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Natural product biosynthesis in Medicago species Cite this: DOI: 10.1039/c3np70104b
Azra Gholami,†ab Nathan De Geyter,†ab Jacob Pollier,†ab Sofie Goormachtigab and Alain Goossens*ab
Covering: up to the end of 2013 The genus Medicago, a member of the legume (Fabaceae) family, comprises 87 species of flowering plants, including the forage crop M. sativa (alfalfa) and the model legume M. truncatula (barrel medic). Medicago species synthesize a variety of bioactive natural products that are used to engage into symbiotic interactions but also serve to deter pathogens and herbivores. For humans, these bioactive natural products often possess promising pharmaceutical properties. In this review, we focus on the two most interesting and well characterized secondary metabolite classes found in Medicago species, the triterpene saponins and the flavonoids, with a detailed overview of their biosynthesis, regulation, and profiling methods. Furthermore, their biological role within the plant as well as their potential utility for Received 26th September 2013 Accepted 15th January 2014
human health or other applications is discussed. Finally, we give an overview of the advances made in metabolic engineering in Medicago species and how the development of novel molecular and omics toolkits can influence a better understanding of this genus in terms of specialized metabolism and
DOI: 10.1039/c3np70104b
chemistry. Throughout, we critically analyze the current bottlenecks and speculate on future directions
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and opportunities for research and exploitation of Medicago metabolism.
1 2 2.1. 2.2. 2.3. 2.4. 2.5. 3 3.1. 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.1.7. 3.1.8. 3.2. 3.3. 3.4.
Introduction Saponins Saponin biosynthesis in M. truncatula Regulation of saponin biosynthesis Biological role of Medicago saponins Commercial potential of Medicago saponins Determination of Medicago saponins Flavonoids Flavonoid biosynthesis in M. truncatula Early steps of avonoid biosynthesis Isoavonoid biosynthesis Flavonol biosynthesis Flavone biosynthesis Aurone biosynthesis Anthocyanin biosynthesis Proanthocyanidin biosynthesis Structural diversication and decoration of avonoids Regulation of avonoid biosynthesis Flavonoid localization Biological function of Medicago avonoids in planta
a
Department of Plant Systems Biology, VIB, Ghent University, Technologiepark 927, B-9052 Gent, Belgium. E-mail: [email protected]; Fax: +32 9 3313809; Tel: +32 9 3313800
b
Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, Belgium † These authors contributed equally to this work
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3.4.1. 3.4.2. 3.4.3. 3.4.4. 3.4.5. 3.5. 3.6. 4 5 5.1. 5.2. 6 7 8
1
Nutrient acquisition Benecial plant-microbe interactions Defense Allelopathy Developmental regulators Pharmacological properties of Medicago avonoids Determination of Medicago avonoids The ‘omics’ toolkit for Medicago research Metabolic engineering Engineering of avonoid biosynthesis in Medicago Engineering of lignin biosynthesis in Medicago Outlook Acknowledgements References
Introduction
Legumes (Fabaceae) constitute a highly diverse plant family that encompasses economically important crops, such as pea and soybean, and that provides about one-third of humankind’s protein intake, fodder and forage for livestock, and raw materials for industries. Legumes are particular among cultivated plants because of their ability to establish symbiotic interactions such as with nitrogen-xing bacteria.1–3 In addition, leguminous plant species are rich sources of health promoting phytochemicals. In particular, the legume subfamily of
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Papilionoideae contains the predominant producers of the very benecial isoavonoids.4–8 Azra Gholami (born 1981) received her Pharm. D degree in Pharmacy from Shiraz University of Medical Sciences and Health Services (Iran, 2006). Aerwards she worked as a researcher in the department of pharmacognosy and medicinal plants of Shiraz University of Medical Science and Health services for two years. In 2009 she started as a predoctoral researcher in the lab of Alain Goossens (VIB-Ghent University, Belgium) to obtain her PhD degree in 2013. Her PhD research focused on the identication of potential regulators of jasmonatemodulated secondary metabolism in Medicago truncatula. Nathan De Geyter (born 1986) graduated as a Master in Sciences, Biology at Ghent University in 2009. In 2010, he started his PhD research in the lab of Alain Goossens and Soe Goormachtig aer he obtained a scholarship from the Agency for Innovation by Science and Technology in Flanders. His work focuses on the Tandem Affinity Purication (TAP) technology in the model legume Medicago truncatula. Using the TAP technique one can identify all unknown protein interactors from a known ‘bait’ protein. The baits that are further investigated are involved in MeJA-dependent regulation of secondary metabolism in M. truncatula. Jacob Pollier (born 1980) obtained his PhD in Biotechnology under the guidance of Alain Goossens at Ghent University (Belgium, 2011). His PhD research focused on the development of a combinatorial biosynthesis platform in plants using triterpene saponins as target compounds. Presently he works as a postdoctoral fellow of the Research Foundation-Flanders in the lab of Alain Goossens. His main research interests are jasmonate signaling and the regulation of triterpene saponin biosynthesis in Medicago truncatula, gene discovery in plant specialized metabolism and combinatorial biosynthesis of triterpenoids in plants and engineered yeast.
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The genus Medicago belongs to this legume subfamily and comprises 87 species of owering plants, including the widely cultivated crop species M. sativa (alfalfa or lucerne).9–11 Alfalfa is the fourth economically most valuable crop in North America (aer corn, soybean, and wheat, respectively) and the temperate world’s most important forage crop. It is a valuable feed source for livestock and alfalfa sprouts are eaten by humans.12 Besides alfalfa, several other Medicago species are used as medicine, human food, green manure, and source of industrial enzymes in biotechnology.10,13 Alfalfa is an obligate outcrossing and tetraploid species, which makes genetic and genomic studies difficult. Therefore, the closely related M. truncatula (barrel medic) was developed as a model legume.14,15 M. truncatula is an annual, diploid and autogamous legume with a moderate genome size (500–550
Soe Goormachtig (born 1969) obtained her PhD in Plant Biotechnology in Marc Van Montagu’s lab at Ghent University (Belgium 1997), studying the symbiotic interaction between the tropical legume Sesbania rostrata and its microsymbiont Azorhizobium caulinodans. Aer a post-doc in the Lab of Prof. Ingo Potrykus (ETH Z¨ urich, Switzerland), she returned to Ghent University where she was appointed as a Professor in functional biology in 2005. She is now also appointed as principal investigator within the VIB Department of Plant Systems Biology at Ghent University. Her research focusses on rhizosphere interactions more specically on metabolites that inuence root organogenesis as well as nodule development in Medicago truncatula. Alain Goossens (born 1971) obtained his PhD in Plant Biotechnology in Marc Van Montagu’s lab at Ghent University (Belgium, 1998), studying plant seed storage protein synthesis. Subsequently, he performed postdoctoral studies at the IBMCP in Valencia (Spain) with Ram´ on Serrano, working on yeast salt tolerance. He returned to Ghent, where he was appointed as a Principle Investigator within the VIB Department of Plant Systems Biology at Ghent University (2003). His research program focuses on jasmonate signaling, gene discovery in plant metabolism and synthetic biology. He has been appointed as a Professor at Ghent University (2010) and is teaching ‘Metabolic Engineering’.
