Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis

BackgroundHolistic profiling and systems biology studies of nutrient availability are providing more and more insight into the mechanisms by which gene expression responds to diverse nutrients and metabolites. Less is known about the mechanisms by which gene expression is affected by endogenous metabolites, which can change dramatically during development. Multivariate statistics and correlation network analysis approaches were applied to non-targeted profiling data to investigate transcriptional and metabolic states and to identify metabolites potentially influencing gene expression during the heterotrophic to autotrophic transition of seedling establishment.ResultsMicroarray-based transcript profiles were obtained from extracts of Arabidopsis seeds or seedlings harvested from imbibition to eight days-old. 1H-NMR metabolite profiles were obtained for corresponding samples. Analysis of transcript data revealed high differential gene expression through seedling emergence followed by a period of less change. Differential gene expression increased gradually to day 8, and showed two days, 5 and 7, with a very high proportion of up-regulated genes, including transcription factor/signaling genes. Network cartography using spring embedding revealed two primary clusters of highly correlated metabolites, which appear to reflect temporally distinct metabolic states. Principle Component Analyses of both sets of profiling data produced a chronological spread of time points, which would be expected of a developmental series. The network cartography of the transcript data produced two distinct clusters comprising days 0 to 2 and days 3 to 8, whereas the corresponding analysis of metabolite data revealed a shift of day 2 into the day 3 to 8 group. A metabolite and transcript pair-wise correlation analysis encompassing all time points gave a set of 237 highly significant correlations. Of 129 genes correlated to sucrose, 44 of them were known to be sucrose responsive including a number of transcription factors.ConclusionsMicroarray analysis during germination and establishment revealed major transitions in transcriptional activity at time points potentially associated with developmental transitions. Network cartography using spring-embedding indicate that a shift in the state of nutritionally important metabolites precedes a major shift in the transcriptional state going from germination to seedling emergence. Pair-wise linear correlations of transcript and metabolite levels identified many genes known to be influenced by metabolites, and provided other targets to investigate metabolite regulation of gene expression during seedling establishment.

[1]  Ayuko Kuwahara,et al.  Gibberellin Biosynthesis and Response during Arabidopsis Seed Germination Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.011650. , 2003, The Plant Cell Online.

[2]  M. Hooks,et al.  Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle* , 2005, Journal of Biological Chemistry.

[3]  I. Graham,et al.  Germination and storage reserve mobilization are regulated independently in Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[4]  Rainer Breitling,et al.  The Potassium-Dependent Transcriptome of Arabidopsis Reveals a Prominent Role of Jasmonic Acid in Nutrient Signaling1[w] , 2004, Plant Physiology.

[5]  Wei Wu,et al.  Gibberellin Mobilizes Distinct DELLA-Dependent Transcriptomes to Regulate Seed Germination and Floral Development in Arabidopsis1[W] , 2006, Plant Physiology.

[6]  Peter McCourt,et al.  The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. , 2003, The Plant journal : for cell and molecular biology.

[7]  F. Carrari,et al.  Metabolic regulation underlying tomato fruit development. , 2006, Journal of experimental botany.

[8]  Alisdair R Fernie,et al.  Plant metabolomics: towards biological function and mechanism. , 2006, Trends in plant science.

[9]  Sjef Smeekens,et al.  SUGAR-INDUCED SIGNAL TRANSDUCTION IN PLANTS. , 2000, Annual review of plant physiology and plant molecular biology.

[10]  E. Baena-González,et al.  Sugar sensing and signaling in plants: conserved and novel mechanisms. , 2006, Annual review of plant biology.

[11]  J. Selbig,et al.  Mode of Inheritance of Primary Metabolic Traits in Tomato[W][OA] , 2008, The Plant Cell Online.

[12]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[13]  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.

[14]  P. Perata,et al.  Digital Object Identifier (DOI) 10.1007/s10265-005-0251-1 REGULAR PAPER , 2022 .

[15]  S Roth,et al.  Regulation of intracellular glutathione levels and lymphocyte functions by lactate. , 1991, Cellular immunology.

[16]  G. Martin,et al.  Transcriptome and Selected Metabolite Analyses Reveal Multiple Points of Ethylene Control during Tomato Fruit Developmentw⃞ , 2005, The Plant Cell Online.

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

[18]  Yves Gibon,et al.  Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in Arabidopsis[W][OA] , 2005, The Plant Cell Online.

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

[20]  Gwénaëlle Le Gall,et al.  Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene , 2005, Metabolomics.

[21]  R. Mache,et al.  Arabidopsis A BOUT DE SOUFFLE, Which Is Homologous with Mammalian Carnitine Acyl Carrier, Is Required for Postembryonic Growth in the Light Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002485. , 2002, The Plant Cell Online.

