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Mol Plant. 2015 Oct 5;8(10):1493-506. doi: 10.1016/j.molp.2015.06.003. Epub 2015 Jun 12.

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

Author information

1
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.
2
Parco Tecnologico Padano, 26900 Lodi, Italy.
3
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, 70125 Bari, Italy.
4
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.
5
Consiglio Nazionale delle Ricerche (CNR), Istituto di Bioscienze e Biorisorse, 06128 Perugia, Italy.

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.

KEYWORDS:

CYP72A67; CYP72A68; Medicago truncatula; TILLING analysis; cytochrome P450; saponin pathway

PMID:
26079384
DOI:
10.1016/j.molp.2015.06.003
[Indexed for MEDLINE]
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