title: Glucosinolate diversity within a phylogenetic framework of the tribe Cardamineae (Brassicaceae) unraveled with HPLC-MS/MS and NMR-based analytical distinction of 70 desulfoglucosinolates.
abstract: As a basis for future investigations of evolutionary trajectories and biosynthetic mechanisms underlying variations in glucosinolate structures, we screened members of the crucifer tribe Cardamineae by HPLC-MS/MS, isolated and identified glucosinolates by NMR, searched the literature for previous data for the tribe, and collected HPLC-MS/MS data for nearly all glucosinolates known from the tribe as well as some related structures (70 in total). This is a considerable proportion of the approximately 142 currently documented natural glucosinolates. Calibration with authentic references allowed distinction (or elucidation) of isomers in many cases, such as distinction of β-hydroxyls, methylthios, methylsulfinyls and methylsulfonyls. A mechanism for fragmentation of secondary β-hydroxyls in MS was elucidated, and two novel glucosinolates were discovered: 2-hydroxy-3-methylpentylglucosinolate in roots of Cardamine pratensis and 2-hydroxy-8-(methylsulfinyl)octylglucosinolate in seeds of Rorippa amphibia. A large number of glucosinolates (ca. 54 with high structural certainty and a further 28 or more suggested from tandem MS), representing a wide structural variation, is documented from the tribe. This included glucosinolates apparently derived from Met, Phe, Trp, Val/Leu, Ile and higher homologues. Normal side chain elongation and side chain decoration by oxidation or methylation was observed, as well as rare abnormal side chain decoration (hydroxylation of aliphatics at the δ rather than β-position). Some species had diverse profiles, e.g. R. amphibia and C. pratensis (19 and 16 individual glucosinolates, respectively), comparable to total diversity in literature reports of Armoracia rusticana (17?), Barbarea vulgaris (20-24), and Rorippa indica (>20?). The ancestor or the tribe would appear to have used Trp, Met, and homoPhe as glucosinolate precursor amino acids, and to exhibit oxidation of thio to sulfinyl, formation of alkenyls, β-hydroxylation of aliphatic chains and hydroxylation and methylation of indole glucosinolates. Two hotspots of apparent biochemical innovation and loss were identified: C. pratensis and the genus Barbarea. Diversity in other species mainly included structures also known from other crucifers. In addition to a role of gene duplication, two contrasting genetic/biochemical mechanisms for evolution of such combined diversity and redundancy are discussed: (i) involvement of widespread genes with expression varying during evolution, and (ii) mutational changes in substrate specificities of CYP79F and GS-OH enzymes.
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
authors.forename: Xiao-Chen
authors.initials: XC
authors.lastname: Huang
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Cecilie I C
authors.initials: CIC
authors.lastname: Hansen
authors.affiliation: Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
authors.forename: Don
authors.initials: D
authors.lastname: Cipollini
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Marian
authors.initials: M
authors.lastname: Ørgaard
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark; Copenhagen Plant Science Center, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Matthes
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark; Copenhagen Plant Science Center, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Fernando
authors.initials: F
authors.lastname: Geu-Flores
authors.affiliation: Biodiversity and Plant Systematics, Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
authors.forename: Marcus A
authors.initials: MA
authors.lastname: Koch
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark; Copenhagen Plant Science Center, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. Electronic address: nia@plen.ku.dk.
authors.forename: Niels
authors.initials: N
authors.lastname: Agerbirk
mesh_terms: D031216:Barbarea / Q000737:chemistry; D019607:Brassicaceae / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D020440:Gene Duplication; D005961:Glucosinolates / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D010802:Phylogeny; D012639:Seeds / Q000737:chemistry
chemical_list: D005961:Glucosinolates
keywords: Aldol cleavage; Barbarea; Cardamine; Fragmentation; Glucosinolate biosynthesis; Ion trap mass spectrometry; Isomer distinction; NMR; Novel glucosinolates; Rorippa