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Review
Mbp). The availability of a wide range of genomic and genetic resources makes it an invaluable model for studying legume secondary metabolism at the molecular genetic level.15 Among the secondary metabolite classes produced by Medicago species, the triterpene saponins and isoavonoids are of the highest interest and have been explored more thoroughly than the others.4 Lignins are other phenolic compounds that are found in all higher plants and are an important factor affecting cell wall digestibility in forage legumes, including alfalfa.4 In addition, the occurrence of alkaloids like stachydrine and trigonelline,16 cyanogenic glycosides (cyanogens) and nonprotein amino acids (NPAAS) such as canavanine17 have also been reported in Medicago species. This review provides a comprehensive overview of the structural variability, biosynthesis, regulation, physiological role in planta and biological activity of the triterpene saponins and the avonoids found in Medicago. For the other classes of Medicago specialized metabolites, we refer to previous reviews on Medicago and plant metabolism.4,17 Finally, the advances made in metabolic engineering of Medicago species are discussed, and the growing molecular and omics toolkits available for the Medicago community are highlighted.
2 Saponins Saponins are a structurally diverse class of amphipathic glycosides with a lipophilic steroid, steroidal alkaloid, or triterpenoid aglycone backbone (also called sapogenin) that is covalently linked to one (monodesmosidic) or more (di- or tridesmosidic) hydrophilic sugar chains via a glycosidic bond. The name saponin is derived from the Latin word for soap, sapo, and points to an important physicochemical property of the compounds, i.e. their ability to form a colloidal solution in water that forms a stable foam when shaken. This makes them useful as emulsiers and foaming agents in food and beverage industries. Saponins also possess various pharmacological properties, making them commonly used in phytotherapy and cosmetics.18–20 The saponins that are present in the various Medicago species all have pentacyclic oleanane-type sapogenins that are characterized by the presence of a C-12–C-13 double bond, and an oxygen atom at the 3b-position.21 The sapogenins are derived from b-amyrin through various oxidative modications, and based on the oxidation pattern, two distinct types of aglycones can be distinguished in Medicago (Fig. 1). A rst class consists of aglycones that are oxidized at the C-28 position, which is frequently accompanied with oxidation at the C-23 position. Aglycones of this class in Medicago are oleanolic acid, queretaroic acid, hederagenin, caulophyllogenin, bayogenin, medicagenic acid, and zanhic acid (Fig. 1). A second class of sapogenins possesses a hydroxy group at the C-24 position, which excludes oxidation at the C-28 position.21 In Medicago, sapogenins of this class include the soyasapogenols A, B, and E (Fig. 1). A comprehensive overview of the various sapogenins and saponins present in the genus Medicago can be found elsewhere.21,22
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2.1. Saponin biosynthesis in M. truncatula The triterpene saponins present in Medicago share a common biogenic origin with the sterols. 2,3-oxidosqualene, the last common precursor molecule between the primary sterol metabolism and the secondary triterpenoid metabolism, is synthesized in the cytosol from isopentenyl pyrophosphate (IPP) derived from the mevalonate (MVA) pathway.23–26 In M. truncatula, ve isoforms of the key MVA pathway enzyme HMGR have been characterized.27 For the remaining steps of the MVA pathway, candidate genes were identied via a functional genomics approach.28 The end-product of the MVA pathway, IPP, is isomerised by IPP isomerase (IPPI) to yield the allylic isomer dimethylallyl pyrophosphate (DMAPP).29 Subsequently, the prenyl transferase farnesyl pyrophosphate synthase (FPS) catalyzes the sequential condensation reactions of DMAPP with two units of IPP to form farnesyl pyrophosphate (FPP).30 Neither IPPI nor FPS have been functionally characterized in M. truncatula, but candidate genes can be found in the genome.28 In the next step of the biosynthetic pathway leading to 2,3-oxidosqualene, two molecules of FPP are coupled head-to-head to form squalene. This reaction is catalyzed by squalene synthase (SQS), for which a single gene copy can be found in the M. truncatula genome.31 Finally, squalene is oxidized by squalene epoxidase (SQE) to 2,3-oxidosqualene. In M. truncatula, at least three SQE genes are present,28 two of which have been characterized.31 The cyclization of 2,3-oxidosqualene forms the branch-point between the primary sterol and the secondary triterpene saponin metabolism. Cycloartenol (Fig. 1), the tetracyclic plant sterol precursor, is synthesized through cyclization of 2,3-oxidosqualene by cycloartenol synthase (CAS),32,33 the ancestral enzyme of all plant oxidosqualene cyclases (OSCs) involved in secondary metabolism.23,34 For the biosynthesis of triterpene saponins in Medicago, 2,3-oxidosqualene is cyclized to the pentacyclic oleanane-type triterpene backbone b-amyrin by the OSC b-amyrin synthase (BAS) (Fig. 1).31,35 Besides this characterized BAS gene, several other OSCs can be identied in the M. truncatula genome, among which genes homologous to OSCs that catalyze the formation of other types of triterpene backbones, such as lupeol. However, lupeol or derivatives thereof have not yet been detected in M. truncatula.28 Aer the synthesis of b-amyrin, the competitive action of two enzymes causes another branching of the saponin biosynthetic pathway. The P450 enzyme CYP716A12 catalyzes the carboxylation of b-amyrin at the C-28 position, which seems to exclude hydroxylation of b-amyrin at the C-24 position catalyzed by the P450 enzyme CYP93E2, or vice versa (Fig. 1).21,36,37 Carboxylation of b-amyrin at the C-28 position leads to oleanolic acid, which is further modied to yield haemolytic saponins, whereas hydroxylation at the C-24 position is essential for the production of the non-haemolytic soyasapogenol glycosides.21,36 Aer the oxidative modications at the C-24 and C-28 positions, several other positions of the b-amyrin backbone are oxidized in Medicago. In the non-haemolytic branch, CYP72A61v2 catalyzes the oxidation at the C-22 position to yield the soyasapogenol B aglycone (Fig. 1).38 In the haemolytic branch, the C-23 position
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Fig. 1 Sapogenins detected in Medicago species according to Tava, et al.21 and their theoretical biosynthetic pathway in M. truncatula. Cycloartenol and the biosynthetic intermediates in grey have not been detected as saponin aglycone in Medicago.
of oleanolic acid is oxidized by CYP72A68v2 (Fig. 1).38 Another M. truncatula P450, CYP72A63, catalyzes the oxidation of the C30 position of b-amyrin,39 leading to queretaroic acid (Fig. 1).
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Aglycones with this type of modication have been detected in M. Arabica,40 however, none have been reported yet in M. truncatula, and thus this enzyme may be involved in the
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biosynthesis of minor saponins and/or may be restricted to specic tissues or organs of M. truncatula plants.39 For the remaining oxidative modications, no enzymes have been characterized yet, but likely they are also catalyzed by P450s. In the haemolytic branch, two enzymes are still missing: an oxidase that catalyzes the 16-a hydroxylation of hederagenin and medicagenic acid, leading to caulophyllogenin and zanhic acid, respectively, and an oxidase that catalyzes the 2-b hydroxylation of the oleanane backbone. In the non-haemolytic branch, the enzymes catalyzing the C-21 oxidation of soyasapogenol B to soyasapogenol A and the C-22 oxidation of soyasapogenol B to soyasapogenol E remain to be discovered. Aer the various oxidative modications of the b-amyrin backbone, the resulting sapogenins are glycosylated at different positions. In Medicago, glycosylation occurs mainly at the C-3 hydroxy and the C-28 carboxy groups of the aglycone. Rarely, additional glycosylation is observed at the C-23 position.22 In M. truncatula, several glucosyltransferases capable of catalyzing the transfer of glucosyl residues to the sapogenins have been identied. UGT71G1 and UGT73K1 were found to transfer glucosyl residues to different sapogenins in vitro, however, the specic position to which the glucosyl residues were transferred was not determined.41 UGT71G1 also recognizes isoavones and the avonol quercetin as substrates. UGT71G1 glucosylates these compounds with higher efficiencies than triterpenes,41,42 suggesting that it might not correspond to a saponin specic biosynthetic enzyme. Another glucosyltransferase, UGT73F3, was found to catalyze the glucosylation of sapogenins at the C-28 carboxy group in vitro. This effect was conrmed in vivo by genetic loss-of-function studies.28 Besides glucose, several other sugars, including glucuronic acid, galactose, rhamnose, arabinose and xylose are found to be part of the sugar chains of Medicago saponins.22 To date however, no enzymes catalyzing the transfer of these sugar residues to the aglycones have been discovered in Medicago. Other types of modications occurring on the Medicago saponins include addition of malonyl and methyl functionalities on the sugar residues. In M. sativa, the occurrence of soyasaponin VI, containing a C-22 maltol functionality has been reported.19,22,43 No specic enzymes for these modications have been reported yet.
