title: The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula.
abstract: Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.).
authors.forename: Jan
authors.identifier: http://orcid.org/0000-0003-1878-9903
authors.initials: J
authors.lastname: Mertens
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.).
authors.forename: Jacob
authors.identifier: http://orcid.org/0000-0002-1134-9238
authors.initials: J
authors.lastname: Pollier
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.).
authors.forename: Robin
authors.identifier: http://orcid.org/0000-0002-4593-9658
authors.initials: R
authors.lastname: Vanden Bossche
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.).
authors.forename: Irene
authors.identifier: http://orcid.org/0000-0001-9362-8662
authors.initials: I
authors.lastname: Lopez-Vidriero
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.).
authors.forename: José Manuel
authors.identifier: http://orcid.org/0000-0001-6769-7349
authors.initials: JM
authors.lastname: Franco-Zorrilla
authors.affiliation: Department of Plant Systems Biology, VIB, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (J.M., J.P., R.V.B., A.G.); andGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain (I.L.-V., J.M.F.-Z.) alain.goossens@psb-vib.ugent.be.
authors.forename: Alain
authors.identifier: http://orcid.org/0000-0002-1599-551X
authors.initials: A
authors.lastname: Goossens
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D003517:Cyclopentanes / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent / Q000235:genetics / Q000378:metabolism; D046913:Medicago truncatula / Q000235:genetics / Q000378:metabolism*; D008798:Mevalonic Acid / Q000378:metabolism; D054883:Oxylipins / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012503:Saponins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014026:Nicotiana / Q000235:genetics; D014315:Triterpenes / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D003517:Cyclopentanes; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D008798:Mevalonic Acid; D054883:Oxylipins; D010940:Plant Proteins; D012503:Saponins; D014315:Triterpenes; C011006:jasmonic acid