The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation

BackgroundIn Arabidopsis, INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5), a putative dual-specificity protein phosphatase, is a positive regulator of auxin response. Mutations in IBR5 result in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the phytohormones auxin and abscisic acid. However, the pathways through which IBR5 influences auxin responses are not fully understood.ResultsWe analyzed double mutants of ibr5 with other mutants that dampen auxin responses and found that combining ibr5 with an auxin receptor mutant, tir1, enhanced auxin resistance relative to either parent. Like other auxin-response mutants, auxin-responsive reporter accumulation was reduced in ibr5. Unlike other auxin-resistant mutants, the Aux/IAA repressor reporter protein AXR3NT-GUS was not stabilized in ibr5. Similarly, the Aux/IAA repressor IAA28 was less abundant in ibr5 than in wild type. ibr5 defects were not fully rescued by overexpression of a mutant form of IBR5 lacking the catalytic cysteine residue.ConclusionOur genetic and molecular evidence suggests that IBR5 is a phosphatase that promotes auxin responses, including auxin-inducible transcription, differently than the TIR1 auxin receptor and without destabilizing Aux/IAA repressor proteins. Our data are consistent with the possibility that auxin-responsive transcription can be modulated downstream of TIR1-mediated repressor degradation.

[2]  Peter McCourt,et al.  Cross-talk in plant hormone signalling: what Arabidopsis mutants are telling us. , 2003, Annals of botany.

[3]  J. L. Key,et al.  Isolation of cloned cDNAs to auxin-responsive poly(A)RNAs of elongating soybean hypocotyl. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[4]  W. Gray,et al.  Arabidopsis ETA2, an Apparent Ortholog of the Human Cullin-Interacting Protein CAND1, Is Required for Auxin Responses Mediated by the SCFTIR1 Ubiquitin Ligase , 2004, The Plant Cell Online.

[5]  G. Muday,et al.  Transport of the Two Natural Auxins, Indole-3-Butyric Acid and Indole-3-Acetic Acid, in Arabidopsis1 , 2003, Plant Physiology.

[6]  K. Mockaitis,et al.  Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings. , 2000, The Plant journal : for cell and molecular biology.

[7]  Jennifer Moon,et al.  A New CULLIN 1 Mutant Has Altered Responses to Hormones and Light in Arabidopsis1[C][OA] , 2006, Plant Physiology.

[8]  Y. Shamoo,et al.  Mutation of E1-CONJUGATING ENZYME-RELATED1 Decreases RELATED TO UBIQUITIN Conjugation and Alters Auxin Response and Development1[C][W][OA] , 2007, Plant Physiology.

[9]  C. Somerville,et al.  Sulfonylurea-resistant mutants of Arabidopsis thaliana , 1986, Molecular and General Genetics MGG.

[10]  Kazuo Shinozaki,et al.  Mitogen-activated protein kinase cascades in plants: a new nomenclature. , 2002, Trends in plant science.

[11]  G. Tena,et al.  Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Shiu,et al.  The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W. Gray,et al.  Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases. , 2005, The Plant journal : for cell and molecular biology.

[14]  J. Callis,et al.  Auxin modulates the degradation rate of Aux/IAA proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Erikson,et al.  Negative regulation of mitosis in fission yeast by catalytically inactive pyp1 and pyp2 mutants. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[16]  P. Green,et al.  Characterization of the Auxin-Inducible SAUR-AC1 Gene for Use as a Molecular Genetic Tool in Arabidopsis , 1994, Plant physiology.

[17]  Michael Sauer,et al.  Molecular and cellular aspects of auxin-transport-mediated development. , 2007, Trends in plant science.

[18]  W. Gray,et al.  Arabidopsis SGT1b is required for SCF(TIR1)-mediated auxin response. , 2003, The Plant cell.

[19]  Chris Somerville,et al.  Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology , 1987, Molecular and General Genetics MGG.

[20]  O. Leyser,et al.  Auxin-induced SCFTIR1-Aux/IAA interaction involves stable modification of the SCFTIR1 complex. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[21]  G. Hagen,et al.  The ARF family of transcription factors and their role in plant hormone-responsive transcription , 1998, Cellular and Molecular Life Sciences CMLS.

[22]  R. Hangarter,et al.  Preventing photochemistry in culture media by long-pass light filters alters growth of cultured tissues. , 1990, Plant physiology.

[23]  B. Bartel,et al.  Auxin: regulation, action, and interaction. , 2005, Annals of botany.

[24]  N. Fedoroff.  Cross-Talk in Abscisic Acid Signaling , 2002, Science's STKE.

[25]  E. P. Maher,et al.  Mutants of Arabidopsis thaliana with altered responses to auxins and gravity , 1980, Biochemical Genetics.

[26]  M. Estelle,et al.  The F-box protein TIR1 is an auxin receptor , 2005, Nature.

[27]  G. Hagen,et al.  Dimerization and DNA binding of auxin response factors. , 1999, The Plant journal : for cell and molecular biology.

[28]  W. Chiu,et al.  Suppression of auxin signal transduction by a MAPK cascade in higher plants , 1998, Nature.

[29]  Alan Marchant,et al.  AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues , 1999, The EMBO journal.

[30]  P. Russell,et al.  Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast , 1995, Nature.

[31]  L. E. Rogg.  Cloning and characterization of IAA28, a gene involved in suppressing lateral root development and mediating auxin responses in Arabidopsis thaliana , 2002 .

[32]  G. Hagen,et al.  The Roles of Auxin Response Factor Domains in Auxin-Responsive Transcription Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.008417. , 2003, The Plant Cell Online.