2.2. Regulation of saponin biosynthesis In Medicago plants, a tissue-specic accumulation of saponins is oen observed. Soyasapogenol glycosides are the main accumulating saponins in M. truncatula roots, whereas none of the major medicagenic acid and zanhic acid glycosides that accumulate in the aerial parts, are present in roots.19,44 This tissue-specic accumulation is likely the result of their tissuespecic biosynthesis, and points to a role for saponins in specic defense mechanisms in different Medicago tissues.19,45 The differential accumulation of specic saponin mixtures in different tissues also suggests the existence of specic regulatory mechanisms that steer the biosynthesis toward the desired biosynthetic end-products. To date, however, no regulators responsible for this tissue-specic saponin compendium have been discovered in Medicago.
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Besides the tissue-specic, constitutive accumulation of saponins, induced saponin biosynthesis is oen observed in response to herbivore feeding or pathogen attack.46 For instance, seven days aer Spodoptera littoralis larvae fed on M. sativa leaves, the total saponin content was increased 84% in the foliage of the damaged plants,47 with doubled concentrations of 3-GlcA,28-AraRhaXyl medicagenic acid and soyasaponin I. Furthermore, the damaged leaves had a deterrent effect on the herbivorous larvae, and when fed on damaged leaves, the larval performance was reduced.48 The increased saponin accumulation is mediated by increased steady-state transcript levels of the corresponding saponin biosynthetic genes. This transcriptional response to pathogen or herbivore attack is controlled by a complex signaling cascade in which jasmonates (JAs) play a crucial role.49,50 As such, treatment of Medicago plants or plant cell cultures with JAs can mimic pathogen or herbivore attack. Accordingly, exposure of M. truncatula cell suspension cultures to methyl jasmonate (MeJA) leads to the transcriptional activation of saponin biosynthetic genes and as a consequence to an increased saponin accumulation.28,31,41,51,52 The JA perception and initial signaling cascades leading to the concerted transcriptional activation of entire secondary metabolic pathways is conserved across the plant kingdom. However, the downstream transcriptional machineries that regulate the specic biosynthetic genes are mostly speciesspecic and remain largely unknown.26,49,50,53 So far, no specic transcription factors that regulate the saponin biosynthesis have been characterized in Medicago. However, another regulatory mechanism, operating at a posttranscriptional level, has recently been discovered. To secure plant development and integrity, the unrestrained production of bioactive saponins upon JA perception needs to be prevented. In M. truncatula, the RING E3 ubiquitin ligase MAKIBISHI1 (MKB1), a functional homolog of the mammalian RMA1 proteins involved in the endoplasmic reticulumassociated degradation (ERAD) protein quality control system, is co-expressed with the saponin biosynthesis enzymes.54 By recruiting the ERAD machinery, MKB1 controls the levels and activity of HMGR, the rate-limiting enzyme in the MVA pathway. M. truncatula roots, in which MKB1 is silenced, overaccumulate bioactive monoglycosylated saponins and lose their integrity, demonstrating the need of this regulatory system to manage saponin biosynthesis.
2.3. Biological role of Medicago saponins The green parts of plants are the main target for herbivores, so the saponins present in the aerial parts of Medicago may exhibit antiherbivory activities by several different mechanisms, thereby inuencing animal metabolism from feed intake to excretion.22,55 First, the accumulating saponins may turn the plants unpalatable for herbivores, for instance due to the bitter avor, as was illustrated with a sensory test performed with human volunteers that showed that the main saponin present in the aerial parts of alfalfa, a tridesmoside of zanhic acid, was the most bitter, astringent and throat-irritating of all tested
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compounds.56 Alfalfa saponins also deter insects from eating, as was shown by larvae of Colorado potato beetles that died in four to six days, mainly because of fasting, when they were fed potato leaves treated with a 0.5% solution of total saponins of alfalfa.57 Second, when herbivores are not deterred and consume Medicago saponins, they may suffer from the various toxic or detrimental effects of the compounds. For instance, intraruminal administration of partially hydrolyzed alfalfa saponins led to reduced microbial fermentation and nutrient degradation in the rumen of sheep.58 Furthermore, the total protozoal count in the rumen was signicantly reduced by the alfalfa saponins, likely due to the disruption of protozoal cell membranes by the formation of water-insoluble addition products of medicagenic acid with membrane sterols.58,59 In addition, alfalfa saponins inhibit rumen motility in sheep and cattle,59 and they appear to be responsible for some cases of rumen bloat.55 Next to ruminants, alfalfa saponins also exhibit toxic effects on monogastric animals55,60 and herbivorous insects.22,61–65 Saponins that accumulate in roots or that are released in the rhizosphere may protect Medicago plants against parasitic nematodes. In vitro assays have shown that saponins from M. arborea, M. arabica, and M. sativa exert toxicity toward the phytoparasitic nematode Xiphinema index.66 Addition of dry plant material from M. arborea and M. sativa roots and tops was shown to suppress root and soil population densities of the rootknot nematode Meloidogyne incognita and the yellow potato cyst nematode Globodera rostochiensis in potting mixes and in eld trials, with saponins being held at least partially responsible for the nematicidal activity.67,68 Saponins released in the rhizosphere may also be involved in negative allelopathic interactions of Medicago plants with surrounding plants.69 It was observed that germination, growth, and yield of cotton was much lower when sown in soil aer alfalfa cultivation, than when sown in soil aer wheat cultivation or in bare soil, likely due to the release of saponins by alfalfa roots.70 The addition of powdered alfalfa roots to sand was shown to inhibit wheat seedling growth.71 Later, a structure-activity relationship study with puried alfalfa saponins showed that medicagenic acid and the monoglycosides 3-O-Glc-medicagenic acid and 3-O-Glc-hederagenin exerted the highest growth inhibitory effects on wheat seedlings. These compounds accumulate in considerable amounts in alfalfa seedlings and roots, whereas they are absent in mature alfalfa tops.72 This underscores that the tissue-specic accumulation of Medicago saponins is correlated with their biological roles. In seeds, saponins may be involved in the protection against insects. For instance, M. truncatula seed our has a strong toxic effect on the rice weevil Sitophilus oryzae, a major pest of stored cereals. Purication and identication of the entomotoxic compound revealed that 3-GlcA,28-AraRhaXyl medicagenic acid protects M. truncatula seeds against the weevil.73 The saponins accumulating in Medicago may also be involved in the protection of the plants against infections by phytopathogenic fungi and microbes. The anti-fungal activity of the saponins present in Medicago is well established, with the medicagenic acid aglycone being the major contributor.74 Besides defense, the saponins present in Medicago roots may also play a role in the interaction of the plant with symbiotic
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partners, such as nitrogen-xing rhizobia and mycorrhizal fungi. For instance, colonization of M. truncatula with the mycorrhizal fungus Glomus intraradices does not lead to quantitative changes in the total saponin content but to a reduced ratio between malonylated and corresponding non-malonylated saponins.46,75 This might hint to a specic, but yet unknown, role of saponins in the mycorrhization process. Intriguingly also the receptor-like kinase NORK, required for Nod factor signaling and root nodule development in M. truncatula, was found to interact with HMGR. Chemical inhibition of HMGR activities or silencing of HMGR expression in M. truncatula roots decreased nodulation, indicating that HMGR and the MVA pathway are essential for nodule development.27 It was postulated that recruitment of HMGR by NORK might modulate the production of specic primary terpenoid compounds, such as particular hormones or sterols, but it is equally possible that it rather affects synthesis of secondary terpenoids such as specic bioactive saponins that might play important roles in the plantmicrobe mutualism.