[22]  K B Singh,et al.  The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element. , 1999, The Plant journal : for cell and molecular biology.

[23]  Fernando Carrari,et al.  On the processing of metabolic information through metabolite-gene communication networks: an approach for modelling causality. , 2007, Phytochemistry.

[24]  Steven Penfield,et al.  Reserve Mobilization in the Arabidopsis Endosperm Fuels Hypocotyl Elongation in the Dark, Is Independent of Abscisic Acid, and Requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1 , 2004, The Plant Cell Online.

[25]  Li Yang,et al.  Large-Scale cis-Element Detection by Analysis of Correlated Expression and Sequence Conservation between Arabidopsis and Brassica oleracea1[W] , 2006, Plant Physiology.

[26]  Catherine Deborde,et al.  Gene and Metabolite Regulatory Network Analysis of Early Developing Fruit Tissues Highlights New Candidate Genes for the Control of Tomato Fruit Composition and Development1[C][W][OA] , 2009, Plant Physiology.

[27]  M. A. Hooks,et al.  Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings , 2004, Molecular Genetics and Genomics.

[28]  J. Derek Bewleyl,et al.  Seed Germination and Dormancy , 2002 .

[29]  C. Daub,et al.  BMC Systems Biology , 2007 .

[30]  F. Rolland,et al.  Sugar sensing and signalling networks in plants. , 2005, Biochemical Society transactions.

[31]  Javier Paz-Ares,et al.  Plant hormones and nutrient signaling , 2009, Plant Molecular Biology.

[32]  Michael J Holdsworth,et al.  Gene Expression Profiling Reveals Defined Functions of the ATP-Binding Cassette Transporter COMATOSE Late in Phase II of Germination1[W][OA] , 2007, Plant Physiology.

[33]  J. Selbig,et al.  Parallel analysis of transcript and metabolic profiles: a new approach in systems biology , 2003, EMBO reports.

[34]  M K Kerr,et al.  Bootstrapping cluster analysis: Assessing the reliability of conclusions from microarray experiments , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Nam-Hai Chua,et al.  ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination. , 2002, The Plant journal : for cell and molecular biology.

[36]  F. Carrari,et al.  Heard it through the grapevine? ABA and sugar cross-talk: the ASR story. , 2004, Trends in plant science.

[37]  G M Coruzzi,et al.  Carbon and nitrogen sensing and signaling in plants: emerging 'matrix effects'. , 2001, Current opinion in plant biology.

[38]  Yuji Kamiya,et al.  Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed. , 2005, The Plant journal : for cell and molecular biology.

[39]  M. Hirai,et al.  Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis , 2007, Proceedings of the National Academy of Sciences.

[40]  S. Mansfield,et al.  The Dynamics of Seedling and Cotyledon Cell Development in Arabidopsis thaliana During Reserve Mobilization , 1996, International Journal of Plant Sciences.

[41]  Alisdair R Fernie,et al.  Regulation of metabolic networks: understanding metabolic complexity in the systems biology era. , 2005, The New phytologist.

[42]  J. Ward,et al.  Assessment of 1H NMR spectroscopy and multivariate analysis as a technique for metabolite fingerprinting of Arabidopsis thaliana. , 2003, Phytochemistry.

[43]  Noah Fahlgren,et al.  Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. , 2007, The Plant journal : for cell and molecular biology.

[44]  O. Fiehn,et al.  Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co‐regulation in biochemical networks , 2004, Proteomics.

[45]  Dennis Shasha,et al.  An integrated genetic, genomic and systems approach defines gene networks regulated by the interaction of light and carbon signaling pathways in Arabidopsis , 2008, BMC Systems Biology.

[46]  Timothy M. D. Ebbels,et al.  springScape: visualisation of microarray and contextual bioinformatic data using spring embedding and an "information landscape" , 2006, ISMB.

[47]  P. León,et al.  Sugar and hormone connections. , 2003, Trends in plant science.

[48]  Dennis Shasha,et al.  A Systems Approach Uncovers Restrictions for Signal Interactions Regulating Genome-wide Responses to Nutritional Cues in Arabidopsis , 2009, PLoS Comput. Biol..

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

[50]  Hur-Song Chang,et al.  Integrative analysis of transcript and metabolite profiling data sets to evaluate the regulation of biochemical pathways during photomorphogenesis. , 2006, Archives of biochemistry and biophysics.

[51]  W. Finch-Savage,et al.  Seed dormancy and the control of germination. , 2006, The New phytologist.

[52]  K. Halliday,et al.  Cold and Light Control Seed Germination through the bHLH Transcription Factor SPATULA , 2005, Current Biology.

[53]  O. Fiehn,et al.  Integrated studies on plant biology using multiparallel techniques. , 2001, Current opinion in biotechnology.

