Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase.

D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the first step in photosynthetic carbon assimilation and represents the largest sink for nitrogen in plants. Improvement of its kinetic properties or the efficiency with which it is used in planta would benefit photosynthesis, nitrogen and water use efficiency, and yield. This paper presents a new non-radioactive microplate-based assay, which determines the product [3-phosphoglycerate (3-PGA)] in an enzymic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase. High sensitivity permits use of highly diluted extracts, and a short reaction time to avoid problems due to fall-off. Throughput was several hundreds of samples per person per day. Sensitivity and convenience compared favourably with radioisotopic assays, which were previously used to assay Rubisco. Its use is illustrated in three applications. (1) Maximal and initial activities and the K(m) for ribulose-1,5-bisphosphate were determined in raw extracts of leaves from several species. Similar values were obtained from those in the literature. (2) Diurnal changes were compared in rosettes of wild-type (WT) Arabidopsis and the starchless pgm mutant. Despite these dramatic differences in carbon metabolism, Rubisco activity and activation were similar in both genotypes. (3) A preliminary association mapping study was performed with 118 Arabidopsis accessions, using 183 markers that probably cover approximately 3-8% of the total genome. At a P-value < 0.005, two, two and no quantitative trait loci (QTL) were found for Rubisco maximal activity, initial activity and activation state, respectively. Inspection of the genomic regions that span these markers revealed these QTL involved genes not previously implicated in the regulation of Rubisco expression or activity.

[1]  A. Portis,et al.  Increased Sensitivity of Oxidized Large Isoform of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase to ADP Inhibition Is Due to an Interaction between Its Carboxyl Extension and Nucleotide-binding Pocket* , 2006, Journal of Biological Chemistry.

[2]  C. Somerville,et al.  Alterations in Growth, Photosynthesis, and Respiration in a Starchless Mutant of Arabidopsis thaliana (L.) Deficient in Chloroplast Phosphoglucomutase Activity. , 1985, Plant physiology.

[3]  T. Mitchell-Olds,et al.  A Multilocus Sequence Survey in Arabidopsis thaliana Reveals a Genome-Wide Departure From a Neutral Model of DNA Sequence Polymorphism , 2005, Genetics.

[4]  I. Andersson,et al.  Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site. , 1990, Journal of molecular biology.

[5]  A. Portis,et al.  Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  G. Lorimer,et al.  Phosphate requirement for the light activation of ribulose‐ 1,5‐biphosphate carboxylase in intact spinach chloroplasts , 1978 .

[7]  Keyan Zhao,et al.  An Arabidopsis Example of Association Mapping in Structured Samples , 2006, PLoS genetics.

[8]  T. Andrews,et al.  Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth. , 1999, Plant physiology.

[9]  Mark Stitt,et al.  The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background , 1999 .

[10]  A unified theory for the basis of the limitations of the primary reaction of photosynthetic CO(2) fixation: was Dr. Pangloss right? , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[11]  A. Makino,et al.  Effects of nitrogen nutrition on the relationships between the levels of rbcS and rbcL mRNAs and the amount of ribulose 1·5‐bisphosphate carboxylase/oxygenase synthesized in the eighth leaves of rice from emergence through senescence , 2005 .

[12]  T. Andrews,et al.  Substrate isomerization inhibits ribulosebisphospate carboxylase‐oxygenase during catalysis , 1990 .

[13]  Mattias Jakobsson,et al.  The Pattern of Polymorphism in Arabidopsis thaliana , 2005, PLoS biology.

[14]  Detlef Weigel,et al.  Large-scale identification of single-feature polymorphisms in complex genomes. , 2003, Genome research.

[15]  Keyan Zhao,et al.  Genome-Wide Association Mapping in Arabidopsis Identifies Previously Known Flowering Time and Pathogen Resistance Genes , 2005, PLoS genetics.

[16]  G. Lorimer,et al.  The Carboxylation and Oxygenation of Ribulose 1,5-Bisphosphate: The Primary Events in Photosynthesis and Photorespiration , 1981 .

[17]  M. Salvucci,et al.  Rubisco: structure, regulatory interactions, and possibilities for a better enzyme. , 2002, Annual review of plant biology.

[18]  D. Jordan,et al.  A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities. , 1981, Plant physiology.

[19]  P. J. Andralojc,et al.  Manipulation of Rubisco: the amount, activity, function and regulation. , 2003, Journal of experimental botany.

[20]  Joachim Selbig,et al.  A Robot-Based Platform to Measure Multiple Enzyme Activities in Arabidopsis Using a Set of Cycling Assays: Comparison of Changes of Enzyme Activities and Transcript Levels during Diurnal Cycles and in Prolonged Darknessw⃞ , 2004, The Plant Cell Online.

[21]  M. Yano,et al.  Are contents of Rubisco, soluble protein and nitrogen in flag leaves of rice controlled by the same genetics? , 2001, Journal of experimental botany.