2.4. Commercial potential of Medicago saponins The combined deterrent and toxic effects on insects make Medicago saponins suitable for use against pest insects in agriculture and horticulture.62 One way to use Medicago saponins against herbivorous insects is by selecting cultivars that accumulate high amounts of saponins.62 For instance, the development, survival, and reproduction of pea aphids fed on an alfalfa line with a high saponin content was reduced as compared to aphids fed on an alfalfa cultivar with a low saponin content. Moreover, it was shown that zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid accumulating in the high saponin cultivar were the main compounds contributing to the resistance of alfalfa against the pea aphid.65,76,77 Besides selecting for Medicago cultivars with a high saponin content, extracted Medicago saponins could be used to spray other plants, as was illustrated with larvae of Colorado potato beetles that died aer feeding on potato leaves treated with a 0.5% solution of total saponins of M. sativa.57 The nematicidal activity of the accumulating saponins allows Medicago biomass to be used as a biological agent to control phytoparasitic nematodes. A eld trial in which soil was amended with pelleted M. sativa reduced the soil population density of the root-knot nematode M. incognita, and tomato plants growing on the amended soil developed less root galls and delivered increased tomato crop yields.67,68 The antifungal activity of alfalfa saponins may also reduce the presence of phytopathogenic fungi in the amended soil. Incorporation of alfalfa plant material in soil inoculated with Phytophthora capsici reduced Phytophthora blight of capsicum in pots and eld trials.78 Total saponins and individual compounds from different Medicago species were shown to prevent fusariosis on tulip bulbs.79,80 Hence, the activity of Medicago saponins on phytopathogenic fungi and nematodes, in combination with the high nitrogen content of the plant material, make Medicago biomass especially useful as agent against soil-born plant pathogens and biological fertilizer.
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Because of the antimicrobial activity, Medicago saponins may also nd applications as antibacterial and antifungal agents. Saponin extracts from M. sativa, M. murex, M. arabica, and M. hybrida inhibit the growth of dermatophytic fungi and monodesmosidic glycosides of medicagenic acid were shown to be the most active compounds.81 Similarly, testing of saponins from M. sativa, M. arborea, and M. arabica against a selection of medically important yeasts and bacteria revealed a high activity against Gram-positive bacteria like Bacillus cereus, B. subtilis, Staphylococcus aureus, and Enterococcus faecalis. M. arabica saponins, in which hederagenin is the dominant aglycone, showed a broad spectrum of action, evidencing the possible application of those compounds as antibacterial agents.82 The ability to rupture erythrocyte membranes (haemolysis) is another important property of several of the saponins present in Medicago. Saponins bind to the sterols present in the erythrocyte membrane, which causes irreversible damage to the lipid bilayer and results in increased membrane permeability and haemoglobin loss.83,84 Recently, it was reported that the M. truncatula mutants lacking hemolytic activity (lha) were devoid of haemolytic activity, because they were unable to produce the aglycones oleanolic acid, hederagenin, bayogenin, medicagenic acid, and zanhic acid. This was caused by mutations that render the CYP716A12 enzyme, which catalyzes the carboxylation of bamyrin at the C-28 position, non-functional,36 and conrms previous reports about the importance of C-28 carboxylation in addition to C-3 glycosylation for haemolytic activity.85 The haemolytic activity has even been used as a detection and quantication method for saponins.55,84,86 Like many triterpenoid saponins, Medicago saponins also exert cytotoxic effects,22 and a toxicity screening of saponins from 12 different Medicago species with the brine shrimp Artemia salina pointed toward hederagenin and bayogenin glycosides as the main cytotoxic compounds.87
2.5. Determination of Medicago saponins The saponins present in Medicago plants occur in complex mixtures of structurally related high-molecular weight compounds and are oen only present in low concentrations.19,22,88,89 This complicates the detection and quantication of saponins and hinders purication of individual compounds, which is necessary for structure elucidation and biological activity testing. In addition, Medicago saponins lack chromophores that allow detection in UV, limiting the choice of detection methods that can be employed for analytical purposes.89,90 The screening of crude extracts using hyphenated techniques, such as LC-MS, provides information about the composition of the saponin mixture present in the investigated plant material. In a pioneering study, a large number of saponins in root extracts of M. sativa and M. truncatula were separated with HPLC, and 15 and 27 saponins of M. sativa and M. truncatula, respectively, were tentatively identied based on fragmentation data under negative ionization.88 More recently, by coupling HPLC to the highly accurate Fourier transform ion cyclotron resonance MS, reliable prediction of the molecular
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formula of the detected saponins was possible. This has led, in combination with the fragmentation spectra under negative ionization, to the tentative identication of 79 saponins in M. truncatula hairy roots,19,54 and further underscores the complexity of the saponin mixture present in Medicago. Such qualitative proling of the saponin mixture, however, does not provide conclusive evidence for the absolute chemical structures of the saponins present in Medicago, which can only be obtained by subjecting puried compounds to a combination of analytical methods, including MS and NMR.22 The purication of saponins from the aerial parts of M. truncatula has led to the chemical characterization of 15 individual compounds.91 Several other saponin molecules have been puried and structurally resolved from M. arborea, M. hybrida, M. arabica, and M. polymorpha.40,92–95 A detailed overview of the different saponin structures reported in various Medicago species can be found elsewhere.22 So far however, the purication of compounds has only led to the identication of the dominant compounds in the saponin mixture; the purication of low abundant compounds remains a serious challenge. The puried and characterized compounds can be used as standards to generate response curves that can be used for the absolute quantication of saponins with HPLC/MS. Differential accumulation of saponins in the various organs of M. truncatula was reported, with higher levels of medicagenic acid conjugates present in leaves and seeds, and higher levels of soyasapogenol conjugates present in the roots.45 In a similar approach, saponins in the aerial parts of three different M. truncatula cultivars were quantied, showing very similar saponin mixtures in the three cultivars, with medicagenic acid, zanhic acid, and soyasapogenol glycosides being the dominant compounds. The observed total saponin concentration in M. truncatula is also very similar to that in M. sativa. However, M. truncatula has a higher concentration of zanhic acid glycosides, and a lower concentration of soyasapogenol glycosides as compared to M. sativa.44 The high levels of zanhic acid glycosides (>40% of the total saponin content) observed in this study are in disagreement with the study by Huhman, et al.,45 which reports that zanhic acid glycosides constitute only 0.6% of the total saponin content. This difference may be attributed to the lack of appropriate standards in the latter study, in which no zanhic acid glycosides were available as standards for absolute quantication.45,91 Besides HPLC/MS, GC/MS can also be used for the quantication of Medicago saponins. However, being high-molecular weight compounds, saponins are not volatile and, consequently, GC/MS analysis can solely be performed on hydrolyzed saponins, thus only providing information on the aglycone structures. Furthermore, hydrolysis is oen not complete or can lead to artefacts or decomposition of aglycones, thereby negatively inuencing the nal result.36,90,96
3 Flavonoids Flavonoids constitute a diverse class of metabolites that are ubiquitously present in the plant kingdom and contains more than 10,000 different structures. The name avonoid is derived
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from the Latin word for yellow, avus, and points to the color of many avonoids. Flavonoids are responsible for much of the yellow, red, blue, and purple plant pigmentation. They play important biochemical and physiological roles in planta and are involved in interactions of the plant with its environment, both for benecial symbioses and for biotic and abiotic stress responses.97–99 Flavonoids also possess various pharmacological properties and are thought to have health-promoting effects.100,101 Flavonoids are characterized by a polyphenolic structure with 15 carbon atoms arranged in a basic C6-C3-C6 structural skeleton, in which two benzene rings (A & B) are linked by a linear three carbon bridge (in the case of chalcones) or by a pyran or pyrone ring (C). According to the different alterations and modications of ring C, avonoids have been classied into different subclasses. All main subclasses of plant avonoids, i.e. anthocyanidins, aurones, chalcones, avanols (proanthocyanidins), avanones, avonols, avones and isoavones are present in the genus Medicago, yielding a complex and branched synthetic network (Fig. 2). In contrast to the triterpene saponins, Medicago avonoids are less genus-specic and generally encountered in many legumes. Nonetheless, bearing the role of Medicago as a model system for legume and avonoid research in mind, we considered it appropriate to provide a comprehensive overview of Medicago avonoid synthesis, by describing the Medicago metabolites, genes and enzymes encountered already and listing the missing gaps in the pathway knowledge.
