May 2013 May I dentification of Potential Regulators of Jasmonate- Regulators ofdentification Potential Identification of Potential Regulators of Jasmonate-Modulated Secondary Metabolism in Medicago truncatula Modulated Secondary Modulated Metabolism in Medicago truncatula truncatula Medicago Azra Gholami Promotor: Professor Alain Goossens Azra Gholami June 2013 Ghent University - Faculty of Sciences Department of Plant Biotechnology and Bioinformatics VIB - Department of Plant Systems Biology Identification of Potential Regulators of Jasmonate-Modulated Secondary Metabolism in Medicago truncatula Azra Gholami Thesis submitted in partial fulfillment of the requirements for the degree of Doctor (PhD) in Sciences: Biotechnology Academic year: 2012-2013 Promotor: Prof. Alain Goossens This work was conducted in the VIB Department of Plant Systems Biology, Ghent University. Board of Examiners Prof. Wout Boerjan (Chair) VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University Prof. Alain Goossens (Promotor) VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University Prof. Sofie Goormachtig * VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University Prof. Bartel Vanholme* VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University Prof. Jan Van Bocxlaer* Department of Bio-analysis, Faculty of Pharmaceutical Sciences, Ghent University Prof. Rosa Maria Cusido* Departament de Productes Naturals, Biologia Vegetal i Edafologia, University of Barcelona Prof. Tom Beeckman VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University Dr. Jacob Pollier VIB Department of Plant Systems Biology, Department of Plant Biotechnology and Bioinformatics, Faculty of Sciences, Ghent University * Members of the Reading Committee Table of contents Commonly used abbreviations and gene names 1 Abstract 7 Transcriptional machineries in jasmonate-elicited plant Chapter 1 9 secondary metabolism Chapter 2 Natural product biosynthesis in Medicago species 35 Chapter 3 Scope of Research 105 Ccr4-associated factor1 (Caf1) is a component of a large Chapter 4 regulatory complex potentially involved in Medicago truncatula 109 secondary metabolism The MYB transcription factor Mt061 modulates defense Chapter 5 137 response mechanisms in Medicago truncatula Functional characterization of small signalling peptides that Chapter 6 potentially steer secondary metabolite biosynthesis in Medicago 165 truncatula Chapter 7 Conclusion and perspectives 201 Summary 207 Abbreviations 1 Commonly used abbreviations and gene names 40S 40S ribosomal protein S8 4CL 4-coumarate: CoA ligase AAC Alcohol acyl-transferase ABA Abscisic acid ABC ATP binding cassette ACC Acetyl-CoA carboxylase ACN Acetonitrile ADS Amorpha-4,11-diene synthase ADH Alcohol dehydrogenase AFLP Amplified fragment length polymorphism AM Arbuscular mycorrhization ANR Anthocyanidin reductase ANS Anthocyanidin synthase AOC Allene oxide cyclase AOS Allene oxide synthase AP2/ERF APETALA2/ETHYLENE Response Factor AVIs Anthocyanic vacuolar inclusions BAN BANYULS gene BAS β-Amyrin synthase bHLH basic helix–loop–helix BLAST Basic local alignment search tool bp Base pair C4H Cinnamate 4-hydroxylase CAD1 (1)- δ -cadinene synthase CAF1 Ccr4-associated factor1 CAS Cycloartenol synthase Ccr4-Not Carbon catabolite repressor protein 4 - Negative on TATA complex complex CHI Chalcone isomerase CHR Chalcone reductase CHS Chalcone synthase Abbreviations 2 CID Collision-induced dissociation CMC Critical micelle concentration COI1 Coronatine insensitive 1 Cou Coumaric acid CPC CAPRICE CYP450 Cytochrome P450 DAT Acetyl-coA:4-O-deacetylvindoline 4-O-acetyltransferase DFR Dihydroflavonol-4-reductase DMAPP Dimethylallyl diphosphate DMID Dihydroxy-4’-methoxy- isoflavanol dehydratase Dof DNA-binding with one finger EAR ERF-associated amphiphilic repression EEP Endonuclease–exonuclease–phophatase EGL3 Enhancer of GL3 ELF1 α Elongation factor 1 α ERF Ethylene response factor ER Endoplasmic reticulum ESI-IT MS Electrospray ionization ion-trap mass spectrometry EST Expressed sequence tag εmax Maximum molar extinction coefficients F3H Flavanone 3-hydroxylase F3 ′5′H Flavonoid 3 ′,5 ′-hydroxylase Fer Ferrulic acid FL- ORF Full length- open reading frame FLS Flavonol synthase FNS Flavone synthase FPKM Fragments per kilobase of transcript per million fragments mapped FPS Farnesyl pyrophosphate synthase FT-ICR MS Fourier transform ion cyclotron resonance mass spectrometry G10H Geraniol 10-hydroxylase GC- MS Gas chromatography- mass spectrometry GL3 Glabra3 Abbreviations 3 Glc Glucose GlcA Glucuronic acid GlcAPyr Glucuronopyranosyl GLS Glucosinolates