title: Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins.
abstract: Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.
authors.affiliation: Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, 26900 Lodi, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Carelli
authors.forename: Elisa
authors.initials: E
authors.lastname: Biazzi
authors.forename: Francesco
authors.initials: F
authors.lastname: Panara
authors.forename: Aldo
authors.initials: A
authors.lastname: Tava
authors.forename: Laura
authors.initials: L
authors.lastname: Scaramelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Porceddu
authors.forename: Neil
authors.initials: N
authors.lastname: Graham
authors.forename: Miriam
authors.initials: M
authors.lastname: Odoardi
authors.forename: Efisio
authors.initials: E
authors.lastname: Piano
authors.forename: Sergio
authors.initials: S
authors.lastname: Arcioni
authors.forename: Sean
authors.initials: S
authors.lastname: May
authors.forename: Carla
authors.initials: C
authors.lastname: Scotti
authors.forename: Ornella
authors.initials: O
authors.lastname: Calderini
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D053878:Hemolytic Agents / Q000378:metabolism; D006801:Humans; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009828:Oleanolic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010084:Oxidation-Reduction; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012502:Sapogenins / Q000378:metabolism; D012503:Saponins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D053878:Hemolytic Agents; D010940:Plant Proteins; D012502:Sapogenins; D012503:Saponins; C077953:erythrodiol; D009828:Oleanolic Acid; D003577:Cytochrome P-450 Enzyme System; C036380:beta-amyrin