Medicagenic Acid Research Articles

Control of plant parasitic nematodes with active saponins and biomass from Medicago sativa

Medicago sativa L., alfalfa, is the most known plant species within the Medicago genus. The plant has been extensively studied for its content of saponins, mainly consisting of triterpene glycosides of medicagenic acid, possessing several biological properties including a biocidal activity on different soil microorganisms. Phytoparasitic nematodes are responsible for heavy economic damages to numerous agricultural crops and, due to their large distribution, they are among the most difficult crop pests to control. Attention on environmental safety and human and animal health has led to the progressive dismission of many synthetic formulations for the control of those pests and to the search of alternative strategies, including the use of natural metabolites from plants. Saponins from M. sativa may be good candidates for natural nematicide formulations, as in our in vitro studies the saponin mixtures from M. sativa tissues have been found effective in vitro against the virus-vector nematode Xiphinema index, the root-knot nematode Meloidogyne incognita and the potato cyst parasite, Globodera rostochiensis. A structure–activity relationship among saponins and related prosapogenins and sapogenin, respectively, has also been analyzed. The nematicidal efficacy differed among the three assayed nematode species, G. rostochiensis being the most susceptible to the active compounds from alfalfa. The in vitro results were also confirmed by experiments in potting mixes infested by M. incognita or G. rostochiensis and amended with dry top and root material from M. sativa, and in field trials on M. incognita and carrot cyst nematode Heterodera carotae with M. sativa pelleted meal. All amendments reduced root and soil population densities of target nematode species compared to non-treated and chemical controls, with a general improvement of plant growth and yield performances.

A new oleanene triterpene from Gladiolus segetum Ker-Gawl

A new pentacyclic oleanene triterpene, 2β, 3β, 16α, 28-tetrahydroxy-olean-12-ene-23-oic acid (1), as well as the known pentacyclic triterpene medicagenic acid (2), have been isolated by different chromatographic techniques from the acid hydrolysate of the saponin fraction of Gladiolus segetum. The identification of these compounds was established by different methods of physical, chemical and spectral evidence.

Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β‐amyrin synthase (AsOXA1) gene

Triterpene saponins are a group of bioactive compounds abundant in the genus Medicago, and have been studied extensively for their biological and pharmacological properties. In this article, we evaluated the effects of the ectopic expression of AsOXA1 cDNA from Aster sedifolius on the production of triterpene saponins in barrel medic (Medicago truncatula Gaertn.). AsOXA1 cDNA encodes beta-amyrin synthase, a key enzyme involved in triterpene saponin biosynthesis. One of the four transgenic lines expressing AsOXA1 accumulated significantly larger amounts of some triterpenic compounds in leaf and root than did control plants. In particular, the leaf exhibited significantly higher levels of bayogenin, medicagenic acid and zanhic acid. The amounts of medicagenic acid and zanhic acid, which represent the core of the M. truncatula leaf saponins, were 1.7 and 2.1 times higher, respectively, than the amounts extracted from the control line. In root, the production of bayogenin, hederagenin, soyasapogenol E and 2beta-hydroxyoleanolic acid was increased significantly. The increase in the total amounts of triterpenic compounds observed in the leaves of transgenic lines correlated with the AsOXA1 expression level. Interestingly, the plants expressing AsOXA1 showed, under different growth conditions, improved nodulation when compared with the control line. Nodulation enhancement was also accompanied by a significant change in the soyasapogenol B content. Our results indicate that the ectopic expression of AsOXA1 in barrel medic leads to a greater accumulation of triterpene saponins and enhanced root nodulation.

Analysis of Saponin Mixtures from Alfalfa (Medicago Sativa L.) Roots using Mass Spectrometry with MALDI Techniques