3.1. Flavonoid biosynthesis in M. truncatula 3.1.1. Early steps of avonoid biosynthesis. The biosynthetic pathway leading to the production of avonoids is derived from the phenylpropanoid pathway (Fig. 2). It starts with the formation of p-coumaric acid and p-coumaroyl-CoA from phenylalanine, catalyzed by the sequential activities of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) on the one hand, and the cytosolic formation of malonyl-CoA from acetyl-CoA by acetyl-CoA carboxylase (ACC) on the other hand. Then, one molecule of p-coumaroyl-CoA and three molecules of malonylCoA serve as substrates for chalcone synthase (CHS) to run a series of sequential decarboxylation and condensation reactions, leading to the formation of a polyketide intermediate. This intermediate undergoes cyclization and aromatization reactions to form the A-ring and the resultant chalcone (naringenin chalcone). In Medicago species., CHS is encoded by multiple (oen 10 or more) gene copies.102–104 Chalcone reductase (CHR), of which the corresponding gene is found only in leguminous plants, removes the hydroxyl group of the second malonyl-CoA during chalcone biosynthesis, which together with the CHS leads to the biosynthesis of isoliquiritigenin, the precursor of the 5-deoxyavonoids.105 In dicotyledonous plants like Medicago, p-coumaric acid and p-coumaroyl-CoA are also the precursors of the monolignols, the monomer building blocks of the lignin heteropolymer. Although engineering of lignin synthesis in Medicago species
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has been pursued (see section 5.2), we will not further discuss this branch of the phenylpropanoid pathway here but instead refer to the literature.106–108 The two avanones, naringenin and liquiritigenin, the precursors of 5-hydroxy- and 5-deoxy-avonoids, respectively, are produced via the enzymatic activity of chalcone isomerase (CHI). Two classes of CHIs have been described. Type I CHIs are found in both legumes and nonlegumes, and convert naringenin chalcone into naringenin. Type II CHIs are legumespecic enzymes that can isomerize both naringenin chalcone and isoliquiritigenin to naringenin and liquiritigenin, respectively (Fig. 2).109,110 Genes encoding enzymes of both classes of CHIs have been identied in Medicago.111 The resulting avanones, naringenin and liquiritigenin, serve as substrates for isoavone synthase (IFS), avone synthase (FNS) and avanone 3-hydroxylase (F3H) to produce isoavonoids, avones and (dihydro)avonols, respectively (Fig. 2). 3.1.2. Isoavonoid biosynthesis. Isoavonoids have the Bring attached to the C-ring via the C-3 instead of the C-2 position (Fig. 2). The rst committed step of isoavonoid biosynthesis begins with an oxidative aryl migration from C-2 to C-3, catalyzed by the cytochrome P450 (P450) enzyme 2-hydroxyisoavanone synthase (2HIS, also known as isoavone synthase, IFS).112 Subsequently, dehydration of the 2-hydroxyisoavanone intermediates, i.e. 2,5,7,40 -tetrahydroxyisoavanone and 2,7,40 -trihydroxyisoavanone, by 2-hydroxyisoavanone dehydratase (2HID) forms the “undecorated” isoavones genistein and daidzein, respectively. The 40 -O-methylated forms of genistein and daidzein, biochanin A and formononetin, respectively, which are the most abundant isoavonoid aglycones in M. truncatula roots,113 are biosynthesized by catalytic action of 2,7,40 -trihydroxyisoavanone-40 -O-methyltransferase (HI40 OMT) on the 2-hydroxyisoavanone intermediates prior to 2HID activity. The isoavonoid aglycones can undergo further modications, such as C-30 hydroxylation of the B-ring. M. truncatula I30 H (CYP81E9) was shown to catalyze the hydroxylation of both biochanin A and formononetin to pratensein and calycosin, respectively.114 In addition, Medicago accumulates isoavonoid aglycones with various modications of the A-ring, such as afrormosin, alfalone, irisolidone, and irilone (Fig. 2). Labeling studies indicated that the rst two are derived from formononetin,115 but the specic enzymes involved in the biosynthesis of these compounds have not been discovered yet. Pterocarpans form a class of isoavonoid derivatives in which the B-ring is coupled to the C-ring via a furan ring. The biosynthesis of medicarpin, the pterocarpan accumulating in Medicago, starts with the conversion of formononetin to 20 hydroxyformononetin, catalyzed by the P450 enzyme I20 H (CYP81E7).114 Next, 20 -hydroxyformononetin is converted to the 20 -hydroxylated isoavanone vestitone by the NADPHdependent isoavone reductase (IFR). Subsequently, vestitone is converted into medicarpin via a 2-step enzymatic conversion catalyzed by vestitone reductase, that reduces vestitone to 7,20 dihydroxy-40 -methoxy-isoavanol (DMI), and by DMI dehydratase, that dehydrates DMI, thereby forming the ether linkage of the furan ring of the pterocarpan skeleton (Fig 1).116,117
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Fig. 2 General phenylpropanoid and (iso)flavonoid biosynthetic pathway in M. truncatula. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; CHR, chalcone reductase IFS, isoflavone synthase; 2HID, 2-hydroxyisoflavanone dehydratase; FNS, flavone synthase; F3H, flavanone 3-hydroxylase; IFR, isoflavone reductase; I20 H, isoflavone 20 -hydroxylase; VR, vestitone reductase; DMID, dihydroxy-40 -methoxy-isoflavanol dehydratase; FLS, flavonol synthase; PRX, peroxidases; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; PTR, pterocarpan reductase.
