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CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis

Molecular Plant 2015年第10期8卷 1493-1506页

作者： Elisa Biazzi Maria Carelli Aldo Tava Pamela Abbruscato ilaria losini Pinarosa Avato Carla Scotti Ornella Calderini Consiglio per la ricerca in agricoltura e I'analisi dell'economia agraria (CRA) Centro di Ricerche per le Produzioni Foraggere e Lattiero Casearie 26900 Lodi Italy Parco Tecnologico Padano 26900 Lodi Italy Dipartimento di Farmacia-Scienze del Farmaco Universita degli Studi di Bad Aldo Moro 70125 Bari Italy Consiglio Nazionale delle Ricerche (CNR) Istituto di Bioscienze e Biorisorse 06128 Perugia Italy These authors contributed equally to this article.

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 ethyl- methanesulfonate （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.

关键词： Medicago truncatula saponin pathway cytochrome P450 CYP72A67 CYP72A68 TILLING analysis

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