The use of metabolomics integrated with transcriptomic and proteomic studies for identifying key steps involved in the control of nitrogen metabolism in crops such as maize.

Linking plant phenotype to gene and protein expression and also to metabolite synthesis and accumulation is one of the main challenges for improving agricultural production worldwide. Such a challenge is particularly relevant to crop nitrogen use efficiency (NUE). Here, the differences in leaf gene transcript, protein, and metabolite accumulation in maize subjected to long-term nitrogen (N)-deficient growth conditions at two important stages of plant development have been studied. The impact of N deficiency was examined at the transcriptomic, proteomic, and metabolomic levels. It was found that a number of key plant biological functions were either up- or down-regulated when N was limiting, including major alterations to photosynthesis, carbon (C) metabolism, and, to a lesser extent, downstream metabolic pathways. It was also found that the impact of the N deficiency stress resembled the response of plants to a number of other biotic and abiotic stresses, in terms of transcript, protein, and metabolite accumulation. The genetic and metabolic alterations were different during the N assimilation and the grain-filling period, indicating that plant development is an important component for identifying the key elements involved in the control of plant NUE. It was also found that integration of the three 'omics' studies is not straightforward, since different levels of regulation seem to occur in a stepwise manner from gene expression to metabolite accumulation. The potential use of these 'omics' studies is discussed with a view to improve our understanding of whole plant nitrogen economics, which should have applications in breeding and agronomy.

[1]  B. Hirel,et al.  New insights towards the function of glutamate dehydrogenase revealed during source-sink transition of tobacco (Nicotiana tabacum) plants grown under different nitrogen regimes. , 2004, Physiologia plantarum.

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

[3]  M. Stitt,et al.  On the Discordance of Metabolomics with Proteomics and Transcriptomics: Coping with Increasing Complexity in Logic, Chemistry, and Network Interactions Scientific Correspondence , 2012, Plant Physiology.

[4]  M. Hirai,et al.  Decoding genes with coexpression networks and metabolomics - 'majority report by precogs'. , 2008, Trends in plant science.

[5]  Jens Nielsen,et al.  The next wave in metabolome analysis. , 2005, Trends in biotechnology.

[6]  F. Mesnard,et al.  NMR analysis of plant nitrogen metabolism , 2004, Photosynthesis Research.

[7]  N. Tuteja,et al.  Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. , 2010, Plant physiology and biochemistry : PPB.

[8]  P. Holm,et al.  Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L.). , 2007, Plant biotechnology journal.

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

[10]  I. Quilléré,et al.  The challenge of remobilisation in plant nitrogen economy. A survey of physio-agronomic and molecular approaches. , 2001 .

[11]  K. Shinozaki,et al.  'Omics' analyses of regulatory networks in plant abiotic stress responses. , 2010, Current opinion in plant biology.

[12]  John P. Moore,et al.  A role for pectin-associated arabinans in maintaining the flexibility of the plant cell wall during water deficit stress , 2008, Plant signaling & behavior.

[13]  Marie-Hélène Mucchielli-Giorgi,et al.  MAnGO: an interactive R-based tool for two-colour microarray analysis , 2007, Bioinform..

[14]  P. Lea,et al.  Improving Nitrogen Use Efficiency in Crops for Sustainable Agriculture , 2011 .

[15]  Oliver Fiehn,et al.  Metabolite profiling in Arabidopsis. , 2006, Methods in molecular biology.

[16]  G. Likens,et al.  Technical Report: Human Alteration of the Global Nitrogen Cycle: Sources and Consequences , 1997 .

[17]  R. Verma,et al.  The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis. , 2003, The Plant cell.

[18]  Kent Vander Velden,et al.  The proteome of maize leaves: Use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints , 2001, Electrophoresis.

[19]  K. Kosová,et al.  Plant proteome changes under abiotic stress--contribution of proteomics studies to understanding plant stress response. , 2011, Journal of proteomics.

[20]  L. N. Ornston,et al.  Regulation of catabolic pathways in Pseudomonas. , 1971, Bacteriological reviews.

[21]  T. McNellis,et al.  Fibrillin protein function: the tip of the iceberg? , 2011, Trends in plant science.

[22]  M. Stitt,et al.  Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco. , 1997, The Plant cell.

[23]  Joachim Kopka,et al.  Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. , 2007, The Plant journal : for cell and molecular biology.

[24]  M. Baier,et al.  Protective function of chloroplast 2-cysteine peroxiredoxin in photosynthesis. Evidence from transgenic Arabidopsis. , 1999, Plant physiology.

[25]  R. Mittler,et al.  Genetic engineering for modern agriculture: challenges and perspectives. , 2010, Annual review of plant biology.

[26]  Carole L. Linster,et al.  L-Ascorbate biosynthesis in higher plants: the role of VTC2. , 2008, Trends in plant science.

[27]  R. Hell Molecular physiology of plant sulfur metabolism , 1997, Planta.

[28]  Yubi Huang,et al.  Differential global gene expression changes in response to low nitrogen stress in two maize inbred lines with contrasting low nitrogen tolerance , 2011, Genes & Genomics.