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Pterocarpans can be further reduced by pterocarpan reductase,118 leading to isoavans, of which vestitol and sativan are compounds accumulating in Medicago. Coumestans, oxidation products of pterocarpans, constitute another class of isoavonoid derivatives. Coumestrol is the main coumestan accumulating in Medicago and is synthesized from the isoavonoid daidzein, likely through dihydrodaidzein and a pterocarpan intermediate.119–121 Up until now, however, the genes and enzymes involved in its biosynthesis have not been elucidated yet. 3.1.3. Flavonol biosynthesis. Flavonols are the most widespread subclass of avonoids in higher plants. They are derived from the dihydroavonols, which are the product of the F3H enzyme, of which the corresponding genes have been identied and characterized both in M. truncatula and M. sativa.122,123 Flavonol synthase (FLS) catalyzes the key step in avonol biosynthesis, converting dihydroavonols, such as dihydrokaempferol, dihydroquercetin, and dihydromyricetin, to the corresponding avonols, kaempferol, quercetin, and myricetin, respectively (Fig. 2). FLS has been characterized in several plant species, including A. thaliana,124,125 Citrus unshiu Marc.,126 parsley,127 soybean,128 and strawberry,129 but not yet in Medicago species. 3.1.4. Flavone biosynthesis. The biosynthesis of avones from avanones occurs by introducing a double bond between C-2 and C-3 that is catalyzed by avone synthase (FNS) (Fig. 2). Two different FNS (FNSI and FNSII) have been characterized in plants. FNSI is mainly found in members of the Apiaceae family as well as monocotyledonous plants,130,131 while FNSII is found widespread among all higher plants.132 All known FNSII proteins belong to the plant P450 subfamily CYP93B and two different mechanisms for their catalytic activity have been reported, i.e. some convert avanone substrates to avones via a 2-hydroxyavanone intermediate, whereas others directly convert avanones to avones.133,134 Two FNSII genes, MtFNSII-1 (CYP93B10) and MtFNSII-2 (CYP93B11), have been characterized in M. truncatula. Both convert avanones to 2-hydroxyavanones and have a distinct tissue-specic expression pattern. MtFNSII-1 is mostly expressed in roots and seeds where the major accumulating avones are 7,40 -dihydroxyavone and apigenin/luteolin, respectively, while MtFNSII-2 is highly expressed in owers and pods.135 Medicago enzyme(s) that catalyze the conversion of the intermediate 2-hydroxyavanones into the accumulating avones have currently not been identied. 3.1.5. Aurone biosynthesis. Structurally, aurones are isomers of avones (Fig. 2). Aurones are widely distributed in fruits and owers, in which they are responsible for yellow pigmentation. Accumulation of the aurone hispidol and its glycoside derivative, hispidol-40 -O-glucoside, is reported in yeast elicitor (YE)-induced cell cultures of M. truncatula. Three peroxidase genes, MtPRX1, MtPRX2 and MtPRX3, were suggested to be involved in hispidol biosynthesis from isoliquiritigenin, based on the observation that their expression proles were correlated with hispidol accumulation. Subsequently, the aurone synthase activity of recombinant MtPRX1 and MtPRX2 proteins was demonstrated in vitro.136
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3.1.6. Anthocyanin biosynthesis. The presence of a positive charge in the anthocyanin structure at acidic pH, called a avylium cation (2-phenylbenzopyrylium), distinguishes anthocyanins from other subgroups of avonoids (Fig. 2). The number and position of hydroxy and methoxy groups on the anthocyanidin skeleton; the identity and number of sugars, and the positions at which they are attached; the extent of sugar acylation and the identity of the acylating agent are all responsible for the variation in the anthocyanin structures.137 Enzymes controlling the B-ring hydroxylation of e.g. naringenin in the early steps of avonoid biosynthesis, such as avonoid 30 hydroxylase (F30 H) and avonoid 30 ,50 -hydroxylase (F30 50 H), are key determinants for the structural fate of the resulting anthocyanins as well as 2,3-cis-avan-3-ols.138 The rst dedicated step toward the biosynthesis of anthocyanins (and proanthocyanidins, see section 3.1.7) is controlled by dihydroavonol-4-reductase (DFR) that uses the same substrates as FLS. Dihydrokaempferol, dihydroquercetin, and dihydromyricetin, which differ only in the hydroxylation pattern of their B-ring, are common substrates of DFR and are converted into leucopelargonidin, leucocyanidin, and leucodelphinidin, respectively.139 In M. truncatula, two DFRs, MtDFR1 and MtDFR2, have been characterized that have distinct substrate preferences. Although the expression pattern of the two characterized MtDFRs is very similar in most tissues tested, it is proposed that MtDFR1 has a more pronounced role than MtDRF2 in anthocyanin (cyanidin glucoside) biosynthesis in leaves.140 The next step in anthocyanin biosynthesis is catalyzed by anthocyanidin synthase (ANS) that converts the leucoanthocyanidins leucocyanidin, leucopelargonin, and leucodelphinidin to the corresponding anthocyanidins cyanidin, pelargonin, and delphinidin, respectively.141 In vitro assays characterized the single MtANS as a bifunctional enzyme involved in the conversion of leucocyanidin to cyanidin during (pro)anthocyanidin biosynthesis, and, more efficiently, dihydroquercetin to quercetin during avonol biosynthesis.142 MtANS is mainly expressed in the seed coat of Medicago species, but also in other tissues to play a role in (pro)anthocyanidin biosynthesis. In addition, downregulation of MtANS resulted in reduced levels of anthocyanins in leaves and of soluble and insoluble proanthocyanidins in seeds of Medicago species.142 3.1.7. Proanthocyanidin biosynthesis. Proanthocyanidins (PAs), also called avanols or condensed tannins, comprise mixtures of oligomers or polymers of avan-3-ol units that share the upstream biosynthetic pathway with the anthocyanins (Fig. 2). The structural diversity of PAs depends on the stereochemistry and hydroxylation pattern of the avan-3-ol units, the position and stereochemistry of the interavanyl linkage between the monomeric units, the extent of polymerization, and the type of modications, like various methyl-, acyl- or glycosyl- substitutions of the monomeric units.103,143 Hydroxylation on the A-ring leads to stereochemistry at C-4, which has great importance in the biosynthesis of PAs, since all chiral intermediates of PA biosynthesis up to (+)-catechin possess the 2,3-trans stereochemistry. Conversely, ()-epicatechin with
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2,3-cis stereochemistry arises from achiral anthocyanidin precursors that provide another starter unit for PA biosynthesis.103,143 Although the only difference between the two PA starter units 2,3-cis-2R,3R-()-epicatechin and 2,3-trans-2R,3S(+)-catechin is the cis- or trans- stereochemical conguration, they are synthesized by two distinct biosynthetic pathway branches (Fig. 2).143 In M. truncatula the structural genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), both involved in PA biosynthesis, have been characterized.142,144–146 LAR is encoded by a member of the plant reductase-epimerase-dehydrogenase (RED) supergene family and is closely related to IFR.147 It uses the same substrates as ANS, i.e. avan-3,4-diols, to convert them to their corresponding PA “starter units”, the 2,3-trans-avan-3-ols catechin, afzelechin and gallocatechin, respectively, in an NADPH-dependent manner. This rst committed step in PA biosynthesis diverges from the pathway common with that of the anthocyanins.145 In M. truncatula, the MtLAR gene is expressed in owers, pods, and seed coats.142 ANR catalyzes the conversion of the anthocyanidins formed by ANS into 2,3-cis-avan-3-ols (()-epicatechin, ()-epiafzelechin and ()-epigallocatechin, respectively). The ANR enzyme is encoded by the BANYULS (BAN) gene, identied rst through characterization of the banyuls locus in Arabidopsis148 and later described in detail in M. truncatula.140,145 Similar to LAR, ANR also belongs to the IFR-like group of the plant RED superfamily. 