[22]  T. Okita,et al.  Interactions of Nitrate and CO2 Enrichment on Growth, Carbohydrates, and Rubisco in Arabidopsis Starch Mutants. Significance of Starch and Hexose1 , 2002, Plant Physiology.

[23]  G. Lorimer,et al.  D-Ribulose-1,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. , 1977, Analytical biochemistry.

[24]  D. Geiger,et al.  Regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase by metabolites , 1995 .

[25]  R. Ellis.  The most abundant protein in the world , 1979 .

[26]  E. Schulze,et al.  Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS , 1992, Planta.

[27]  R. Lilley,et al.  An improved spectrophotometric assay for ribulosebisphosphate carboxylase. , 1974, Biochimica et biophysica acta.

[28]  Frédérique Bitton,et al.  Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis , 2004, The Plant Cell Online.

[29]  T. Shikanai,et al.  A Rapid and Sensitive Method for Determination of Relative Specificity of RuBisCO from Various Species by Anion-Exchange Chromatography , 1996 .

[30]  S. Long,et al.  What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. , 2004, The New phytologist.

[31]  J. Seemann,et al.  Sucrose cycling, Rubisco expression, and prediction of photosynthetic acclimation to elevated atmospheric CO2 , 1998, Plant, Cell & Environment.

[32]  B. Hirel,et al.  Rubisco synthesis, turnover and degradation: some new thoughts on an old problem. , 2006, The New phytologist.

[33]  M. Salvucci,et al.  Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves. , 1986, Plant physiology.

[34]  A. Portis,et al.  Kinetic Analysis of the Slow Inactivation of Rubisco During Catalysis: Effects of Temperature, O2 and Mg++ , 2006, Photosynthesis Research.

[35]  T. Mitchell-Olds,et al.  The molecular basis of quantitative genetic variation in central and secondary metabolism in Arabidopsis. , 1998, Genetics.

[36]  Wilkin,et al.  Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate , 1998, Plant physiology.

[37]  Jungwon Yoon,et al.  The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community , 2003, Nucleic Acids Res..

[38]  A. Portis.  Rubisco activase – Rubisco's catalytic chaperone , 2004, Photosynthesis Research.

[39]  E. Buckler,et al.  Plant molecular diversity and applications to genomics. , 2002, Current opinion in plant biology.

[40]  J. David,et al.  MSTRAT: an algorithm for building germ plasm core collections by maximizing allelic or phenotypic richness. , 2001, The Journal of heredity.

[41]  J. Fisahn,et al.  Adjustment of diurnal starch turnover to short days: depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period. , 2004, The Plant journal : for cell and molecular biology.

[42]  T. Mitchell-Olds,et al.  Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis. , 2003, The Plant journal : for cell and molecular biology.

[43]  E. Schulze,et al.  Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with ‘antisense’ rbcS , 1991, Planta.

[44]  Thomas Altmann,et al.  Variation of Enzyme Activities and Metabolite Levels in 24 Arabidopsis Accessions Growing in Carbon-Limited Conditions1[W] , 2006, Plant Physiology.

[45]  Alice Barkan,et al.  A Pentatricopeptide Repeat Protein Facilitates the trans-Splicing of the Maize Chloroplast rps12 Pre-mRNA[W] , 2006, The Plant Cell Online.

[46]  M. Nei.  Analysis of gene diversity in subdivided populations. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Y. F. Wang,et al.  Properties of hybrid enzymes between Synechococcus large subunits and higher plant small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli. , 2001, Archives of biochemistry and biophysics.

[48]  Joachim Selbig,et al.  Extension of the Visualization Tool MapMan to Allow Statistical Analysis of Arrays, Display of Coresponding Genes, and Comparison with Known Responses1 , 2005, Plant Physiology.

[49]  E. Schulze,et al.  Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS , 1991, Planta.

[50]  Ning Sun,et al.  Evaluation of light regulatory potential of Calvin cycle steps based on large-scale gene expression profiling data , 2003, Plant Molecular Biology.

[51]  A. Dhingra,et al.  Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[52]  Yves Gibon,et al.  Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system. , 2002, The Plant journal : for cell and molecular biology.

[53]  P. Donnelly,et al.  Inference of population structure using multilocus genotype data. , 2000, Genetics.

[54]  J. Berry,et al.  Regulation of ribulose bisphosphate carboxylase activity in vivo by a light-modulated inhibitor of catalysis. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[55]  M. Stitt,et al.  Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology , 1994 .

[56]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[57]  S. Bhattacharya,et al.  A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). , 2002, Journal of biochemical and biophysical methods.

[58]  X. Ruan,et al.  An improved equation and assay for determining the CO2/O2 specificity for Rubisco , 2004, Photosynthesis Research.

[59]  R. Sage.  Variation in the kcat of Rubisco in C3 and C4 plants and some implications for photosynthetic performance at high and low temperature , 2002 .