Elisa Biazzi

Medicago truncatula CYP716A12 Is a Multifunctional Oxidase Involved in the Biosynthesis of Hemolytic Saponins

Triterpenic saponins participate in plant defense mechanisms and possess a broad spectrum of biological and pharmacological properties. This article identifies the P450 cytochrome CYP716A12 as a key gene in the synthesis of hemolytic saponins in Medicago truncatula by a combination of genetic and biochemical analyses. Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

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.

New triterpenic saponins from the aerial parts of Medicago arabica (L.) huds.

The reinvestigation of saponin composition from Medicago arabica from Italy allowed the detection of nineteen (1-19) saponins. All of them were purified by reverse-phase chromatography and their structures elucidated by spectroscopic and spectrometric (1D and 2D NMR; ESI-MS/MS) and chemical methods. Fourteen were known saponins, previously found in other plants including other Medicago species. They have been identified as glycosides of oleanolic acid, 2beta,3beta-dihydroxyolean-12-en-28-oic acid, hederagenin, bayogenin and soyasapogenol B. Five saponins, identified as 3-O-[-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl]-30-O-beta-D-glucopyranosyl 2beta,3beta,30-trihydroxyolean-12-en-28-oic acid (1), 3-O-[alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl]-30-O-[beta-D-glucopyranosyl] 3beta,30-dihydroxyolean-12-en-28-oic acid (2), 3-O-[beta-D-glucuronopyranosyl]-30-O-[alpha-L-arabinopyranosyl(1-->2)-beta-d-glucopyranosyl] 2beta,3beta,30-trihydroxyolean-12-en-28-oic acid (3), 3-O-[beta-D-glucuronopyranosyl]-30-O-[alpha-L-arabinopyranosyl(1-->2)-beta-D-glucopyranosyl] 3beta,30-dihydroxyolean-12-en-28-oic acid (4) and 3-O-[beta-D-glucuronopyranosyl]-30-O-[beta-D-glucopyranosyl] 2beta,3beta,30-trihydroxyolean-12-en-28-oic acid (5), are reported here as new natural compounds. These new saponins, possessing a hydroxy group at the 30-methyl position of the triterpenic skeleton, have never been previously found in the genus Medicago.

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

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.

Variation in saponin content during the growing season of spotted medic [Medicago arabica (L.) Huds.]

BACKGROUND Spotted medic [Medicago arabica (L.) Huds.] is a minor forage species containing saponins which are reported to be biologically active. This study assessed the concentration and composition pattern of spotted medic saponins during the growing season and at senescence. The pattern of saponins was based on identification and quantification of their constituent sapogenins. At senescence, individual saponin concentrations of aerial and subterranean plant organs were also determined. RESULTS Leaf total saponin content did not vary during the growing season and decreased remarkably at senescence. Seven sapogenins were identified and quantified during the season, bayogenin and hederagenin being the most abundant ones throughout. Total saponin content varied among plant organs at senescence, with the highest concentration in roots. A variable number of saponins from one (in seeds) to 19 (in leaves) were quantified. A clear relationship between leaf concentrations of sapogenins and those of their derivative saponins was revealed by correlation analysis. CONCLUSION The species displayed a sapogenins/saponins pattern markedly different from those of other perennial or annual Medicago species. Saponins of queretaroic acid and 2β-hydroxy queretaroic acid had no precedent in the Leguminosae. The high concentration of biologically active hederagenin suggested further assessment of possible effects on feeding animals.

Genome-Wide Association Mapping and Genomic Selection for Alfalfa ( Medicago sativa ) Forage Quality Traits

Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits (by genomic selection or MAS) and forage yield.

Triterpenoid glycosides from Medicago sativa as antifungal agents against Pyricularia oryzae.

The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.) tops and roots, the corresponding mixtures of prosapogenins from tops, and purified saponins and sapogenins against the causal agent of rice blast Pyricularia oryzae isolates are presented. In vitro experiments highlighted a range of activities, depending upon the assayed metabolite. The antifungal effects of the most promising prosapogenin mixture from alfalfa tops were confirmed by means of in planta tests using three different Italian cultivars of rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low blast resistance. The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure-activity relationship. Results indicate that prosapogenins are active compounds to prevent the fungal attack of P. oryzae on different rice cultivars. Therefore, if properly formulated, these substances could represent a promising and environmentally friendly treatment to control rice blast.

Characterization of Two Agrostis−Festuca Alpine Pastures and Their Influence on Cheese Composition

Recently, there has been a renewed interest in mountain farming, and several studies have been carried out on milk and cheese obtained in the unique environmental conditions of the Alps, a 1300 km mountain chain, located in the north of Italy. In this paper, the influence, on some cheese constituents, of two very similar mountain grasslands, both dominated by Festuca - Agrostis , was investigated. The two pastures were located in the same area in the southeastern Italian alpine region and differed in sunshine orientation and exposure. Milk obtained from cows grazing on these pastures was used to produce a semi-hard traditional cheese. The differences observed between the cheeses of the two areas for both some hydrocarbons (1-phytene and 2-phytene) and trans-fatty acids can be explained by a different rumen environment created by the botanical composition of the two pastures. The multidisciplinary approach can be considered a successful strategy, suitable for studying markers of authenticity.

Collection of mutants for functional genomics in the legume Medicago truncatula

We have established mutant collections of the model species Medicago truncatula according to current protocols. In particular, we used a transposon ( Tnt1 ) tagging method and an ethyl methanesulfonate (EMS) mutagenesis approach (TILLING). The collections were subjected to both forward and reverse genetics screenings, and several mutants were isolated that affect plant traits (e.g. shoot, root developments, flower morphology, etc.) and also biosynthetic pathways of secondary compounds (saponins and tannins). Genes responsible for some of the mutations were cloned and further characterized.

Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and activity.

In the present work, eleven saponins and three sapogenins purified from Medicago sativa were tested for their cytotoxicity against highly proliferating white poplar (Populus alba L.) cell suspension cultures. After preliminary screening, four saponins with different structural features in terms of aglycone moieties and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5, monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a bidesmoside of zanhic acid) and different cytotoxicity were selected and used for further investigation on their structure-activity relationship. Transmission Electron Microscopy (TEM) analyses provided for the first time evidence of the effects exerted by saponins on plant cell wall integrity. Exposure to saponin 3 and saponin 10 resulted into disorganization of the outer wall layer and the effect was even more pronounced in white poplar cells treated with the two medicagenic acid derivatives, saponins 4 and 5. Oxidative burst and nitric oxide accumulation were common hallmarks of the response of white poplar cells to saponins. When DNA damage accumulation and DNA repair profiles were evaluated by Single Cell Gel Electrophoresis, induction of single and double strand breaks followed by effective repair was observed within 24h. The reported data are discussed in view of the current issues dealing with saponin structure-activity relationship.