3.1.8. Structural diversication and decoration of avonoids. Flavonoids oen accumulate as malonylated or acetylated glycoconjugates. For instance, almost all anthocyanidin aglycones are conjugated with one or more sugar moieties, mainly at the C-3 position. Glycosylation enhances the watersolubility of anthocyanidins and is critical for the transport and sequestration of these compounds in the vacuole. Of the more than 100 identied UDP-glycosyltransferases (UGTs) in the M. truncatula genome, UGT71G1, UGT85H2, UGT78G1, and UGT72L1 have been shown to possess activity on avonoid aglycones.144,41,149–151 UGT71G1 was shown to be capable of glycosylating avonols (quercetin), isoavonoids (genistein), and saponins.41,42 UGT85H2 is a multifunctional avonoid glycosyltransferase with the ability to glycosylate various avonoids, such as isoavones (biochanin A), avonols (kaempferol), and chalcones (isoliquiritigenin).150 Also UGT78G1 has a broad substrate range, i.e. on formononetin, kaempferol, and the anthocyanidins pelargonidin and cyanidin.151 Although it had been shown that isoavones were the preferred in vitro substrates, yielding the corresponding 7-Oglucosides,152 the phenotypes observed by UGT78G1 overexpression in alfalfa, as well as in M. truncatula retrotransposon insertion lines, rather support a role in anthocyanidin glycosylation. Expression of UGT78G1 is strongly upregulated by Legume Anthocyanin Production 1 (LAP1), a MYB-family transcription factor (TF) involved in anthocyanin biosynthesis but not isoavone biosynthesis.153 Correspondingly, UGT78G1 is expressed in anthocyanin accumulating sites, i.e. owers, leaves, and buds, rather than in roots, which are the major sites for isoavone accumulation in Medicago.152 Since PAs are
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typically sequestered in vacuoles in malonylated and glycosylated forms, glycosylation has been proposed to be critical for the transport and storage of these compounds at their nal destination in the vacuole. UGT72L1 is a Medicago glycosyltransferase responsible for the glycosylation of the PA precursor ()-epicatechin, leading to epicatechin 30 -O-glucoside. The expression pattern of UGT72L1 in developing seeds correlates with the presence of epicatechin 30 -O-glucoside and PA accumulation.144 However, since it is not proven that UGT72L1 by itself is essential for PA biosynthesis, redundant pathways for glycosylation and subsequent PA polymerization may exist.146 Malonylated and acetylated isoavonoids are considered as ‘storage forms’ that accumulate in the vacuoles, serving as a pool of biosynthetic precursors or inactive forms of phytoalexins.154,155 The malonyl residues substituted on the sugar moiety protect the isoavonoid glycoconjugates from enzymatic degradation, change their lipophilicity, and act as molecular tags promoting efficient vacuolar uptake of the conjugates.156,157 Three malonyltransferases from M. truncatula, named MtMaT1, MtMaT2, and MtMaT3, have been shown to catalyze the malonylation of a range of isoavone 7-O-glucosides in vitro and MtMaT1 and/or MtMaT2 were suggested to function as malonyl CoA:isoavone 7-O-malonyltransferases in vivo.158 3.2. Regulation of avonoid biosynthesis Flavonoids play a signicant role in responses to several environmental factors, including both biotic, e.g. pathogen attack and herbivory, and abiotic stresses, e.g. ultraviolet (UV)-light, salt stress, and nutrient deciencies.13,15,97,98,159 In Medicago, isoavonoid phytoalexins and related compounds accumulate in response to fungal or yeast elicitors (YE).51,52,110,115,155,160 In agreement, fungal pathogen infections are capable of massively changing the expression of phenylpropanoid biosynthesis genes, such as Phymatotrichopsis omnivore, responsible for destructive root rot disease in many dicot species, in M. sativa.161 In M. truncatula, increased levels of the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside were observed aer infection with the fungal pathogen Phoma medicaginis.162 Intriguingly, in M. truncatula cell cultures, genes involved in the early steps of phenylpropanoid/isoavonoid biosynthesis are induced by YE treatment but not by MeJA elicitation, while downstream pathway genes specic for medicarpin formation were induced by both.155 The difference in outcome is due to the different triggers applied. Indeed, YE mimics pathogen attack in the plant cell, while MeJA induces wound signaling cascades. Two different strategies have been proposed for the induction of medicarpin in response to pathogen (YE) or wound signals (MeJA) in M. truncatula. In non-stress conditions, glycosylated and malonylated formononetin conjugates are sequestered in the vacuoles. When MeJA or wound stresses are applied, formononetin conjugates are converted to free formononetin isoavones and transferred to the cytosol. Concomitantly, the downstream enzymes are induced by MeJA and formononetin is converted to medicarpin. Elicitation with YE on the other hand leads to the elevation of medicarpin levels via de novo
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biosynthesis.155 Also the phytoalexin accumulation pattern during fungal infection reect the existence of different regulatory systems that orchestrate metabolic uxes through specic metabolic pathways.51,52,115,155,160 In M. truncatula, four YE-induced WRKY TFs (W100577, W100630, W108715, and W109669) were identied, of which the expression was correlated with that of the genes involved in the central phenylpropanoid pathway and in the downstream steps of medicarpin biosynthesis. Ectopic expression of these WRKY TFs in tobacco (Nicotiana tabacum) led to higher levels of avonoids and other phenolic compounds.160 In addition, studies about the regulation of anthocyanin and PA biosynthesis revealed additional ternary protein complexes composed of three families of TFs, i.e. the R2R3-MYB domain, the bHLH (basic helix–loop–helix) domain, and the WD40 repeat proteins.163 So far in Medicago, only the LAP1 R2R3-MYB from M. truncatula was described to be involved in the orchestration of anthocyanin biosynthesis.153 Overexpression of LAP1 in M. truncatula and several closely related species, including alfalfa and white clover, led to the accumulation of anthocyanin pigments, which was reective of the upregulation of a large number of genes associated with anthocyanin biosynthesis, including the glucosyltransferase UGT78G1.153 Also two major components of the regulatory complex involved in the regulation of PA biosynthesis have been found in M. truncatula, namely M. truncatula WD40-1 (MtWD40-1), which is a singlecopy WD40-repeat protein orthologous to Arabidopsis Transparent Testa Glabra 1 (TTG1) and designated as a positive regulator of PA biosynthesis in M. truncatula seeds,164 and MtPAR (M. truncatula proanthocyanidin regulator), which is an R2R3 MYB-type TF that plays a positive regulatory role on genes involved in the PA pathway.165 The gene encoding MtPAR is only expressed in the seed coat where PAs accumulate. In addition, ectopic expression of MtPAR in M. sativa was shown to cause accumulation of PAs in the shoots.165
3.3. Flavonoid localization Flavonoids can be transported within and between cells and even between tissue layers, oen leading to active or passive release at the plant surface to carry out their role in regulating interactions of the plant with its environment. In specic tissues, avonoid synthesis and accumulation is oen located in distinct cells. In M. truncatula roots, avonoids accumulate in specic cell types, i.e. at the root tip and in root cap cells, from where they can be exuded or sloughed off into the soil.99 With regard to the active intercellular transport of avonoids, ABC (ATP binding cassette) transporters are likely involved, since the M. truncatula ABC transporter MtABCG10 is located in the plasma membrane and active in the vascular tissue of most organs. Silencing MtABCG10 in M. truncatula hairy roots resulted in a lower accumulation and exudation of medicarpin and its isoavone precursors.166 At the cellular level, avonoids are found in the extracellular space and in most plant cell compartments, i.e. the cytosol, vacuole, endoplasmic reticulum, chloroplast, nucleus and small vesicles.167 However, most conjugated avonoids are
