Abstract
RuBisCO enables net carbon fixation through the carboxylation of RuBP during photosynthesis. Its complex biochemistry and catalytic diversity found among different plants make characterization of RuBisCO properties useful for investigations aimed at improving photosynthetic performance. This chapter reports methods for rapid extraction of soluble proteins to examine RuBisCO catalytic properties, and for large-scale purification of RuBisCO from leaves to measure the specificity of the enzyme toward its gaseous substrates.
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References
Tcherkez G (2013) Modelling the reaction mechanism of ribulose-1,5-bisphosphate carboxylase/oxygenase and consequences for kinetic parameters. Plant Cell Environ 36:1586–1596
Tcherkez GG, Farquhar GD, Andrews TJ (2006) Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Natl Acad Sci U S A 103:7246–7251
Raines CA (2003) The Calvin cycle revisited. Photosynth Res 75:1–10
Andersson I (2008) Catalysis and regulation in Rubisco. J Exp Bot 59:1555–1568
Carmo-Silva E, Scales JC, Madgwick PJ, Parry MAJ (2015) Optimizing Rubisco and its regulation for greater resource use efficiency. Plant Cell Environ 38:1817–1832
Galmés J, Kapralov MV, Andralojc PJ et al (2014) Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends. Plant Cell Environ 37:1989–2001
Orr DJ, Alcântara A, Kapralov MV et al (2016) Surveying Rubisco diversity and temperature response to improve crop photosynthetic efficiency. Plant Physiol 172:707–717
Sharwood RE, Ghannoum O, Kapralov MV et al (2016) Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C3 photosynthesis. Nat Plants 2:16186
Carmo-Silva E, Andralojc PJ, Scales JC et al (2017) Phenotyping of field-grown wheat in the UK highlights contribution of light response of photosynthesis and flag leaf longevity to grain yield. J Exp Bot 68:3473–3486
Parry MAJ, Andralojc PJ, Scales JC et al (2013) Rubisco activity and regulation as targets for crop improvement. J Exp Bot 64:717–730
Sharwood RE, Sonawane BV, Ghannoum O, Whitney SM (2016) Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry. J Exp Bot 67:3137–3148
Prins A, Orr DJ, Andralojc PJ et al (2016) Rubisco catalytic properties of wild and domesticated relatives provide scope for improving wheat photosynthesis. J Exp Bot 67:1827–1838
Perdomo JA, Sales CRG, Carmo-Silva E (2018) Quantification of photosynthetic enzymes in leaf extracts by immunoblotting. In: Covshoff S (ed) Photosynthesis: methods and protocols, Methods in molecular biology, vol 1770. Springer, New York
Carmo-Silva E, Salvucci ME (2013) The regulatory properties of Rubisco activase differ among species and affect photosynthetic induction during light transitions. Plant Physiol 161:1645–1655
Parry MAJ, Andralojc PJ, Parmar S et al (1997) Regulation of Rubisco by inhibitors in the light. Plant Cell Environ 20:528–534
Whitney SM, Von Caemmerer S, Hudson GS, Andrews TJ (1999) Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth. Plant Physiol 121:579–588
Wishnick M, Lane MD (1971) Ribulose diphosphate carboxylase from spinach leaves. Methods Enzymol 23:570–577
McCurry SD, Gee R, Tolbert NE (1982) Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach, tobacco, or tobacco leaves. Methods Enzymol 90:515–521
Barta C, Carmo-Silva E, Salvucci ME (2011) Purification of Rubisco activase from leaves or after expression in Escherichia coli. In: Carpentier R (ed) Methods in molecular biology, Photosynthesis research protocols, vol 684, 2nd edn. Humana Press, New York, pp 363–374
Carmo-Silva E, Barta C, Salvucci ME (2011) Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves. In: Carpentier R (ed) Methods in molecular biology, Photosynthesis research protocols, vol 684, 2nd edn. Humana Press, New York, pp 339–347
Acknowledgments
The authors thank André Alcântara and Dr. Karen Harper (Lancaster University) for technical assistance that led to method improvements, and Dr. Cristina Sales (Lancaster University) for providing the gel image. The authors acknowledge funding through a sub-contract from the University of Illinois as part of the Bill & Melinda Gates Foundation award RIPE: Realizing Increased Photosynthetic Efficiency.
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Orr, D.J., Carmo-Silva, E. (2018). Extraction of RuBisCO to Determine Catalytic Constants. In: Covshoff, S. (eds) Photosynthesis. Methods in Molecular Biology, vol 1770. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7786-4_13
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DOI: https://doi.org/10.1007/978-1-4939-7786-4_13
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