Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action

Heterotrimeric G‐proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G‐protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G‐protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G‐protein × hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G‐protein signaling are well represented. Conversely, some theoretical regulatory modes of the G‐protein are not supported; (2) a new mechanism of G‐protein signaling is revealed, in which Gβ regulates gene expression identically in the presence or absence of Gα; (3) guard cells and leaves favor different G‐protein modes in transcriptome regulation, supporting system specificity of G‐protein signaling. Our method holds significant promise for analyzing analogous ‘switch‐like’ signal transduction events in any organism.

[1]  Alain Vavasseur,et al.  Arabidopsis OST1 Protein Kinase Mediates the Regulation of Stomatal Aperture by Abscisic Acid and Acts Upstream of Reactive Oxygen Species Production Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.007906. , 2002, The Plant Cell Online.

[2]  Satoru Miyano,et al.  Identification of Genetic Networks from a Small Number of Gene Expression Patterns Under the Boolean Network Model , 1998, Pacific Symposium on Biocomputing.

[3]  Jean YH Yang,et al.  Bioconductor: open software development for computational biology and bioinformatics , 2004, Genome Biology.

[4]  S. Assmann,et al.  Heterotrimeric G proteins regulate reproductive trait plasticity in response to water availability. , 2010, The New phytologist.

[5]  Alan M. Jones,et al.  The plant heterotrimeric G-protein complex. , 2007, Annual review of plant biology.

[6]  Tsuyoshi Nakagawa,et al.  The Plastid Protein THYLAKOID FORMATION1 and the Plasma Membrane G-Protein GPA1 Interact in a Novel Sugar-Signaling Mechanism in Arabidopsis[W] , 2006, The Plant Cell Online.

[7]  J. Giraudat,et al.  ABSCISIC ACID SIGNAL TRANSDUCTION. , 1998, Annual review of plant physiology and plant molecular biology.

[8]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Robitaille,et al.  The role of Gbetagamma subunits in the organization, assembly, and function of GPCR signaling complexes. , 2009, Annual review of pharmacology and toxicology.

[10]  M. Lohse,et al.  G Protein Activation without Subunit Dissociation Depends on a Gαi-specific Region* , 2005, Journal of Biological Chemistry.

[11]  J. Slusser,et al.  Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings. , 2008, Plant, cell & environment.

[12]  D. Siderovski,et al.  G-protein signaling: back to the future , 2005, Cellular and Molecular Life Sciences.

[13]  A. Levitzki,et al.  Signal transduction by a nondissociable heterotrimeric yeast G protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Schroeder,et al.  Guard cell ABA and CO2 signaling network updates and Ca2+ sensor priming hypothesis. , 2006, Current opinion in plant biology.

[15]  B. Charrier,et al.  Real-time PCR: what relevance to plant studies? , 2004, Journal of experimental botany.

[16]  John T. Dimos,et al.  A Stem Cell Molecular Signature , 2002, Science.

[17]  Sarah M Assmann,et al.  Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling , 2008, Genome Biology.

[18]  Alan M. Jones,et al.  Differential Roles of Arabidopsis Heterotrimeric G-Protein Subunits in Modulating Cell Division in Roots1[W] , 2006, Plant Physiology.

[19]  E. Liscum,et al.  A mutant Arabidopsis heterotrimeric G-protein beta subunit affects leaf, flower, and fruit development. , 2001, The Plant cell.

[20]  Dmitrij Frishman,et al.  MIPS: analysis and annotation of proteins from whole genomes in 2005 , 2006, Nucleic Acids Res..

[21]  Alan M. Jones,et al.  Abscisic acid regulation of guard-cell K+ and anion channels in Gβ- and RGS-deficient Arabidopsis lines , 2008, Proceedings of the National Academy of Sciences.

[22]  M. Robitaille,et al.  The Role of G βγ Subunits in the Organization, Assembly, and Function of GPCR Signaling Complexes , 2009 .

[23]  J. Flexas,et al.  Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid1[C][W][OA] , 2009, Plant Physiology.

[24]  R. Finkelstein,et al.  Abscisic Acid Signaling in Seeds and Seedlings Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010441. , 2002, The Plant Cell Online.

[25]  S. Assmann,et al.  Hormone interactions in stomatal function , 2009, Plant Molecular Biology.

[26]  Alan M. Jones,et al.  GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination1[w] , 2004, Plant Physiology.

[27]  William Stafford Noble,et al.  Matrix2png: a utility for visualizing matrix data , 2003, Bioinform..

[28]  Alan M. Jones,et al.  G-Protein Complex Mutants Are Hypersensitive to Abscisic Acid Regulation of Germination and Postgermination Development1[W] , 2006, Plant Physiology.

[29]  Alan M. Jones,et al.  A Reevaluation of the Role of the Heterotrimeric G Protein in Coupling Light Responses in Arabidopsis1 , 2003, Plant Physiology.

[30]  S. Lateef,et al.  G-Protein-Coupled Receptor 1, G-Protein Gα-Subunit 1, and Prephenate Dehydratase 1 Are Required for Blue Light-Induced Production of Phenylalanine in Etiolated Arabidopsis1 , 2006, Plant Physiology.

[31]  S. Assmann,et al.  Light regulation of stomatal movement. , 2007, Annual review of plant biology.

[32]  S. Spiegel,et al.  Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins , 2003, Nature.