[29]  B. Ney,et al.  The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. , 2007, Journal of experimental botany.

[30]  and H Reinbothe,et al.  Urea, Ureides, and Guanidines in Plants , 1962 .

[31]  E. Kok,et al.  Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics. , 2010, Plant biotechnology journal.

[32]  Charles Auffray,et al.  Deciphering cellular states of innate tumor drug responses , 2006, Genome Biology.

[33]  M. Taylor,et al.  Recent advances in polyamine research , 1997 .

[34]  J. Araus,et al.  Comparative genomic and physiological analysis of nutrient response to NH4+, NH4+:NO3- and NO3- in barley seedlings. , 2008, Physiologia plantarum.

[35]  F. Kaplan,et al.  β-Amylase Induction and the Protective Role of Maltose during Temperature Shock1 , 2004, Plant Physiology.

[36]  K. Dietz Peroxiredoxins in plants and cyanobacteria. , 2011, Antioxidants & redox signaling.

[37]  B. Hirel,et al.  Physiology of maize I: A comprehensive and integrated view of nitrogen metabolism in a C4 plant , 2005 .

[38]  Weiwen Zhang,et al.  Integrating multiple 'omics' analysis for microbial biology: application and methodologies. , 2010, Microbiology.

[39]  E. Cowling,et al.  The Nitrogen Cascade , 2003 .

[40]  Mari L. Salmi,et al.  Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. , 2009, Journal of experimental botany.

[41]  J. Prioul,et al.  Developmental Analysis of Maize Endosperm Proteome Suggests a Pivotal Role for Pyruvate Orthophosphate Dikinase1[W][OA] , 2007, Plant Physiology.

[42]  S. Filleur,et al.  Signaling mechanisms integrating root and shoot responses to changes in the nitrogen supply , 2004, Photosynthesis Research.

[43]  Terry Speed,et al.  Normalization of cDNA microarray data. , 2003, Methods.

[44]  X. Yang,et al.  Gene Expression Biomarkers Provide Sensitive Indicators of in Planta Nitrogen Status in Maize[W][OA] , 2011, Plant Physiology.

[45]  S. Rothstein,et al.  Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. , 2011, Journal of experimental botany.

[46]  N. Kruger,et al.  Pathways and fluxes: exploring the plant metabolic network. , 2012, Journal of experimental botany.

[47]  Luca Espen,et al.  Evaluation of protein pattern changes in roots and leaves of Zea mays plants in response to nitrate availability by two-dimensional gel electrophoresis analysis , 2009, BMC Plant Biology.

[48]  S. Polasky,et al.  Agricultural sustainability and intensive production practices , 2002, Nature.

[49]  R. Molinié,et al.  Metabolic profiling of maize mutants deficient for two glutamine synthetase isoenzymes using 1H-NMR-based metabolomics. , 2010, Phytochemical analysis : PCA.

[50]  R. D. Slocum Genes, enzymes and regulation of arginine biosynthesis in plants. , 2005, Plant physiology and biochemistry : PPB.

[51]  J. Ribaut,et al.  Quantitative trait loci for yield and correlated traits under high and low soil nitrogen conditions in tropical maize , 2007, Molecular Breeding.

[52]  D. M. Pharr,et al.  Mannitol metabolism in plants: a method for coping with stress , 1996 .

[53]  G. Glevarec,et al.  Genetic and Physiological Analysis of Germination Efficiency in Maize in Relation to Nitrogen Metabolism Reveals the Importance of Cytosolic Glutamine Synthetase , 2002, Plant Physiology.

[54]  J. Prioul,et al.  From QTLs for enzyme activity to candidate genes in maize , 1999 .

[55]  Joachim Selbig,et al.  Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations , 2007, The Plant journal : for cell and molecular biology.

[56]  B. Andrieu,et al.  Nitrogen management and senescence in two maize hybrids differing in the persistence of leaf greenness: agronomic, physiological and molecular aspects. , 2005, The New phytologist.

[57]  K. Keegstra,et al.  Biosynthesis of plant cell wall polysaccharides - a complex process. , 2006, Current opinion in plant biology.

[58]  Rongchen Wang,et al.  Microarray Analysis of the Nitrate Response in Arabidopsis Roots and Shoots Reveals over 1,000 Rapidly Responding Genes and New Linkages to Glucose, Trehalose-6-Phosphate, Iron, and Sulfate Metabolism1[w] , 2003, Plant Physiology.

[59]  Kazuki Saito,et al.  Metabolomic approaches toward understanding nitrogen metabolism in plants. , 2011, Journal of experimental botany.

[60]  M. Hirai,et al.  Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[61]  A. Hoekema,et al.  A small-scale procedure for the rapid isolation of plant RNAs. , 1989, Nucleic acids research.

[62]  H. Rosen,et al.  A modified ninhydrin colorimetric analysis for amino acids. , 1957, Archives of biochemistry and biophysics.

[63]  Xianghua Li,et al.  Expression Profiles of 10,422 Genes at Early Stage of Low Nitrogen Stress in Rice Assayed using a cDNA Microarray , 2006, Plant Molecular Biology.

