Vered Tzin1,2, John H Snyder3,4,5, Dong Sik Yang3,6, David V Huhman3, Bonnie S Watson3, Stacy N Allen3, Yuhong Tang3, Karel Miettinen7,8, Philipp Arendt7,8, Jacob Pollier7,8, Alain Goossens7,8, Lloyd W Sumner9,10. 1. Plant Biology Division, Noble Research Institute, Ardmore, OKLA, 73401, USA. vtzin@bgu.ac.il. 2. French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Israel. vtzin@bgu.ac.il. 3. Plant Biology Division, Noble Research Institute, Ardmore, OKLA, 73401, USA. 4. Department of Plant Biology, Cornell University, Ithaca, NY, 14850, USA. 5. National Institute of Biological Sciences, Beijing, China. 6. Biomaterials Laboratory, Material Research Center, Samsung Advanced Institute of Technology, Suwon, South Korea. 7. Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Ghent, Belgium. 8. VIB Center for Plant Systems Biology, 9052, Ghent, Belgium. 9. Plant Biology Division, Noble Research Institute, Ardmore, OKLA, 73401, USA. sumnerlw@missouri.edu. 10. Department of Biochemistry, Interdisciplinary Plant Group, Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA. sumnerlw@missouri.edu.
Abstract
INTRODUCTION: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. OBJECTIVES: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. METHODS: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. RESULTS: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of β-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2β-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2β-hydroxyoleanolic acid, hederagenin and total saponin levels. CONCLUSIONS: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2β-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.
INTRODUCTION:Triterpenesaponins are important bioactive plant natural products found in many plant families including the Leguminosae. OBJECTIVES: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. METHODS: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. RESULTS: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of β-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2β-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2β-hydroxyoleanolic acid, hederagenin and total saponin levels. CONCLUSIONS: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2β-hydroxylase, both of which function during the early steps of triterpene-oleanatesapogenin biosynthesis.
Authors: Jerome Verdier; Jian Zhao; Ivone Torres-Jerez; Shujun Ge; Chenggang Liu; Xianzhi He; Kirankumar S Mysore; Richard A Dixon; Michael K Udvardi Journal: Proc Natl Acad Sci U S A Date: 2012-01-17 Impact factor: 11.205
Authors: Antoine Branca; Timothy D Paape; Peng Zhou; Roman Briskine; Andrew D Farmer; Joann Mudge; Arvind K Bharti; Jimmy E Woodward; Gregory D May; Laurent Gentzbittel; Cécile Ben; Roxanne Denny; Michael J Sadowsky; Joëlle Ronfort; Thomas Bataillon; Nevin D Young; Peter Tiffin Journal: Proc Natl Acad Sci U S A Date: 2011-09-26 Impact factor: 11.205
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