This model study demonstrates the value of Matrix Assisted Laser Desorption Ionization (MALDI) MS in global analysis of mixtures of saponin isolates. Unfractionated saponin extract derived from alfalfa [Medicago sativa L.] roots was analyzed. In addition, a few saponin fractions that were purified from the same extract by a series of chromatographic steps were also studied. MALDI mass spectrometry utilized TOF, TOF/TOF and QqTOF analyzers. Low-resolution fingerprints of the mixture characterized the sample in terms of a minimal number of distinct saponin components. The main species observed under MALDI-TOF and oMaldi-QqTOF MS positive ion mode conditions were sodiated pseudomolecular ions [M+Na]+. No protonated molecular ions [M+H]+ were detected. The MS/MS spectra acquired on [M+Na]+ precursor ions under conditions of collision induced dissociation (CID) were dominated by cleavages at glycosidic bonds. Product ions representing free aglycones were either absent or present at low intensities and were never observed for structures carrying an oligosaccharide bound to a sapogenin at the C3- O-position. In general, product ion series generated from CID fragmentation of glycans bound via an ether bond ( e.g., C3- O-position in medicagenic acid) were consistent with the gas phase cleavages of each of the glycosidic bonds within the oligosaccharide, thus revealing the primary structure. In contrast, glycans bound via an ester bond ( e.g., C28- O-position in MA) were released as intact sodiated species. A total of 78 pseudomolecular ions demonstrating signal-to-noise ratios above 5 were observed in the MALDI-TOF mass spectrum of unfractionated root extract from M. sativa. Molecular masses of 52 out of 78 were consistent with at least one known or novel saponin structure, with 10 of those 52 likely representing doubly sodiated saponin species. Calculated masses of the majority of the known M. sativa saponin structures were matched to experimental pseudomolecular ion masses. MS/MS analysis of unfractionated extract allowed us to propose putative structures for 51 saponins: 15 of these corresponded to the known M. sativa species, 8 to other Medicago genus species and 26 were not reported before for Medicago genus. We submit that the approach described here might serve as a high throughput strategy for evaluating effects of stressors or genetic manipulation on the overall composition of the saponin content of an organism.

Effect of alfalfa saponins and flavonoids on pea aphid

AbstractWe studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa (Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3‐GlcA,28‐AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7‐O‐β‐D‐glucuronopyranosyl‐4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, 7‐O‐{2‐O‐E‐feruloyl‐[β‐D‐glucuronopyranosyl(1→3)]‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside} apigenin, and 4′‐O‐[2′‐O‐E‐feruloyl‐O‐β‐D‐glucuronopyranosyl(1→2)‐O‐β‐D‐glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre‐reproductive period on cv. Radius was prolonged and the reproductive and post‐reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.

Evaluation of nematicidal properties of saponins from Medicago spp.

The nematicidal activity of saponins from Medicago arborea (tops), M. arabica (tops and roots) and M. sativa (tops and roots) against the plant-parasitic nematode Xiphinema index was investigated. Nematicidal activity of related prosapogenins and sapogenins on X. index is also described. Saponins from Medicago spp. at different concentrations were all nematicidal, those from M. arborea tops being the less effective. In general, saponins induced 100% mortality at 500 μg ml−1 between 8 and 48 h, while prosapogenins resulted in toxicity starting at 125 μg ml−1. Differences in the effects on X. index induced by prosapogenins and sapogenins were less pronounced, although prosapogenins displayed a larger range of activity. Assays with purified sapogenins demonstrated the relationship of the observed nematicidal activity of M. sativa and M. arborea to the content of the main aglycones (medicagenic acid and hederagenin, respectively) in the saponin extracts. Hederagenin displayed the highest bioactivity, giving 38% mortality after 1 h at 125 μg ml−1.

Deterrence and Toxicity of Plant Saponins for the Pea Aphid Acyrthosiphon Pisum Harris

Plant saponins are widely distributed among plants and have a wide range of biological properties. Three alfalfa saponins - zanhic acid tridesmoside, 3GlcA, 28AraRhaXyl medicagenic acid glycoside, and 3GlcA, 28AraRha medicagenic acid glycoside - were tested for their settling inhibition effects on feeding behavior of the aphid Acyrthosiphon pisum using the electrical penetration graph method. Application of saponins to artificial diets affected the insects' probing behavior. In general, saponins incorporated into sucrose-agarose gels significantly reduced the number of aphid probes and extended their duration. Lower saponin concentrations (50 ppm) extended aphid activity and corresponded to phloem sap ingestion. In contrast, higher concentrations (100 ppm) strongly reduced aphid ability to ingest phloem and xylem sap.

Variety and environment effects on the dynamics of saponins in lucerne ( Medicago sativa L.)