GLVs Green leaf volatiles GST Glutathione S-transferase HD- ZIP Homeodomain-leucine zipper HMGR 3-hydroxy-3-methylglutaryl-CoA reductase HDAC Histone deacetylases HPL Hydroperoxide lyase HPLC High-performance liquid chromatography HPOT Hydroperoxy linolenic acid hpRNAi Hairpin RNA-mediated Interference I2’H Isoflavone 2’-hydroxylase I3’H Isoflavone 3’-hydroxylase IFR Isoflavone reductase IFS Isoflavone synthase IPP Isopentenyl pyrophosphate JA Jasmonate JA–Ile JA–isoleucine JAM1 JA factor-stimulating MAPKK 1 JAZ Jasmonate ZIM-domain JMT Jasmonate O-methyltransferase LAP1 Legume anthocyanin production 1 LAR Leucoanthocyanidin reductase LIP Lipase LiF Laser-induced fluorescence LOX Lipoxygenase m/z Mass-to-charge ratio Mal Malonic acid MAPKK Mitogen-activated protein kinase kinase MaT Malonyltransferases Abbreviations 4 MATE Vacuole-localized multidrug and toxic extrusion MeJA Methyl jasmonate MEKK1 MAPK/ERK kinase kinase 1 MEP pathway 2-C-methyl-D-erythritol 4-phosphate pathway MAPK Mitogen-activated protein kinase MtPAR M. truncatula proanthocyanidin regulator MVA Mevalonic acid pathway NFs Nod factors Nod gene Nodulin gene NINJA Novel Interactor of JAZ NMR Nuclear magnetic resonance NPAAS Nonprotein amino acids ORCA Octadecanoid-derivative responsive catharanthus AP2-domain OSC Oxidosqualene cyclase PAL Phenylalanine ammonia-lyase PA Proanthocyanidin PAP1 Production of anthocyanin pigment 1 PCA Principal component analysis PMT Putrescine N-methyltransferase PR1a Pathogenesis-related protein 1a PRX Peroxidase PSK-α Phytosulfokine-α PVCs Prevacuolar compartments PVs Plant volatiles QRT-PCR Quantitative Real-time Polymerase Chain Reaction QToF Quadrupole time-of-flight SCF COI1 Skp–Cullin–F-box-type E3 ubiquitin ligase complex SD-U Synthetic defined medium lacking URA SGD Strictosidine β-d-glucosidase SQE Squalene epoxidase SEM Standard error of the mean SQS Squalene synthase Abbreviations 5 SNAREs Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptors TAP Tandem affinity purification TCP Teosinte Branched/Cycloidea/Pcf TDC Tryptophan decarboxylase TFs Transcription factors TGN Trans-Golgi network TIA Terpenoid indole alkaloid TIC Total ion current TPI Trypsin protease inhibitor TPL Topless Trp Tryptophan TT8 Transparent testa 8 TTG1 Transparent Testa Glabra 1 UGT UDP-dependent glycosyltransferase UPLC Ultra performance liquid chromatography UTR Untranslated region VR Vestitone reductase YE Yeast elicitor Abstract 7 Abstract Accumulation of secondary metabolites often occurs in plants imposed to biotic and abiotic stress signals. Perception of the stress signals triggers a signal transduction network that leads to the activation or de novo biosynthesis of transcription factors. Then transcription factors regulate the expression of the genes encoding enzymes that catalyze the biosynthesis of target secondary metabolites. Here, we focused on the identification of the regulators of secondary metabolite biosynthesis in the model legume Medicago truncatula . Considering the conserved role of JAs in the induction of secondary metabolite biosynthesis in plants, we exploited a gene list generated previously through a genome-wide transcript profiling of MeJA-elicited cell cultures of M. truncatula . In this PhD thesis, several of the potential regulators identified in the previous study were selected for further characterization. Through a reverse genetics approach two regulatory proteins were found with a putative role in secondary metabolite production in M. truncatula hairy roots. Overexpression of Mt148 encoding a CCR4-associate factor1 (Caf1) protein led to the downregulation of some secondary metabolism genes in M. truncatula hairy roots. Overexpression of Mt061 codifying for an R2R3-type MYB family transcription factor, demonstrated that it is a regulator of green leaf volatiles (GLV) biosynthesis. This MYB- transcription factor can also modulate defense response processes in M.truncatula . Furthermore, we studied the putative role of small signaling peptides in the signaling transduction pathways towards the regulation secondary metabolism in M. truncatula . We found that Taximin, a small signaling peptide previously identified in Taxus baccata , as well as the M. truncatula taximin homologs can affect secondary metabolite biosynthesis in M. truncatula . 1 Transcriptional machineries in jasmonate- elicited plant secondary metabolism Nathan De Geyter*, Azra Gholami*, Sofie Goormachtig and Alain Goossens Published in Trends in Plant Science (IF: 11.047) De Geyter, N.*, Gholami, A.*, Goormachtig, S., and Goossens, A. (2012). Transcriptional