[33]  A. Webb,et al.  ABI1 Protein Phosphatase 2C Is a Negative Regulator of Abscisic Acid Signaling , 1999, Plant Cell.

[34]  Jean-Loup Faulon,et al.  Boolean dynamics of genetic regulatory networks inferred from microarray time series data , 2007, Bioinform..

[35]  Alan M. Jones,et al.  Plant heterotrimeric G protein function: insights from Arabidopsis and rice mutants. , 2004, Current opinion in plant biology.

[36]  C. Patrick Doncaster,et al.  Analysis of Variance and Covariance: How to Choose and Construct Models for the Life Sciences , 2007 .

[37]  S. Merlot,et al.  The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. , 2001, The Plant journal : for cell and molecular biology.

[38]  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.

[39]  C. Malbon,et al.  Physiological regulation of G protein-linked signaling. , 1999, Physiological reviews.

[40]  Anbupalam Thalamuthu,et al.  Gene expression Evaluation and comparison of gene clustering methods in microarray analysis , 2006 .

[41]  H. Hamm,et al.  Heterotrimeric G protein activation by G-protein-coupled receptors , 2008, Nature Reviews Molecular Cell Biology.

[42]  Dmitrij Frishman,et al.  MIPS: analysis and annotation of proteins from whole genomes in 2005 , 2005, Nucleic Acids Res..

[43]  Alan M. Jones,et al.  The beta-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. , 2003, The Plant cell.

[44]  X. Cui,et al.  Statistical tests for differential expression in cDNA microarray experiments , 2003, Genome Biology.

[45]  Lingang Zhang,et al.  Heterotrimeric G protein alpha and beta subunits antagonistically modulate stomatal density in Arabidopsis thaliana. , 2008, Developmental biology.

[46]  Alan M. Jones,et al.  GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling , 2007, Proceedings of the National Academy of Sciences.

[47]  George Casella,et al.  Assessing agreement of clustering methods with gene expression microarray data , 2008, Comput. Stat. Data Anal..

[48]  Alan M. Jones,et al.  G Protein Regulation of Ion Channels and Abscisic Acid Signaling in Arabidopsis Guard Cells , 2001, Science.

[49]  Yannick Marrari,et al.  Assembly and trafficking of heterotrimeric G proteins. , 2007, Biochemistry.

[50]  Rainer Hedrich,et al.  In the light of stomatal opening: new insights into 'the Watergate'. , 2005, The New phytologist.

[51]  Julian I Schroeder,et al.  Microarray Expression Analyses of Arabidopsis Guard Cells and Isolation of a Recessive Abscisic Acid Hypersensitive Protein Phosphatase 2C Mutant Online version contains Web-only data. , 2004, The Plant Cell Online.

[52]  P. Schenk,et al.  Heterotrimeric G Proteins Facilitate Arabidopsis Resistance to Necrotrophic Pathogens and Are Involved in Jasmonate Signaling1 , 2005, Plant Physiology.

[53]  J. Mesirov,et al.  Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. , 1999, Science.

[54]  N. Fedoroff,et al.  Heterotrimeric G protein signaling in the Arabidopsis unfolded protein response , 2007, Proceedings of the National Academy of Sciences.

[55]  Lingang Zhang,et al.  Activation of the heterotrimeric G protein alpha-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis. , 2009, The Plant journal : for cell and molecular biology.

[56]  Alan M. Jones,et al.  Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors , 2006, Genome Biology.

[57]  S. He,et al.  The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation. , 2008, The Plant journal : for cell and molecular biology.

[58]  C. Hung,et al.  Transgenic Arabidopsis Plants Expressing the Type 1 Inositol 5-Phosphatase Exhibit Increased Drought Tolerance and Altered Abscisic Acid Signaling[W] , 2008, The Plant Cell Online.

[59]  I. Feussner,et al.  The a-subunit of the heterotrimeric G-protein affects jasmonate responses in Arabidopsis thaliana , 2009 .

[60]  B. Charrier,et al.  Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain Reaction1 , 2002, Plant Physiology.

[61]  S. Assmann,et al.  Regulation of root-wave response by extra large and conventional G proteins in Arabidopsis thaliana. , 2008, The Plant journal : for cell and molecular biology.

[62]  Lingang Zhang,et al.  Heterotrimeric G protein α and β subunits antagonistically modulate stomatal density in Arabidopsis thaliana , 2008 .

[63]  N. Lambert,et al.  Differential dissociation of G protein heterotrimers , 2008, The Journal of physiology.

[64]  S. He,et al.  Role of stomata in plant innate immunity and foliar bacterial diseases. , 2008, Annual review of phytopathology.

[65]  A. Tinker,et al.  The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[66]  S. Assmann,et al.  Two Novel GPCR-Type G Proteins Are Abscisic Acid Receptors in Arabidopsis , 2009, Cell.

[67]  S. Assmann G Proteins Go Green: A Plant G Protein Signaling FAQ Sheet , 2005, Science.

[68]  E. Grill,et al.  Regulators of PP2C Phosphatase Activity Function as Abscisic Acid Sensors , 2009, Science.

[69]  X. Q. Wang,et al.  Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. , 2000, Science.

[70]  K. Shinozaki,et al.  ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. , 2002, Plant & cell physiology.

[71]  Benjamin M. Bolstad,et al.  affy - analysis of Affymetrix GeneChip data at the probe level , 2004, Bioinform..

[72]  Sarah M Assmann,et al.  Plants: the latest model system for G‐protein research , 2004, EMBO reports.