In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in plant aerial and subterranean part from the isoprenoid pathway. The exploitation of saponins as pharmaceutics, agrochemicals and in 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 CYP72A67 is responsible for the hydroxylation at 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, also in relation with 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 sapogenin profile, expression patterns of genes involved in sapogenin synthesis and response to the inoculation with Sinorhizobium meliloti. The results obtained allow to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.