Lucerne saponins are important antinutritional factors, which may hinder the nutritive value of this forage species. Little information exists on possible environmental influences on their accumulation in plants. This study assessed the effect of variety and climatic factors possibly influencing the saponin content. Saponins are glycosides deriving from various sapogenins (aglycone moieties). Being sapogenins a necessary constituent of saponins, saponin evaluation was based on identification and quantification of their aglycones, which are easier to detect than saponins per se. From analytical determinations, medicagenic acid and zanhic acid resulted the most abundant sapogenins, and their variation in concentration was monitored at weekly intervals along the vegetative season of 2 consecutive years. The results suggested the difficulty in outlining a consistent model of saponin accumulation across various harvests within years. Sapogenin concentrations increased from the second to the third year of growth, possibly more as a consequence of the sward age than climatic conditions in the 2 years. The variety Sapko (selected on the basis of a test assessing biologically-active saponins) showed concentrations consistently low across seasons in each year and from year to year, and differed markedly from the two other cultivars. There was a slight, general trend of positive correlation between concentrations and maximum daily temperatures across the year, but the patterns of concentration did not match necessarily those of temperatures, suggesting that these compounds do not have just a cause-effect relationship with summer temperatures as previously reported. While the variety confirmed to be a key factor in the saponin content, the role of other factors, such as temperature, radiation or sward age (inferred from some of the results), is probably dimmed by complex interactions. On the basis of the present findings, some caution could be recommended in feeding animals with lucerne harvested in late summer and, particularly, on aged swards.

Effect of low and high-saponin lines of alfalfa on pea aphid

Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction and survival, and disturbance in development of its population. This line negatively influenced aphid probing behaviour, particularly prolonging the non-probing period and probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The high-saponin line of alfalfa differed from the low-saponin one by the presence of zanhic acid tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The saponins incorporated into sucrose–agarose gels significantly reduced number of the aphid probes into the gels and extended their duration in comparison to the control gels (without tested compounds). Role of zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential factors for partial resistance of alfalfa towards the pea aphid is discussed.

Antimicrobial activity of saponins from Medicago sp.: structure‐activity relationship

The antimicrobial activity of saponins from Medicago sativa, M. arborea and M. arabica against a selection of medically important yeasts, Gram-positive and -negative bacteria was investigated. Structure-activity growth inhibitory effects of related prosapogenins and sapogenins are also described. Increasing antibiotic activity was observed going from the saponin extracts to the sapogenin samples, suggesting that the sugar moiety is not important for the antimicrobial efficacy. Activity was especially high against Gram-positive bacteria (Bacillus cereus, B. subtilis, Staphylococcus aureus and Enterococcus faecalis) with M. arabica being the species showing a broader spectrum of action. Discrete antifungal activity was also observed, mainly against Saccharomyces cerevisiae. The observed antimicrobial properties of M. sativa and M. arborea were related to the content of medicagenic acid, while hederagenin seems to contribute to the bioactivity of M. arabica total sapogenins.

Triterpene Saponins from the Roots of Medicago hybrida

Fourteen triterpene saponins (1-14) have been isolated from the roots of Medicago hybrida and their structures elucidated by FAB-MS and NMR analysis. Two of them are new compounds and were identified as hederagenin 3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-28-O-beta-D-glucopyranoside (7) and oleanolic acid 3-O-[beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranosyl]-28-O-[alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranoside] (14). Seven saponins being mono- and bidesmosides of hederagenin (1, 5, 6, 9), one bidesmoside of bayogenin (2), and two bidesmosides of 2beta,3beta-dihydroxyolean-12-en-23-al-28-oic acid (11) and oleanolic acid (13) are known compounds but not previously reported as saponin constituents of Medicago, whereas five other saponins, being mono- and bidesmosides of medicagenic acid (3, 4, 8, 10, 12), and one monodesmoside of hederagenin (8) have been previously isolated from other Medicago species. The presence of 2beta,3beta-dihydroxyolean-12-en-23-al-28-oic acid might represent an interesting intermediate in the biosynthesis of these substances.

Two New Triterpenoid Saponins Isolated from Polygala japonica

Bioassay guided investigation of whole parts of Polygala japonica afforded two new triterpenoid saponins, characterized as 3-O-beta-D-glucopyranosyl medicagenic acid 28-O-{beta-D-xylopyranosyl(1-->4)-[beta-D-apiofuranosyl(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl} ester (1), 3-O-beta-D-glucopyranosyl 2-oxo-olean-12-en-23, 28-dioic acid 28-O-{beta-D-xylopyranosyl(1-->4)-[beta-D-apiofuranosyl(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl} ester (2), together with four known triterpenoid saponins (3-6). Their structures were elucidated by spectroscopic and chemical methods. Saponins 3, 4 and 5 showed significant anti-inflammation effects on carrageenan-induced acute paw edema in mouse.

