?

WHAT CONSTITUTES A BETTER RUBISCO FOR C3 PLANTS? Improving Rubisco performance is not solely dependent upon increasing its specificity for CO2 as opposed to O2 (Sc/o). When photosynthesis in C3 leaves is limited by Rubisco c catalysis, CO2 assimilation rates are dependent upon kcat and/or the CO2 affinity of their Rubisco under atmospheric levels of O2 [i.e. their Km for CO2 under 20.6% (v/v) O2; Kc21% O2 ] (Andrews & Whitney 2003). Increases in Sc/o therefore need to be accompanied by enhancements in c carboxylation efficiency ( kcat Kc21% O2 ) for C3 photosynthesis to benefit (Whitney et al. 2001). Rubisco in some red algae already have these desired catalytic properties, with one in particular from Griffithsia monilis having a twofold higher c Sc/o and 20% increase in kcat Kc21% O2 relative to plant Rubisco (Whitney et al. 2001). While functionally, this Rubisco would be ideal for transplanting into a C3 plant, with 30% growth improvements predicted (Long et al. 2006), the biogenesis requirements of algae Rubisco are incompatible with leaf chloroplasts. While there is some glimmer of hope that land plants themselves have evolved catalytic diversity of potential benefit to C3 crops (Galmes et al. 2005), there still remains a paucity of data on Rubisco catalysis, especially under varying temperature. Initial studies of catalytic diversity for Rubisco from C3 and C4 plants highlighted significant natural variation (Yeoh et al. 1981), but the last 30 years of escalating genomic information has left us ‘data rich’ with regard to Rubisco sequence information but ‘insight poor’ with regard to fully appreciating catalytic diversity. Galmes et al. (2014) begin to bridge this information disparity by examining LSu sequence with variations in catalysis for Rubisco from C3

As noted earlier, bioengineering Rubisco improvements in C3 crops such as wheat and rice requires changes that c increase carboxylation efficiency ( kcat Kc21% O2 ) and Sc/o. While the catalysis study of Galmes et al. (2014) unfortunately lacks measurements of Sc/o, we used the parameters in c for all Table 1 to calculate Kc21% O2 and re-plot it against kcat 28 C3 species (Fig. 2a). Given the evolutionary divergence of the species, no correlation was readily identifiable between these two parameters (black dashed line, Fig. 2a; r2 = 0.12). However, when only Rubisco from the CAM, carnivorous c Table 1. Comparison of kcat Kc21% O2 from the taxomic group

classifications of Galmes et al. (2014)

Taxa grouping

n

c kcat Kc21% O2

Bryophytes Ferns Basal angiosperm Aquatic macrophyte Gymnosperms CAM Carnivorous C3 low stress C3 water stress

2 2 1 1 2 3 3 7 7

130.3 ± 15.9 147.4 ± 39.1 136.0 143.4 178.9 ± 83.6 210.7 ± 4.0 213.1 ± 48.1 199.7 ± 34.7 189.9 ± 35.8

c kcat Kc21% O2 ratio to C3 low stress taxa

0.65 0.74 0.68 0.72 0.90 1.06 1.07 1.00 0.95

c Improvements in kcat Kc21% O2 mM-1.s-1 were determined by normalizing to values for C3 low stress which contains wheat. n is the number of biological replicates included in each taxa grouping. Kc21% O2 calculated from table 1 values in Galmes et al. (2014) using the equation Kc(1 + O/KO), where O is the O2 concentration in airsaturated H2O; 252 μm.

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1981–1984

Atmospheric CO2 effects on Rubisco in C3 plants

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c ) versus the calculated affinity for CO Figure 2. Comparison of Rubisco catalytic parameters across taxa. (a) Rubisco catalytic turnover ( kcat 2

in the presence of atmospheric O2 concentrations using the formula Kc(1 + O/KO) where O is the O2 concentration in air-saturated H2O; 252 μm from table 1 of Galmes et al. (2014) for C3 plants grouped into bryophytes ( ), ferns ( ), gymnosperms ( ), basal angiosperm ( ), aquatic macrophyte ( ), CAM ( ), carnivorous ( ), C3 low stress ( ) and C3 water stress ( ). Linear regressions were applied to all data (black dashed line) and a subset of ‘higher’ plants, including CAM, carnivorous, C3 low stress and C3 water stress (orange dashed line). The quality of linear fits c and K from table 2 of Galmes et al. (2014). Linear regressions were applied to separate is indicated by the r2 value. (b) Plot of the averages of kcat c c and Kc21% O2 for each taxa grouping. Linear grouping of taxa [coloured as in (a)] excluding the bryophytes. (c) Re-plot of the averaged kcat regressions applied to the groupings as above except bryophytes were included in the lower carboxylation efficiency group. The quality of linear fits is indicated by the r2 value.

