Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids1[OPEN]

A metabolic network-guided genome-wide association study of seed free amino acids facilitates the identification of a histidine-specific transporter in Arabidopsis. Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed’s nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants.

[1]  Tomoo Shimada,et al.  Plant Vacuoles. , 2018, Annual review of plant biology.

[2]  Aaron Fait,et al.  Correlation-Based Network Generation, Visualization, and Analysis as a Powerful Tool in Biological Studies: A Case Study in Cancer Cell Metabolism , 2016, BioMed research international.

[3]  J. Gershenzon,et al.  Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays[OPEN] , 2016, Plant Cell.

[4]  M. Gore,et al.  ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed1[OPEN] , 2016, Plant Physiology.

[5]  A. Fernie,et al.  Genomics-based strategies for the use of natural variation in the improvement of crop metabolism. , 2016, Plant science : an international journal of experimental plant biology.

[6]  Yan Lu,et al.  Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents1[OPEN] , 2015, Plant Physiology.

[7]  Shin-Han Shiu,et al.  The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis1[OPEN] , 2015, Plant Physiology.

[8]  Daniel S. Himmelstein,et al.  Understanding multicellular function and disease with human tissue-specific networks , 2015, Nature Genetics.

[9]  Y. Stierhof,et al.  The putative Cationic Amino Acid Transporter 9 is targeted to vesicles and may be involved in plant amino acid homeostasis , 2015, Front. Plant Sci..

[10]  Alexander Platt,et al.  Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana , 2015, Proceedings of the National Academy of Sciences.

[11]  E. Buckler,et al.  Genome-Wide Association Study Based on Multiple Imputation with Low-Depth Sequencing Data: Application to Biofuel Traits in Reed Canarygrass , 2015, G3: Genes, Genomes, Genetics.

[12]  A. Fernie,et al.  Combined correlation-based network and mQTL analyses efficiently identified loci for branched-chain amino acid, serine to threonine, and proline metabolism in tomato seeds. , 2015, The Plant journal : for cell and molecular biology.

[13]  Kazuki Saito,et al.  Metabolome-genome-wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism , 2014, The Plant journal : for cell and molecular biology.

[14]  Réjane Pratelli,et al.  Regulation of amino acid metabolic enzymes and transporters in plants. , 2014, Journal of experimental botany.

[15]  T. Rocheford,et al.  A Foundation for Provitamin A Biofortification of Maize: Genome-Wide Association and Genomic Prediction Models of Carotenoid Levels , 2014, Genetics.

[16]  Wei Chen,et al.  Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism , 2014, Nature Genetics.

[17]  S. Postel,et al.  Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1. , 2014, Plant, cell & environment.

[18]  Melanie Krebs,et al.  Characterization of the putative amino acid transporter genes AtCAT2, 3 &4: the tonoplast localized AtCAT2 regulates soluble leaf amino acids. , 2014, Journal of plant physiology.

[19]  M. Tegeder.  Transporters involved in source to sink partitioning of amino acids and ureides: opportunities for crop improvement. , 2014, Journal of experimental botany.

[20]  M. Gore,et al.  CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds[W] , 2013, Plant Cell.

[21]  T. Juenger,et al.  Genome-Wide Association Mapping Combined with Reverse Genetics Identifies New Effectors of Low Water Potential-Induced Proline Accumulation in Arabidopsis1[W][OPEN] , 2013, Plant Physiology.

[22]  Michael A. Gore,et al.  Genome-Wide Association Study and Pathway-Level Analysis of Tocochromanol Levels in Maize Grain , 2013, G3: Genes, Genomes, Genetics.

[23]  A. Korte,et al.  The advantages and limitations of trait analysis with GWAS: a review , 2013, Plant Methods.

[24]  Yang Li,et al.  University of Groningen Identifying Genotype-by-Environment Interactions in the Metabolism of Germinating Arabidopsis Seeds Using Generalized Genetical Genomics , 2012 .

[25]  Moudud Alam,et al.  A Novel Generalized Ridge Regression Method for Quantitative Genetics , 2013, Genetics.

[26]  G. Galili,et al.  Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality. , 2013, Plant biotechnology journal.

[27]  Zoran Nikoloski,et al.  Network analysis: tackling complex data to study plant metabolism. , 2013, Trends in biotechnology.

[28]  Christian Gieger,et al.  Mining the Unknown: A Systems Approach to Metabolite Identification Combining Genetic and Metabolic Information , 2012, PLoS genetics.

[29]  Bjarni J. Vilhjálmsson,et al.  An efficient multi-locus mixed model approach for genome-wide association studies in structured populations , 2012, Nature Genetics.

[30]  M. Stitt,et al.  Genome-wide association mapping of leaf metabolic profiles for dissecting complex traits in maize , 2012, Proceedings of the National Academy of Sciences.

[31]  Jianbing Yan,et al.  Genome-Wide Association Studies Identified Three Independent Polymorphisms Associated with α-Tocopherol Content in Maize Kernels , 2012, PloS one.