Antidermatophyte activity of medicago extracts and contained saponins and their structure-activity relationships

Saponin-rich extracts of Medicago sativa L. have been shown to display antifungal activity against a range of common fungibut only a few individual saponins have been tested against dermatophytes. Total saponins were separately obtained from roots and aerial parts of Medicago sativa, M. murex, M. arabica and M. hybrida. Nineteen saponins and three parent triterpenoids, were tested against three dermatophytic fungi Microsporum gypseum, Trichophyton interdigitale and T. tonsurans using mircowell plate serial dilution assay to determine the Minimum Inhibitory Concentration (MIC). Miconazole was used as a positive control. T. tonsurans appeared to be the most sensitive of the dermatophytes to the active compounds. Aglycones showed less antifungal effect than the glycosides, which displayed a range of activities. Monodesmosidic glycosides of medicagenic acid were the most active compounds, especially the 3-O-beta-D-glucopyranoside, which displayed MIC less than 0.09mm against all three fungi, although those of hederagenin and zanhic acid also showed weak activity (MIC 3.3mm against two fungal species). Bidesmosidic saponins had weaker activity than monodesmosidic ones. This corresponds to activity of these and similar compounds against other fungal species but this is the first comprehensive report of activity of Medicago saponins against dermatophytes.

Triterpenoid Glycosides from Leaves of Medicago arborea L.

Eighteen triterpene saponins (1-18) from Medicago arborea leaves have been isolated and their structures elucidated by spectroscopic, spectrometric (1D and 2D NMR, FAB-MS, ESI-MS/MS), and chemical methods. They have been identified as glycosides of medicagenic, zanhic, and 2beta-hydroxyoleanolic acids, soyasapogenol B, bayogenin, and 2beta,3beta-dihydroxyolean-12-en-23-al-28-oic acid. Twelve of them, identified as 3-O-beta-D-glucopyranosyl-28-O-[alpha-L-arabinopyranosyl(1-->3)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside] zanhic acid (3), 3-O-beta-D-glucopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-[alpha-L-arabinopyranosyl-(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside] zanhic acid (4), 3-O-[alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucopyranosyl]-2beta-hydroxyoleanolic acid (5), 3-O-beta-D-glucuronopyranosyl-28-O-[alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]medicagenic acid (6), 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]bayogenin (9), 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]-2beta,3beta-dihydroxyolean-12-en-23-al-28-oic acid (10), 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-[beta-D-apiofuranosyl(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]zanhic acid (12), 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-[alpha-L-arabinopyranoside(1-->3)]-alpha-L-rhamnopyrano-syl(1-->2)-alpha-L-arabinopyranoside]zanhic acid (13), 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyrano-syl(1-->4)-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]zanhic acid (14), 3-O-[alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-28-O-[beta-D-xylopyranosyl(1-->4)-[beta-D-apiofurano-syl(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranoside]zanhic acid (16), 3-O-[beta-D-glucopyrano-syl(1-->2)-beta-D-glucopyranosyl]-28-O-[beta-D-xylopyranosyl(1-->4)-[alpha-L-arabinopyranosyl(1-->3)]-alpha-L-rhamno-pyranosyl (1-->2)-alpha-L-arabinopyranoside]zanhic acid (17), and 3-O-beta-D-glucuronopyranosyl-28-O-[beta-D-xylopyranosyl(1-->4)-[beta-D-apiofuranosyl(1-->3)]-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyrano-side]medicagenic acid (18), are reported as new natural compounds. The presence of the aldehydic group on the sapogenin moiety of saponin 10 is discussed in the framework of a possible elucidation of the biosynthesis of these metabolites.

Determination of Saponins in Aerial Parts of Barrel Medic (Medicago truncatula) by Liquid Chromatography−Electrospray Ionization/Mass Spectrometry

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.

Triterpene Saponins from Polygalaceae

The aim of this review is to give updated results on the chemistry and biological activities of Polygalaceae saponins from literature data and from authors’ studies. During the last decade the improvement of isolation and structure elucidation techniques allowed the characterization of about 150 complex triterpene saponins from Polygalaceae having mainly preatroxigenin, medicagenic acid and presenegenin as aglycone. The sequence 3-O-(β-d-glucopyranosyl)-presenegenin 28-[O-α-L-rhamnopyranosyl-(1→2)-β-d-fucopyranosyl] ester with additional acylations with p-methoxy-, di- or trimethoxycinnamoyl groups at the position 4 of the fucopyranosyl moiety often encountered in the genera Polygala, Muraltia, Carpolobia, and Securidaca may represent a chemotaxonomic marker for the Polygalaceae family. The biological and pharmacological properties of some Polygalaceae species such as in vitro activity on the central nervous system, the hypoglycemic, cytotoxic, immunomodulatory activities and in vivo immunoadjuvant properties are highlighted.

