CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin

ABSTRACT In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthe-sized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponinsaspharmaceutics, agrochemicalsand inthe food and cosmetic industries hasraised interestin identifyingthe enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved inhemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethyl-methanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complemen-tation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible forhydroxylationattheC-2positiondownstreamofoleanolicacidsynthesis.TheaffinityofCYP72A67forsub-strates with different substitutions at multiple carbon positions was investigated in the same in vitro yeastsystem, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenicacid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants werecompared 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 thehemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities(leaves/roots) of sapogenin synthesis.Key words: Medicago truncatula, saponin pathway, cytochrome P450, CYP72A67, CYP72A68, TILLING analysisBiazzi E., Carelli M., Tava A., Abbruscato P., Losini I., Avato P., Scotti C., and Calderini O. (2015). CYP72A67CatalyzesaKeyOxidativeStepinMedicagotruncatulaHemolyticSaponinBiosynthesis.Mol.Plant.8,1493–1506.