Metabolite Profiling of Triterpene Saponins in Medicago truncatula Hairy Roots by Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

L egumes (Fabaceae) are important agricultural crops, characterized by the presence of root nodules that contain symbiotic nitrogen-fixing bacteria. Fixation of atmospheric nitrogen allows reduced fertilizer costs and makes legumes a key player in crop rotation to replenish nitrogen-depleted soil. Furthermore, legumes serve as a major source of proteins and oil for human and animal nutrition. Leguminous crops with a significant economical value include oilseed crops such as soybean (Glycine max) and peanut (Arachis hypogaea), forage and soil-conditioning crops such as alfalfa (Medicago sativa) and clover (Trifolium spp.), and pulses such as beans (Phaseolus spp.), peas (Pisum spp.), and lentils (Lens spp.).The more than 19 400 leguminous species, distributed over 730 genera, produce a vast array of metabolites with diverse structures and biological activities. An important group of legume natural products are triterpene saponins, a class of secondary metabolites that display a wide range of biological activities.1 In plants they serve as allelopathic, antipalatability, anti-insect, or antifungal agents.2 Saponins also have a broad range of pharmaceutical properties, such as anti-inflammatory, antimicrobial, anticancer, or adjuvant activities.3,4 Besides saponins, isoflavonoids, alkaloids, and nonprotein amino acids commonly occur as natural products in several legume species. 1,5 As a model species for legume biology, barrel medic (Medicago truncatula), a close relative of alfalfa, is an excellent system to study secondary metabolism of legumes. 1,6 Consequently, several studies about the metabolite composition of M. truncatula have been published over the past decade. Metabolite profiling using LC-MS-based methods revealed the presence of a complex mixture of triterpene saponins in various M. truncatula tissues. 2,7À9 In accordance with other legumes from the Papilionoideae subfamily, M. truncatula is rich in isoflavonoids. 10,11 Hairy roots are generated by an infection of healthy plant tissue, not necessarily roots, with Agrobacterium rhizogenes. They are characterized by fast growth rates and do not require growth regulators for their cultivation. Furthermore, they have a high genetic stability and a high biosynthetic potential for a longer time than natural roots.12,13 Their potential to produce compounds that are also naturally occurring in wild-type roots in combination with their tolerance to cultivation in large-scale bioreactors makes hairy roots an appealing alternative for the destructive harvesting of wild-type roots, especially those of endangered plant species, to obtain valuable phytochemicals. 14,15 Hairy roots are also a valuable tool to investigate the secondary metabolism in M. truncatula. A prerequisite for the study of secondary metabolism and its application in metabolic engineering projects is the possibility to generate transgenic plant tissue. ABSTRACT: Triterpenes are one of the largest classes of plant natural products, with an enormous variety in structure and bioactivities. Here,...

UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance

“…In contrast, nonhemolytic saponins were unaffected. The nonhemolytic saponins are derived from sapogenins that are not carboxylated at the C28 position, and MS n fragmentation of these revealed an aglycone fragment ion with a deduced mass of 474 D (Pollier et al, 2011). A similar fragmentation product was observed for P-type saponins and suggests that structurally similar sapogenins, with four hydroxyl groups but no C28 carboxylation, are present in this plant type.…”

The bHLH Transcription Factors TSAR1 and TSAR2 Regulate Triterpene Saponin Biosynthesis in Medicago truncatula

“…Sterilization of M. truncatula seeds (ecotype Jemalong J5), transformation of seedlings by Agrobacterium rhizogenes (strain LBA 9402/12), and the subsequent generation of hairy roots were carried out as described previously (Pollier et al, 2011). Hairy roots were cultivated for 21 d in liquid medium to provide proper amounts to be used for RNA and metabolite extraction.…”

“…A total of 400 mg of the ground material was used for metabolite extractions as described previously (Pollier et al, 2011). Liquid chromatography-electrospray ionization-mass spectrometry analysis was performed using an Acquity UPLC BEH C18 column (150 3 2.1 mm, 1.7 mm; Waters) mounted on an Acquity UPLC system (Waters).…”

Rapid annotation and structural characterization of saponins in the active fraction of Albizia julibrissin by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry based on accurate mass database

“…Peak 13 (julibroside J31) was taken as an example to clarify the fragmentation pathway in Figure as previously described . The fragment ions at m/z 1009.9552, 1000.9639, 918.9296, 699.8374, 601.2049, 499.2102 corresponded to [M–2H] 2– , [M–H 2 O–2H] 2– , [M–Glc–2H] 2– , [M–C3 chain–2H] 2– , [Glc‐Ara‐Rha‐Glc–H] – , and [M–C3 chain–C21 chain–C28 chain+H 2 O–H] – .…”