Quantification of Saponins in Aerial and Subterranean Tissues of Medicago truncatula

Triterpene saponins from aerial and subterranean organs of Medicago truncatula cv. Jemalong A-17 were qualitatively profiled and quantified using reverse-phase HPLC with on-line photodiode array detection and electrospray-ionization mass spectrometry (HPLC/PDA/ESI/MS). Absolute quantifications were performed for 3-O-beta-D-glucopyranosyl-medicagenic acid and soyasaponin 1 (3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucuronopyranoside] soyasapogenol B), whereas relative quantifications were determined for 29 other saponins in root, stem, leaf, seedpod, and seed. Roots contained the greatest total amount of saponins followed by leaf and seed, respectively. The quantitative data also reveal the differential accumulation of triterpene saponins in the various organs of M. truncatula. Specifically, relatively higher quantities of medicagenic acid conjugates accumulated in leaf and seed, whereas relatively higher levels of soysapogenol conjugates were observed in root. The differential accumulation of specific triterpene saponins is suggestive of spatially differentiated biosynthesis and/or biological function.

Triterpene Saponins from Barrel Medic (Medicago truncatula) Aerial Parts

Triterpene saponins from Medicago truncatula aerial parts have been separated and their structures determined by the extensive use of 1D- and 2D-NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS. Fifteen individual compounds were isolated that included seven medicagenic acid and eight zanhic acid glycosides. Additionally, two soyasapogenol B and soyasapogenol E glycosides were identified by MS/MS and TLC. Four medicagenic acid glycosides (5, 11, 12, 14) and eight zanhic acid glycosides (1-4, 6-9) are reported here for the first time. The common feature of M. truncatula aerial part saponins is the (1-->3) linkage between the two glucose units at C-3 of medicagenic and zanhic acids, which is different from that found in alfalfa (Medicago sativa), where this linkage was always (1-->2). This may suggest differences in glucosyltransferases between these two Medicago species.

Genomics‐based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula

The biosynthesis of triterpene saponins is poorly characterized in spite of the importance of these glycosylated secondary metabolites for plant defense and animal health. The model legume Medicago truncatula synthesizes more than 30 different saponins based on at least five triterpene aglycones; soyasapogenols B and E, medicagenic acid, hederagenin and bayogenin. We have employed an inducible cell culture system, DNA array-based and in silico transcript profiling, and targeted metabolite profiling, to identify triterpene glycosyltransferases (GTs) from among the more than 300 GTs expressed in M. truncatula. Two uridine diphosphate glucosyltransferases were functionally characterized; UGT73K1 with specificity for hederagenin and soyasapogenols B and E, and UGT71G1 with specificity for medicagenic acid. The latter enzyme also glycosylated certain isoflavones and the flavonol quercetin with higher efficiency than triterpenes; however, integrated transcript and metabolite profiling supported a function for UGT71G1 in terpenoid but not (iso)flavonoid biosynthesis in the elicited cell cultures.

Combined Effects of Elevated Co2and Herbivore Damage on Alfalfa and Cotton

We examined herbivore-induced responses of alfalfa (Medicago sativa) and cotton (Gossypium hirsutum) under different CO2 conditions. Plants were grown under ambient (350 ppm) or elevated (700 ppm) CO2 levels, and were either damaged or undamaged by Spodoptera littoralis larvae. At harvest, growth of undamaged (control) plants was determined, and foliar chemical composition of both undamaged and damaged plants was analyzed. Cotton grew faster overall and showed a greater increase in growth in response to CO2 enrichment than did alfalfa. Elevated CO2 levels increased starch and decreased nitrogen levels in damaged alfalfa and undamaged cotton plants. Alfalfa saponin levels were significantly increased by elevated CO2 and damage. Regarding specific saponins, medicagenic acid bidesmoside (3GlcA,28AraRhaXyl medicagenate) concentrations were reduced by high CO2, whereas zanhic acid tridesmoside (3GlcGlcGlc,23Ara,28AraRhaXylApi Za) levels were unaffected by the treatments. Soyasaponin I (3GlcAGalRha soyasapogenol B) was only detected in minute amounts. Alfalfa flavonoid analyses showed that total flavonoid levels were similar between treatments, although free apigenin increased and apigenin glucoside (7-O-[2-O-feruloyl-beta-D-glucuronopyranozyl (1-->2)-O-8-D-glucuronopyranozyl]-4'-O-beta-D-glucuronopyranozide apigenin) decreased in CO2-enriched plants. In cotton, herbivore damage increased levels of total terpenoid aldehydes, gossypol, hemigossypolone, the heliocides H1 and H4, but not H2 and H3, whereas CO2 enrichment had no effect. These results demonstrate that combined effects of CO2 and herbivore damage vary between plant species, which has implications for the competitive balance within plant communities.