[32]  Katherine E. Guill,et al.  The relationship between parental genetic or phenotypic divergence and progeny variation in the maize nested association mapping population , 2011, Heredity.

[33]  A. Fernie,et al.  Metabolic Profiling of a Mapping Population Exposes New Insights in the Regulation of Seed Metabolism and Seed, Fruit, and Plant Relations , 2012, PLoS genetics.

[34]  Ratnakar Vallabhaneni,et al.  Maize Provitamin A Carotenoids, Current Resources, and Future Metabolic Engineering Challenges , 2012, Front. Plant Sci..

[35]  A. Auton,et al.  Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel , 2011, Nature Genetics.

[36]  Qian Qian,et al.  Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm , 2011, Nature Genetics.

[37]  Detlef Weigel,et al.  Natural Variation in Arabidopsis: From Molecular Genetics to Ecological Genomics1[W][OA] , 2011, Plant Physiology.

[38]  Magali Schnell Ramos,et al.  Toward the Storage Metabolome: Profiling the Barley Vacuole1[W][OA] , 2011, Plant Physiology.

[39]  Jason A. Corwin,et al.  Combining Genome-Wide Association Mapping and Transcriptional Networks to Identify Novel Genes Controlling Glucosinolates in Arabidopsis thaliana , 2011, PLoS biology.

[40]  E. Marcotte,et al.  Prioritizing candidate disease genes by network-based boosting of genome-wide association data. , 2011, Genome research.

[41]  O. Loudet,et al.  What does Arabidopsis natural variation teach us (and does not teach us) about adaptation in plants? , 2011, Current opinion in plant biology.

[42]  A. Fernie,et al.  The genetic architecture of branched-chain amino acid accumulation in tomato fruits , 2011, Journal of experimental botany.

[43]  R. Ingle.  Histidine Biosynthesis , 2011, The arabidopsis book.

[44]  A. Fernie,et al.  A seed high-lysine trait is negatively associated with the TCA cycle and slows down Arabidopsis seed germination. , 2011, The New phytologist.

[45]  M. Nordborg,et al.  Conditions Under Which Genome-Wide Association Studies Will be Positively Misleading , 2010, Genetics.

[46]  J. Holland,et al.  Estimating and Interpreting Heritability for Plant Breeding: An Update , 2010 .

[47]  M. Scott,et al.  Quantitative Trait Loci for Endosperm Modification and Amino Acid Contents in Quality Protein Maize , 2010 .

[48]  A. Fernie,et al.  Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain of Arabidopsis Mitochondria[W][OA] , 2010, Plant Cell.

[49]  A. Fernie,et al.  Characterization of the Branched-Chain Amino Acid Aminotransferase Enzyme Family in Tomato1[W][OA] , 2010, Plant Physiology.

[50]  A. Fernie,et al.  Seed desiccation: a bridge between maturation and germination. , 2010, Trends in plant science.

[51]  T. Rocheford,et al.  Rare genetic variation at Zea mays crtRB1 increases β-carotene in maize grain , 2010, Nature Genetics.

[52]  A. D. Jones,et al.  Broad connections in the Arabidopsis seed metabolic network revealed by metabolite profiling of an amino acid catabolism mutant. , 2010, The Plant journal : for cell and molecular biology.

[53]  Detlef Weigel,et al.  The Scale of Population Structure in Arabidopsis thaliana , 2010, PLoS genetics.

[54]  S. Binder.  Branched-Chain Amino Acid Metabolism in Arabidopsis thaliana , 2010, The arabidopsis book.

[55]  Gad Galili,et al.  The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana , 2010, The arabidopsis book.

[56]  Bjarni J. Vilhjálmsson,et al.  Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines , 2010 .

[57]  A. Fernie,et al.  Deciphering Transcriptional and Metabolic Networks Associated with Lysine Metabolism during Arabidopsis Seed Development1[C][W][OA] , 2009, Plant Physiology.

[58]  M. Tegeder,et al.  AAP1 regulates import of amino acids into developing Arabidopsis embryos. , 2009, The Plant journal : for cell and molecular biology.

[59]  R. Mott,et al.  A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana , 2009, PLoS genetics.

[60]  Ratnakar Vallabhaneni,et al.  Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize1[C][W][OA] , 2009, Plant Physiology.

[61]  Detlef Weigel,et al.  QTL Mapping in New Arabidopsis thaliana Advanced Intercross-Recombinant Inbred Lines , 2009, PloS one.

[62]  Sébastien Baud,et al.  Storage Reserve Accumulation in Arabidopsis: Metabolic and Developmental Control of Seed Filling , 2008, The arabidopsis book.

[63]  Christoph Benning,et al.  New Connections across Pathways and Cellular Processes: Industrialized Mutant Screening Reveals Novel Associations between Diverse Phenotypes in Arabidopsis1[W][OA] , 2008, Plant Physiology.

[64]  A. D. Jones,et al.  LC-MS/MS assay for protein amino acids and metabolically related compounds for large-scale screening of metabolic phenotypes. , 2007, Analytical chemistry.