and both C3-plant stress grouping are considered, some indication of a linear correlation is detected (orange dashed line, Fig. 2a; r2 = 0.45). Further comparison of the averaged Kc and Kc21% O2 values for each taxonomic and environmental groupc identified two ing (table 2 in Galmes et al. 2014) with kcat collective groups (that omitted bryophytes in the Kc versus c relationship) on distinct trajectories with regard to how kcat reductions in Kc and/or Kc21% O2 were accompanied by reducc (Fig. 2b,c). As shown in Table 1, these alternate tions in kcat linear relationships result in comparable lower carboxylation efficiencies for Rubisco from bryophytes, ferns, the macrophytes, basal angiosperm and gymnosperms, and c slightly higher, but very similar, values for kcat Kc21% O2 among the other C3 samples. One could interpret from this finding that investigating C3 plants for natural improvements in Rubisco might be of limited merit. However, including measurements of Sc/o diversity may allay such an interpretation as beneficial variations in Sc/o have been measured in response to adaptation to environmental cues (Galmes et al. 2005) that would provide significant benefit in the context of C3 crop photosynthesis (Parry et al. 2007).

NEW RUBISCO STRUCTURE-FUNCTION INSIGHT FOR TESTING Galmes et al. (2014) incorporated into their catalytic survey a correlative analysis with LSu amino acid variations.Although a relatively small dataset comprise a highly divergent array of C3 species, their analyses uncover an array of residues tentatively ascribed as potential catalytic switches. Of particular

interest are the amino acids correlated with Rubisco isoforms c with a kcat > 4 (R258Y and A228S) that might be worthwhile targets for transplastomic testing in tobacco (Whitney & Sharwood 2008). As shown by Whitney et al. (2011b), such testing is important for experimentally deciphering what sequence differences truly influence Rubisco catalysis as opposed to those involved in facilitating evolved complementarity with ancillary protein function. Robert E. Sharwood & Spencer M. Whitney Research School of Biology, Australian National University, Canberra, ACT 0200, Australia

REFERENCES Andrews T.J. (1996) The bait in the Rubisco mousetrap. Nature Structural Biology 3, 3–7. Andrews T.J. & Whitney S.M. (2003) Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants [Review]. Archives of Biochemistry and Biophysics 414, 159–169. Cleland W.W., Andrews T.J., Gutteridge S., Hartman F.C. & Lorimer G.H. (1998) Mechanism of Rubisco – the carbamate as general base [Review]. Chemical Reviews 98, 549–561. Evans J.R. & von Caemmerer S. (2011) Enhancing photosynthesis. Plant Physiology 155, 19. Galmes J., Flexas J., Keys A.J., Cifre J., Mitchell R.A.C., Madgwick P.J., . . . Parry M.A.J. (2005) Rubisco specificity factor tends to be larger in plant species from drier habitats and in species with persistent leaves. Plant, Cell & Environment 28, 571–579. Galmes J., Kapralov M.V., Andralojc P.J., Conesa M.A., Keys A.J., Parry M.A. & Flexas J. (2014) Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends. Plant, Cell and Environment. doi: 10.1111/pce.12335. [Epub ahead of print]. Ghannoum O., Evans J.R., Chow W.S., Andrews T.J., Conroy J.P. & von Caemmerer S. (2005) Faster Rubisco is the key to superior nitrogen-use

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efficiency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses. Plant Physiology 137, 638–650. Long S.P., Zhu X.G., Naidu S.L. & Ort D.R. (2006) Can improvement in photosynthesis increase crop yields? Plant, Cell and Environment 29, 315– 330. Parry M.A.J., Madgwick P.J., Carvalho J.F.C. & Andralojc P.J. (2007) Prospects for increasing photosynthesis by overcoming the limitations of Rubisco. The Journal of Agricultural Science 145, 31–43. Sharwood R.E., von Caemmerer S., Maliga P. & Whitney S.M. (2008) The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large Subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth. Plant Physiology 146, 83–96. Tcherkez G.G., Farquhar G.D. & Andrews T.J. (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proceedings of the National Academy of Sciences of the United States of America 103, 7246–7251. Whitney S.M. & Sharwood R.E. (2008) Construction of a tobacco master line to improve Rubisco engineering in chloroplasts. Journal of Experimental Botany 59, 1909–1921.

Whitney S.M., Baldet P., Hudson G.S. & Andrews T.J. (2001) Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. The Plant Journal 26, 535–547. Whitney S.M., Houtz R.L. & Alonso H. (2011a) Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, Rubisco. Plant Physiology 155, 27–35. Whitney S.M., Sharwood R.E., Orr D., White S.J., Alonso H. & Galmes J. (2011b) Isoleucine 309 acts as a C4 catalytic switch that increases ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate in Flaveria. Proceedings of the National Academy of Sciences of the United States of America 108, 14688–14693. Yeoh H.H., Badger M.R. & Watson L. (1981) Variations in kinetic properties of Ribulose-1,5-bisphosphate carboxylases among plants. Plant Physiology 67, 1151–1155. Zelitch I., Schultes N.P., Peterson R.B., Brown P. & Brutnell T.P. (2009) High glycolate oxidase activity is required for survival of maize in normal air. Plant Physiology 149, 195–204.

Received 10 June 2014; accepted for publication 10 June 2014

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1981–1984

Correlating Rubisco catalytic and sequence diversity within C3 plants with changes in atmospheric CO2 concentrations.

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