[54]  Stefan Walenta,et al.  Lactate: mirror and motor of tumor malignancy. , 2004, Seminars in radiation oncology.

[55]  Stephanie Schulte,et al.  Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-beta1. , 2007, Experimental cell research.

[56]  George W Bassel,et al.  Elucidating the Germination Transcriptional Program Using Small Molecules1[W][OA] , 2008, Plant Physiology.

[57]  L. Barantin,et al.  Concentration Measurement by Proton NMR Using the ERETIC Method. , 1999, Analytical chemistry.

[58]  H. Jarmer,et al.  Genome-Wide Analysis of the Arabidopsis Leaf Transcriptome Reveals Interaction of Phosphate and Sugar Metabolism1[W] , 2006, Plant Physiology.

[59]  T. Schroeder,et al.  Lactate in solid malignant tumors: potential basis of a metabolic classification in clinical oncology. , 2004, Current medicinal chemistry.

[60]  M. Hirai,et al.  Elucidation of Gene-to-Gene and Metabolite-to-Gene Networks in Arabidopsis by Integration of Metabolomics and Transcriptomics* , 2005, Journal of Biological Chemistry.

[61]  Martin M. Sachs,et al.  Anaerobic gene expression and flooding tolerance in maize , 1996 .

[62]  I. Graham,et al.  Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana. , 2001, Biochemical Society transactions.

[63]  V. Germain,et al.  Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[64]  B Zhang,et al.  ocs element promoter sequences are activated by auxin and salicylic acid in Arabidopsis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[65]  E. Huq,et al.  PHYTOCHROME-INTERACTING FACTOR 1 Is a Critical bHLH Regulator of Chlorophyll Biosynthesis , 2004, Science.

[66]  R. Crawford,et al.  Tolerance of anoxia and ethanol metabolism in germinating seeds. , 1977 .

[67]  Eunkyoo Oh,et al.  PIL5, a Phytochrome-Interacting bHLH Protein, Regulates Gibberellin Responsiveness by Binding Directly to the GAI and RGA Promoters in Arabidopsis Seeds[W] , 2007, The Plant Cell Online.

[68]  Joshua S Yuan,et al.  Plant systems biology comes of age. , 2008, Trends in plant science.

[69]  Lothar Willmitzer,et al.  Integrative gene-metabolite network with implemented causality deciphers informational fluxes of sulphur stress response. , 2005, Journal of experimental botany.

[70]  Yves Gibon,et al.  Integration of metabolite with transcript and enzyme activity profiling during diurnal cycles in Arabidopsis rosettes , 2006, Genome Biology.

[71]  Valérie Laval,et al.  Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement. , 2007, Molecular plant-microbe interactions : MPMI.

[72]  J. W. Allwood,et al.  1H NMR, GC-EI-TOFMS, and data set correlation for fruit metabolomics: application to spatial metabolite analysis in melon. , 2009, Analytical chemistry.

[73]  Naohide Taniguchi,et al.  PICKLE is required for SOLITARY-ROOT/IAA14-mediated repression of ARF7 and ARF19 activity during Arabidopsis lateral root initiation. , 2006, The Plant journal : for cell and molecular biology.

[74]  B. Denoyes-Rothan,et al.  Quantitative metabolic profiling by 1-dimensional 1H-NMR analyses: application to plant genetics and functional genomics. , 2004, Functional plant biology : FPB.

[75]  P. Toorop,et al.  Gene expression profiles of Arabidopsis Cvi seeds during dormancy cycling indicate a common underlying dormancy control mechanism. , 2006, The Plant journal : for cell and molecular biology.

[76]  Rainer Hoefgen,et al.  Metabolomics integrated with transcriptomics: assessing systems response to sulfur-deficiency stress. , 2007, Physiologia plantarum.

[77]  P. Zimmermann,et al.  GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox1[w] , 2004, Plant Physiology.

[78]  Yuji Kamiya,et al.  CHOTTO1, a double AP2 domain protein of Arabidopsis thaliana, regulates germination and seedling growth under excess supply of glucose and nitrate. , 2009, Plant & cell physiology.

[79]  Steven Penfield,et al.  Arabidopsis ABA INSENSITIVE4 Regulates Lipid Mobilization in the Embryo and Reveals Repression of Seed Germination by the Endosperm[W] , 2006, The Plant Cell Online.

[80]  J. Sheen,et al.  Metabolic repression of transcription in higher plants. , 1990, The Plant cell.

[81]  Rodrigo A Gutiérrez,et al.  Systems approach identifies an organic nitrogen-responsive gene network that is regulated by the master clock control gene CCA1 , 2008, Proceedings of the National Academy of Sciences.