AGAMOUS-LIKE 6 is a floral promoter that negatively regulates the FLC/MAF clade genes and positively regulates FT in Arabidopsis.

MADS-box genes encode a family of transcription factors that regulate diverse developmental programs in plants. The present work shows the regulation of flowering time by AGL6 through control of the transcription of both a subset of the FLOWERING LOCUS C (FLC) family genes and FT, two key regulators of flowering time. The agl6-1D mutant, in which AGL6 was activated by the 35S enhancer, showed an early flowering phenotype under both LD and SD conditions. Its early flowering was additively accelerated by CONSTANS (CO) overexpression. The agl6-1D mutation strongly suppressed the late flowering of fve-4 and fca-9 mutants. Endogenous AGL6 transcript accumulation was photoperiod-independent and the AGL6:GFP protein was preferentially localized in the nucleus. In agl6-1D plants, the expression of FLC, MADS AFFECTING FLOWERING (MAF) 4, and MAF5 was downregulated. Interestingly, late flowering of a functional FRIGIDA (FRI) FLC allele was dramatically suppressed by the agl6-1D mutation. AGL6 activation in the flc-3 background further enhanced FT expression, suggesting that AGL6 also regulates FT expression independently of FLC mRNA level. A near RNA-null ft-10 mutation completely suppressed early flowering of the agl6-1D plants, suggesting that FT is a major downstream output of AGL6. Transgenic plants overexpressing an artificial microRNA targeting AGL6 showed a late-flowering phenotype. In these plants, FT expression was downregulated, whereas FLC expression was upregulated. The present results suggest that AGL6 acts as a floral promoter with a dual role, the inhibition of the transcription of the FLC/MAF genes and the promotion of FT expression in Arabidopsis.

[1]  Y. Niwa,et al.  Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. , 2007, Journal of bioscience and bioengineering.

[2]  Neff Walker,et al.  A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development , 2004 .

[3]  Detlef Weigel,et al.  Comprehensive Interaction Map of the Arabidopsis MADS Box Transcription Factorsw⃞ , 2005, The Plant Cell Online.

[4]  J. Ecker,et al.  Small RNA-mediated chromatin silencing directed to the 3′ region of the Arabidopsis gene encoding the developmental regulator, FLC , 2007, Proceedings of the National Academy of Sciences.

[5]  R. Poethig,et al.  EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis , 2006, Development.

[6]  Baohong Zhang,et al.  Plant microRNA: a small regulatory molecule with big impact. , 2006, Developmental biology.

[7]  L. Sieburth,et al.  Widespread Translational Inhibition by Plant miRNAs and siRNAs , 2008, Science.

[8]  J. Riechmann,et al.  Analysis of the Arabidopsis MADS AFFECTING FLOWERING Gene Family: MAF2 Prevents Vernalization by Short Periods of Cold Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.009506. Online version contains Web-only data. , 2003, The Plant Cell Online.

[9]  Javier F. Palatnik,et al.  Specific effects of microRNAs on the plant transcriptome. , 2005, Developmental cell.

[10]  Kerstin Kaufmann,et al.  Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne. , 2006, Journal of experimental botany.

[11]  Claire Périlleux,et al.  Mutagenesis of Plants Overexpressing CONSTANS Demonstrates Novel Interactions among Arabidopsis Flowering-Time Genes , 2000, Plant Cell.

[12]  A. Berr,et al.  The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana. , 2009, The Plant journal : for cell and molecular biology.

[13]  Ilha Lee,et al.  SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy. , 2008, The Plant journal : for cell and molecular biology.

[14]  W. Peacock,et al.  Epigenetic regulation of flowering. , 2007, Current opinion in plant biology.

[15]  C. Dean,et al.  The Timing of Developmental Transitions in Plants , 2006, Cell.

[16]  D. Weigel,et al.  A thermosensory pathway controlling flowering time in Arabidopsis thaliana , 2003, Nature Genetics.