[65]  Nicholas J. Provart,et al.  An “Electronic Fluorescent Pictograph” Browser for Exploring and Analyzing Large-Scale Biological Data Sets , 2007, PloS one.

[66]  Daniel J. Kliebenstein,et al.  Linking Metabolic QTLs with Network and cis-eQTLs Controlling Biosynthetic Pathways , 2007, PLoS genetics.

[67]  J. Balk,et al.  Functional analysis of Arabidopsis genes involved in mitochondrial iron–sulfur cluster assembly , 2007, Plant Molecular Biology.

[68]  S. Binder,et al.  Branched‐chain amino acid metabolism in higher plants , 2007 .

[69]  S. Chander,et al.  Using molecular markers to identify two major loci controlling carotenoid contents in maize grain , 2007, Theoretical and Applied Genetics.

[70]  Alisdair R. Fernie,et al.  Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches1[W] , 2006, Plant Physiology.

[71]  I. Kranner,et al.  Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds. , 2006, Phytochemical analysis : PCA.

[72]  E. Buckler,et al.  Genetic association mapping and genome organization of maize. , 2006, Current opinion in biotechnology.

[73]  D. Robinson,et al.  Subcellular volumes and metabolite concentrations in spinach leaves , 1994, Planta.

[74]  A. Stepansky,et al.  Histidine biosynthesis in plants , 2006, Amino Acids.

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

[76]  J. A. Smith,et al.  Constitutively High Expression of the Histidine Biosynthetic Pathway Contributes to Nickel Tolerance in Hyperaccumulator Plantsw⃞ , 2005, The Plant Cell Online.

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

[78]  Mark Daly,et al.  Haploview: analysis and visualization of LD and haplotype maps , 2005, Bioinform..

[79]  James K. M. Brown,et al.  QTL analysis of flowering time inArabidopsis thaliana , 1995, Molecular and General Genetics MGG.

[80]  Thomas Girke,et al.  The Vegetative Vacuole Proteome of Arabidopsis thaliana Reveals Predicted and Unexpected Proteinsw⃞ , 2004, The Plant Cell Online.

[81]  Thomas Altmann,et al.  Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. , 2004, Genome research.

[82]  O. Fiehn,et al.  Differential metabolic networks unravel the effects of silent plant phenotypes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[83]  G. Coruzzi,et al.  Correlation of ASN2 Gene Expression with Ammonium Metabolism in Arabidopsis1 , 2004, Plant Physiology.

[84]  V. Rai.  Role of Amino Acids in Plant Responses to Stresses , 2002, Biologia Plantarum.

[85]  A. Paterson,et al.  QTL mapping of naturally-occurring variation in flowering time of Arabidopsis thaliana , 1994, Molecular and General Genetics MGG.

[86]  B. Miflin,et al.  Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine , 1983, Planta.

[87]  B. Miflin,et al.  Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase , 1982, Biochemical Genetics.

[88]  O. Fiehn,et al.  Interpreting correlations in metabolomic networks. , 2003, Biochemical Society transactions.

[89]  W. Weckwerth.  Metabolomics in systems biology. , 2003, Annual review of plant biology.

[90]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[91]  O. Loudet,et al.  Bay-0 × Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis , 2002, Theoretical and Applied Genetics.

[92]  Sébastien Baud,et al.  An integrated overview of seed development in Arabidopsis thaliana ecotype WS , 2002 .

[93]  P. Donnelly,et al.  Case-control studies of association in structured or admixed populations. , 2001, Theoretical population biology.

[94]  R. Vierstra,et al.  The ubiquitin-specific protease family from Arabidopsis. AtUBP1 and 2 are required for the resistance to the amino acid analog canavanine. , 2000, Plant physiology.

[95]  U. Sauer,et al.  Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism , 1999, Journal of bacteriology.

[96]  B. Larkins,et al.  Protein Storage Bodies and Vacuoles , 1999, Plant Cell.

[97]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[98]  G. Coupland,et al.  Analysis of natural allelic variation at flowering time loci in the Landsberg erecta and Cape Verde Islands ecotypes of Arabidopsis thaliana. , 1998, Genetics.

[99]  C. Stanley,et al.  Plant Cells Contain Two Functionally Distinct Vacuolar Compartments , 1996, Cell.

[100]  A. Melchinger,et al.  PLABQTL: a program for composite interval mapping of QTL. , 1996 .

[101]  G. Galili.  Regulation of Lysine and Threonine Synthesis. , 1995, The Plant cell.

[102]  M. Jacobs,et al.  Threonine Accumulation in a Mutant of Arabidopsis thaliana (L.) Heynh. with an Altered Aspartate Kinase , 1995 .

[103]  D. Guyer,et al.  Evidence for cross-pathway regulation of metabolic gene expression in plants. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[104]  H. Höfte,et al.  Protein sorting to the vacuolar membrane. , 1992, The Plant cell.

[105]  K. Dietz,et al.  Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine. , 1990, Plant physiology.