[64]  A. Altman,et al.  Interactions of polyamines and nitrogen nutrition in plants , 1993 .

[65]  M. Stitt,et al.  “Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes , 1991, Planta.

[66]  Identifying pre-post chemotherapy differences in gene expression in breast tumours: a statistical method appropriate for this aim , 2002, British Journal of Cancer.

[67]  L. Quek,et al.  C4GEM, a Genome-Scale Metabolic Model to Study C4 Plant Metabolism1[W][OA] , 2010, Plant Physiology.

[68]  J. Flexas,et al.  Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. , 2009, Annals of botany.

[69]  H. Vanacker,et al.  Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. , 2006, The New phytologist.

[70]  M. Stitt,et al.  Regulation of secondary metabolism by the carbon-nitrogen status in tobacco: nitrate inhibits large sectors of phenylpropanoid metabolism. , 2006, The Plant journal : for cell and molecular biology.

[71]  C. Witte Urea metabolism in plants. , 2011, Plant science : an international journal of experimental plant biology.

[72]  W. Martin,et al.  Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts , 1996, Plant Molecular Biology.

[73]  Jan Szopa,et al.  Catecholamines are active compounds in plants , 2007 .

[74]  Charles Auffray,et al.  Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces , 2005, Nucleic acids research.

[75]  R. Rakwal,et al.  Gel-based proteomics approach for detecting low nitrogen-responsive proteins in cultivated rice species , 2009, Physiology and Molecular Biology of Plants.

[76]  William R. Raun,et al.  Improving Nitrogen Use Efficiency for Cereal Production , 1999 .

[77]  M. Bernards Plant natural products: a primer. , 2010 .

[78]  Kazuki Saito,et al.  Members of the LBD Family of Transcription Factors Repress Anthocyanin Synthesis and Affect Additional Nitrogen Responses in Arabidopsis[W][OA] , 2009, The Plant Cell Online.

[79]  Joachim Selbig,et al.  The metabolic signature related to high plant growth rate in Arabidopsis thaliana , 2007, Proceedings of the National Academy of Sciences.

[80]  Francesca Chiaromonte,et al.  Qualitative network models and genome-wide expression data define carbon/nitrogen-responsive molecular machines in Arabidopsis , 2007, Genome Biology.

[81]  F. Daniel-Vedele,et al.  Arabidopsis Roots and Shoots Show Distinct Temporal Adaptation Patterns toward Nitrogen Starvation1[W] , 2011, Plant Physiology.

[82]  J. Eberwine,et al.  Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA. , 1996, BioTechniques.

[83]  L. Negroni,et al.  Differential protein expression assessed by two‐dimensional gel electrophoresis for two wheat varieties grown at four nitrogen levels , 2004, Proteomics.

[84]  J. Galloway,et al.  Transformation of the Nitrogen Cycle: Recent Trends, Questions, and Potential Solutions , 2008, Science.

[85]  Guo-ping Zhang,et al.  Proteomic analysis of nitrogen stress-responsive proteins in two rice cultivars differing in N utilization efficiency , 2011 .

[86]  K. Edwards,et al.  Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production[W][OA] , 2006, The Plant Cell Online.

[87]  T. Dyson,et al.  World food trends and prospects to 2025. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[88]  D. Arnon COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. , 1949, Plant physiology.

[89]  L. Schrader,et al.  Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid , 1975 .

[90]  J. Morot-Gaudry,et al.  Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence , 2000, Planta.

[91]  B. Hirel,et al.  An approach to the genetics of nitrogen use efficiency in maize. , 2004, Journal of experimental botany.

[92]  Tong Zhu,et al.  Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis , 2007, BMC Genomics.

[93]  Rafael A. Cañas,et al.  Can genetic variability for nitrogen metabolism in the developing ear of maize be exploited to improve yield? , 2012, The New phytologist.

[94]  A. Fernie,et al.  Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants , 2005 .

[95]  R. Hell,et al.  Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties. , 2006, Journal of plant physiology.

[96]  Michael E Webb,et al.  Elucidating biosynthetic pathways for vitamins and cofactors. , 2007, Natural product reports.

[97]  S. Smeekens,et al.  Photosynthesis, sugars and the regulation of gene expression. , 2000, Journal of experimental botany.

[98]  T. Sugiyama,et al.  Photosynthetic Determinants of Growth in Maize Plants: Effects of Nitrogen Nutrition on Growth, Carbon Fixation and Photochemical Features , 1989 .

[99]  J. Lunn Gene families and evolution of trehalose metabolism in plants. , 2007, Functional plant biology : FPB.

[100]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[101]  L. Miguet,et al.  Proteins of Diverse Function and Subcellular Location Are Lysine Acetylated in Arabidopsis1[W][OA] , 2011, Plant Physiology.

[102]  Kunpeng Li,et al.  Proteome profile of maize (Zea Mays L.) leaf tissue at the flowering stage after long-term adjustment to rice black-streaked dwarf virus infection. , 2011, Gene.