[17]  W. Scheible,et al.  Eleven Golden Rules of Quantitative RT-PCR , 2008, The Plant Cell Online.

[18]  C. Vincent,et al.  The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. , 2006, Genes & development.

[19]  R. Martienssen,et al.  Vernalization requires epigenetic silencing of FLC by histone methylation , 2004, Nature.

[20]  H. Sommer,et al.  Characterization of the Antirrhinum floral homeotic MADS‐box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. , 1992, The EMBO journal.

[21]  R. Simon,et al.  The CONSTANS gene of arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors , 1995, Cell.

[22]  R. Amasino,et al.  FLOWERING LOCUS C Encodes a Novel MADS Domain Protein That Acts as a Repressor of Flowering , 1999, Plant Cell.

[23]  Gerco C. Angenent,et al.  Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis , 2005, Development.

[24]  Fabio Fornara,et al.  FT Protein Movement Contributes to Long-Distance Signaling in Floral Induction of Arabidopsis , 2007, Science.

[25]  J. Görlach,et al.  Growth Stage–Based Phenotypic Analysis of Arabidopsis , 2001, The Plant Cell Online.

[26]  D. Weigel,et al.  Cell-cell signaling and movement by the floral transcription factors LEAFY and APETALA1. , 2000, Science.

[27]  M. Robertson,et al.  Post-translational modifications of the endogenous and transgenic FLC protein in Arabidopsis thaliana. , 2008, Plant & cell physiology.

[28]  A. Moorman,et al.  Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data , 2003, Neuroscience Letters.

[29]  W. Peacock,et al.  The FLF MADS Box Gene: A Repressor of Flowering in Arabidopsis Regulated by Vernalization and Methylation , 1999, Plant Cell.

[30]  Wolfgang Busch,et al.  Integration of Spatial and Temporal Information During Floral Induction in Arabidopsis , 2005, Science.

[31]  Detlef Weigel,et al.  Highly Specific Gene Silencing by Artificial MicroRNAs in Arabidopsis[W][OA] , 2006, The Plant Cell Online.

[32]  L. Hennig,et al.  FLC or not FLC: the other side of vernalization. , 2008, Journal of experimental botany.

[33]  F. Turck,et al.  The impact of chromatin regulation on the floral transition. , 2008, Seminars in cell & developmental biology.

[34]  Youn Kyung Kim,et al.  Isolation of 151 mutants that have developmental defects from T-DNA tagging. , 2007, Plant & cell physiology.

[35]  J. Mathieu,et al.  Export of FT Protein from Phloem Companion Cells Is Sufficient for Floral Induction in Arabidopsis , 2007, Current Biology.

[36]  Adam M. Gustafson,et al.  microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants , 2005, Cell.

[37]  M. Purugganan,et al.  Complex rearrangements lead to novel chimeric gene fusion polymorphisms at the Arabidopsis thaliana MAF2-5 flowering time gene cluster. , 2008, Molecular biology and evolution.

[38]  S. van Nocker,et al.  A Mechanism Related to the Yeast Transcriptional Regulator Paf1c Is Required for Expression of the Arabidopsis FLC/MAF MADS Box Gene Familyw⃞ , 2004, The Plant Cell Online.

[39]  J. Zethof,et al.  The petunia AGL6 gene has a SEPALLATA-like function in floral patterning. , 2009, The Plant journal : for cell and molecular biology.

[40]  H. Sommer,et al.  GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. , 1992, The EMBO journal.

[41]  Z. Schwarz‐Sommer,et al.  Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. , 2000, Science.

[42]  M. Cho,et al.  Control of lateral organ development and flowering time by the Arabidopsis thaliana MADS-box Gene AGAMOUS-LIKE6. , 2010, The Plant journal : for cell and molecular biology.

[43]  Koji Goto,et al.  TERMINAL FLOWER2, an Arabidopsis Homolog of HETEROCHROMATIN PROTEIN1, Counteracts the Activation of FLOWERING LOCUS T by CONSTANS in the Vascular Tissues of Leaves to Regulate Flowering Time Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016 , 2003, The Plant Cell Online.

