title: CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis.
abstract: In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.
authors.affiliation: Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria (CRA), Centro di Ricerche per le Produzioni Foraggere e Lattiero Casearie, 26900 Lodi, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Biazzi
authors.affiliation: Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria (CRA), Centro di Ricerche per le Produzioni Foraggere e Lattiero Casearie, 26900 Lodi, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Carelli
authors.affiliation: Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria (CRA), Centro di Ricerche per le Produzioni Foraggere e Lattiero Casearie, 26900 Lodi, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Tava
authors.affiliation: Parco Tecnologico Padano, 26900 Lodi, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Abbruscato
authors.affiliation: Parco Tecnologico Padano, 26900 Lodi, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Losini
authors.affiliation: Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, 70125 Bari, Italy.
authors.forename: Pinarosa
authors.initials: P
authors.lastname: Avato
authors.affiliation: Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria (CRA), Centro di Ricerche per le Produzioni Foraggere e Lattiero Casearie, 26900 Lodi, Italy. Electronic address: carla.scotti@entecra.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Scotti
authors.affiliation: Consiglio Nazionale delle Ricerche (CNR), Istituto di Bioscienze e Biorisorse, 06128 Perugia, Italy.
authors.forename: Ornella
authors.initials: O
authors.lastname: Calderini
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D046913:Medicago truncatula / Q000378:metabolism*; D009828:Oleanolic Acid / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D012503:Saponins / Q000096:biosynthesis*; D014315:Triterpenes / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D012503:Saponins; D014315:Triterpenes; D009828:Oleanolic Acid; C005982:medicagenic acid; D003577:Cytochrome P-450 Enzyme System
keywords: CYP72A67; CYP72A68; Medicago truncatula; TILLING analysis; cytochrome P450; saponin pathway