[33]  G. Hagen,et al.  Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. , 1997, The Plant cell.

[34]  K. Struhl,et al.  Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience). Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram-negative Bacteria. , 1988 .

[35]  K. Feldmann,et al.  Arabidopsis AUX1 Gene: A Permease-Like Regulator of Root Gravitropism , 1996, Science.

[36]  S. Luan,et al.  Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis. , 1998, The Plant journal : for cell and molecular biology.

[37]  B. Bartel,et al.  A library of Arabidopsis 35S-cDNA lines for identifying novel mutants , 2001, Plant Molecular Biology.

[38]  K. Dreher,et al.  The Arabidopsis Aux/IAA Protein Family Has Diversified in Degradation and Auxin Responsiveness[W] , 2006, The Plant Cell Online.

[39]  G. Hagen,et al.  Activation and repression of transcription by auxin-response factors. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[40]  M. Estelle,et al.  The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p. , 1998, Genes & development.

[41]  M. Estelle,et al.  Growth and development of the axr1 mutants of Arabidopsis. , 1990, The Plant cell.

[42]  M. Estelle,et al.  The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. , 1998, Science.

[43]  J. S. Lee,et al.  Arabidopsis MAPK Phosphatase 2 (MKP2) Positively Regulates Oxidative Stress Tolerance and Inactivates the MPK3 and MPK6 MAPKs* , 2007, Journal of Biological Chemistry.

[44]  Ottoline Leyser,et al.  Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins , 2001, Nature.

[45]  Candace Timpte,et al.  Arabidopsis auxin-resistance gene AXR1 encodes a protein related to ubiquitin-activating enzyme E1 , 1993, Nature.

[46]  G. Fink,et al.  Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Estelle,et al.  The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Amasino,et al.  A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. , 1998, The Plant journal : for cell and molecular biology.

[49]  Michael Grüninger,et al.  Introduction , 2002, CACM.

[50]  O. Leyser,et al.  Changes in auxin response from mutations in an AUX/IAA gene. , 1998, Science.

[51]  Filip Rolland,et al.  Role of the Arabidopsis Glucose Sensor HXK1 in Nutrient, Light, and Hormonal Signaling , 2003, Science.

[52]  F. B. Pickett,et al.  The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways. , 1995, The Plant journal : for cell and molecular biology.

[53]  Daniel P. Schachtman,et al.  The Arabidopsis Transcription Factor MYB77 Modulates Auxin Signal Transduction[W] , 2007, The Plant Cell Online.

[54]  B. Neel,et al.  Protein tyrosine phosphatases in signal transduction. , 1997, Current opinion in cell biology.

[55]  J Chory,et al.  dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. , 1998, The Plant journal : for cell and molecular biology.

[56]  M. Estelle,et al.  AXR1-ECR1–Dependent Conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 Is Required for Auxin Response Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010282. , 2002, The Plant Cell Online.

[57]  G. Fink,et al.  Tryptophan-Requiring Mutants of the Plant Arabidopsis thaliana , 1988, Science.

[58]  A. Theologis,et al.  Early Genes and Auxin Action , 1996, Plant physiology.

[59]  D. Schachtman,et al.  High-Affinity Auxin Transport by the AUX1 Influx Carrier Protein , 2006, Current Biology.

[60]  G. Hagen,et al.  Auxin-regulated gene expression in intact soybean hypocotyl and excised hypocotyl sections , 1984, Planta.

[61]  Masashi Yamada,et al.  Plant development is regulated by a family of auxin receptor F box proteins. , 2005, Developmental cell.

[62]  T. Baskin,et al.  A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid. , 2006, The Plant journal : for cell and molecular biology.

[63]  S. Tiwari,et al.  Aux/IAA Proteins Contain a Potent Transcriptional Repression Domain , 2004, The Plant Cell Online.

[64]  M. Yamamoto,et al.  Differential effects of 1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on the gravitropic response of roots in an auxin-resistant mutant of arabidopsis, aux1. , 1998, Plant & cell physiology.

[65]  B. Bartel,et al.  A Gain-of-Function Mutation in IAA28 Suppresses Lateral Root Development , 2001, Plant Cell.

[66]  B. Bartel,et al.  IBR5, a Dual-Specificity Phosphatase-Like Protein Modulating Auxin and Abscisic Acid Responsiveness in Arabidopsis Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017046. , 2003, The Plant Cell Online.

[67]  Michal Sharon,et al.  Mechanism of auxin perception by the TIR1 ubiquitin ligase , 2007, Nature.

[68]  Ming-Ming Zhou,et al.  Structure and regulation of MAPK phosphatases. , 2004, Cellular signalling.

[69]  Ottoline Leyser,et al.  The Arabidopsis F-box protein TIR1 is an auxin receptor , 2005, Nature.

[70]  B. Bartel,et al.  Genetic analysis of indole-3-butyric acid responses in Arabidopsis thaliana reveals four mutant classes. , 2000, Genetics.

[71]  A. Theologis,et al.  The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana. , 1995, Journal of molecular biology.

[72]  G. Hagen,et al.  AUX/IAA Proteins Are Active Repressors, and Their Stability and Activity Are Modulated by Auxin Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010289. , 2001, The Plant Cell Online.

[73]  Travis W. Banks,et al.  Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana. , 1999, Genes & development.

[74]  A. Theologis,et al.  Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum sativum). , 1993, Journal of molecular biology.

[75]  J. Reed,et al.  Roles and activities of Aux/IAA proteins in Arabidopsis. , 2001, Trends in plant science.

[76]  P. Oeller,et al.  Early auxin-induced genes encode short-lived nuclear proteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.