[44]  Y. Kobayashi,et al.  A pair of related genes with antagonistic roles in mediating flowering signals. , 1999, Science.

[45]  S. Davis Faculty Opinions recommendation of Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. , 2006 .

[46]  R. Amasino,et al.  Loss of FLOWERING LOCUS C Activity Eliminates the Late-Flowering Phenotype of FRIGIDA and Autonomous Pathway Mutations but Not Responsiveness to Vernalization , 2001, Plant Cell.

[47]  Song Tan,et al.  Structure of serum response factor core bound to DNA , 1995, Nature.

[48]  S. Park,et al.  Role of SVP in the control of flowering time by ambient temperature in Arabidopsis. , 2007, Genes & development.

[49]  E. Meyerowitz,et al.  Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. , 1994, Genes & development.

[50]  J. Bowman,et al.  Early flower development in Arabidopsis. , 1990, The Plant cell.

[51]  K. Gaston,et al.  Transcriptional repression in eukaryotes: repressors and repression mechanisms , 2003, Cellular and Molecular Life Sciences CMLS.

[52]  P. Huijser,et al.  Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. , 2000, The Plant journal : for cell and molecular biology.

[53]  Hajime Sakai,et al.  Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238. , 2003, The Plant Cell Online.

[54]  M. Robertson,et al.  The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. , 2006, The Plant journal : for cell and molecular biology.

[55]  Jason S. Cumbie,et al.  High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes , 2007, PloS one.

[56]  R. Amasino,et al.  PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis. , 2004, Genes & development.

[57]  J. Zeevaart Leaf-produced floral signals. , 2008, Current opinion in plant biology.

[58]  Joonki Kim,et al.  CONSTANS Activates SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 through FLOWERING LOCUS T to Promote Flowering in Arabidopsis1[w] , 2005, Plant Physiology.

[59]  K. Goto,et al.  FD, a bZIP Protein Mediating Signals from the Floral Pathway Integrator FT at the Shoot Apex , 2005, Science.

[60]  Ilha Lee,et al.  SUPPRESSOR OF FRIGIDA3 Encodes a Nuclear ACTIN-RELATED PROTEIN6 Required for Floral Repression in Arabidopsisw⃞ , 2005, The Plant Cell Online.

[61]  Yaoguang Liu,et al.  Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. , 1995, Genomics.

[62]  N. Chua,et al.  The GIGANTEA-Regulated MicroRNA172 Mediates Photoperiodic Flowering Independent of CONSTANS in Arabidopsis[W][OA] , 2007, The Plant Cell Online.

[63]  J. Chory,et al.  Activation tagging of the floral inducer FT. , 1999, Science.

[64]  Katja E. Jaeger,et al.  FT Protein Acts as a Long-Range Signal in Arabidopsis , 2007, Current Biology.

[65]  Jong Seob Lee,et al.  Integration of floral inductive signals by flowering locus T and suppressor of overexpression of Constans 1 , 2006 .

[66]  R. Dixon,et al.  Activation tagging in Arabidopsis. , 2000, Plant physiology.

[67]  J. S. Lee,et al.  The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. , 2000, Genes & development.

[68]  Linda Gruffman Post-transcriptional regulation of the expression of the flowering time gene FT in different light conditions , 2006 .

[69]  J. Riechmann,et al.  Regulation of flowering in Arabidopsis by an FLC homologue. , 2001, Plant physiology.

[70]  R. Deal,et al.  The Nuclear Actin-Related Protein ARP6 Is a Pleiotropic Developmental Regulator Required for the Maintenance of FLOWERING LOCUS C Expression and Repression of Flowering in Arabidopsisw⃞ , 2005, The Plant Cell Online.

[71]  Yang Wu,et al.  A repressor complex governs the integration of flowering signals in Arabidopsis. , 2008, Developmental cell.