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sequestered in the vacuole, where for instance the vacuolar acidic environment and co-pigments determine anthocyaninmediated oral pigmentation.168 In addition, PA subunits are polymerized and subsequently converted to brown oxidation products in the vacuoles. Isoavonoids, such as formononetin, were also found to be sequestrated in the vacuoles in glycosylated and malonylated forms that were only converted to free formononetin and translocated to the cytosol for conversion to medicarpin in response to certain stresses.155 Two mechanisms have been proposed for intracellular avonoid movement: (i) vesicle trafficking-mediated transport and (ii) membrane transporter-mediated transport. Although most studies have been performed on anthocyanins only, this likely holds true for other avonoid classes as well.167,169 Via the rst mechanism, anthocyanins rst accumulate in anthocyanoplasts, which are vesicle-like structures, or in membrane-less proteinaceous matrices, called anthocyanic vacuolar inclusions (AVIs).156,170 Then, anthocyanoplasts and AVIs are covered by prevacuolar compartments (PVCs), i.e. endocytic multi-vesicle compartments involved in ER-Golgivacuole vesicle trafficking, and subsequently imported into the central vacuole.171 In addition, a direct trans-Golgi networkindependent trafficking pathway from the ER to PVCs exists that enables the transport of anthocyanins by the interaction of protein storage vacuoles (PSVs) and anthocyanin-containing vesicle-like structures.167 Membrane transporter-mediated transport of anthocyanins can happen in two ways: (i) primary transport mediated by multidrug resistance-associated protein (MRP)-type ABC transporters172,173 and (ii) proton gradient-dependent secondary transport that is mainly driven by V-ATPase and vacuolar H+pyrophosphatase.167 Furthermore, vacuole-localized multidrug and toxic extrusion (MATE) transporters have been identied as a transporter involved in avonoid/H+ exchange.174 In M. truncatula two MATE-type transporters, MATE1 and MATE2, have been identied as H+-gradient-dependent transporters of PAs and anthocyanin/avonols, respectively.158,175 MATE1 was shown to transport epicatechin 30 -O-glucoside and its expression is conned to the seed coat of M. truncatula. It is an ortholog of Arabidopsis Transparent Testa (TT12) and can complement the seed PA deciency phenotype of the tt12 mutation in Arabidopsis.158 MATE2 is produced primarily in leaves and owers and is involved in vacuolar sequestration of anthocyanins and other avonoids in owers and leaves. It has a higher transport capacity for anthocyanins than for other avonoid glycosides and in spite of its high similarity to MATE1 it cannot effectively transport PA precursors.175 Instead, MATE2 transports glycosylated and malonyl-glycosylated avonoids.175 Malonylation increases both the affinity and transport efficiency of MATE2 for glycosylated avonoid compounds.175 Coregulation of MATE2 with UGT78G1 and MaT4 by LAP1 in M. truncatula152,153 further supports the role of MATE2 in the transport of malonylated anthocyanin glycosides into the vacuole.175 Glutathione S-transferases (GSTs) are involved in both vesicle trafficking and membrane transporter-mediated transport of avonoids. Although their exact role in avonoid This journal is © The Royal Society of Chemistry 2014
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transport has not been determined yet, it has been suggested that they can bind to PAs, anthocyanins or avonols to form a GST-anthocyanin or GST-avonol complex, protecting them from oxidation and/or guiding them to the central vacuole.167,176
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3.4. Biological function of Medicago avonoids in planta Phenolic compounds were already present in the most early plant lineages and contributed to the successful evolutionary adaptation of plants from an aquatic environment to the land. Phenolics have acquired miscellaneous and sometimes crucial functions in plants. In particular in legumes, such as Medicago, avonoids perform many functions (Fig. 3).99,113 Flavonoids, especially avonols, play an important role as energy-escape valves, UV screens, and antioxidants in protecting the plant against abiotic stresses, e.g. oxidative damage associated with exposure to highly energetic, short wavelength solar light. Furthermore, avonoids, and especially anthocyanins, are involved in reproduction and seed dispersal via ower and fruit pigmentation, which attracts pollinators and herbivores. The above-mentioned roles are not truly specic for Medicago species though, and will not be reviewed here. We will rather focus on other roles, in nutrient acquisition, development, allelopathy, and response to biotic stress, on which specic studies on Medicago avonoids have been reported. 3.4.1. Nutrient acquisition. Flavonoids can affect plant nutrient acquisition through chemical changes of the soil, either via direct or indirect contributions to the availability of nitrogen (N), phosphorous (P) and iron (Fe).177 Conversely, N, P, and Fe supply in the soil can affect avonoid biosynthesis.178,179 It has been shown that avonoid exudation from the host is Pregulated and that avonoid accumulation is affected upon Nlimitation in nodulating legumes.99 Since avonoids can act as metal chelators, a putative function in the chemical mobilization of scarcely soluble soil Pforms has been postulated.177 For example, an isoavonoid identied in root exudates of M. sativa was able to dissolve ferric phosphate, thus making both phosphate and Fe available to the plant.180 Flavonoids, including the isoavone genistein and the avonols quercetin and kaempferol, can alter Fe availability by reducing Fe(III) to Fe(II) and by chelating otherwise unavailable iron into iron oxides and/or (poorly soluble) iron minerals.177 Although the role of avonoids in nutrient acquisition has been established already longtime, a lot of questions remain. Indeed, detailed information on the dynamic composition, concentrations, microbial modications, and persistence of the avonoid proles released in the rhizosphere, is oen lacking or contradicting.181 3.4.2. Benecial plant-microbe interactions. Flavonoids also serve an ‘indirect’, signaling-based role in enhancing the uptake of two of the most important macronutrients, N and P, by stimulating benecial plant-microbe symbioses, via the processes of nodulation and arbuscular mycorrhization (AM), respectively. Nodulation is a symbiosis that occurs predominantly between legumes and Gram-negative a/b-proteobacteria, collectively referred to as rhizobia. In this intimate interaction,
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bacteria x atmospheric nitrogen for the plant in exchange for organic sugars. The bidirectional communication during nodulation involves two main groups of molecules: the plant produces nodulation (nod) gene-inducing avonoids, and in response the bacteria secrete mitogenic lipo-chitooligosaccharide (LCO) Nod factors (NFs) that trigger early plant responses.1,177,182 About 30 nod gene-inducing avonoids have been isolated from nine legume genera. One of the rst avonoids discovered to possess nod gene-inducing activities was luteolin, isolated from M. sativa.177,182,183 In general, avones were shown to be the most potent inducers of nod genes in Sinorhizobium meliloti, which colonizes M. truncatula184 and hydroxylation at the C-4 and the C-7 positions is important for this activity.185,186 Mixtures can be more effective than single compounds though, and some avonoids act as inducers for certain rhizobia species and as anti-inducers for others.178,182 Previously, it was shown that some isoavonoids from M. sativa, such as medicarpin and coumestrol, even repress NF production.187 Besides their nod gene-inducing activity, most of these avonoids, especially luteolin and apigenin, also act as chemoattractants, thereby concentrating compatible rhizobia at the root surface.99,182 Flavonoid-decient transgenic M. truncatula roots, in which CHS was silenced, exhibited a near complete loss of nodulation, whereas avone-depleted roots had reduced nodulation and isoavone-decient roots nodulated normally.113,184 These differences could point to an important role of avonols, in particular kaempferol, during nodulation, more particular to cause an inhibition in auxin transport, resulting in local auxin accumulation, which precedes the initiation of cell division and is thus needed for correct nodule primordium development.113,186 Also the M. sativa isoavone formononetin and its 7-O-glucoside ononin were shown to be involved in nodulation, since they were able to counteract the autoregulation of nodulation, the process in which more extensive nodulation is suppressed when the plant is already fully engaged.188 The other important type of plant-microbe mutualism is the process of AM, which occurs between almost all land plants and fungi of the phylum Glomeromycota. In AM, the fungal partner extends the underground root system of the host, thereby greatly enhancing the uptake of water and nutrients, such as P and N, whereas the plant invests up to 20% of its xed carbon into the fungus.189 Some of the host plant exudates that stimulate processes promoting AM, e.g. spore germination, hyphal growth, hyphal branching in the soil, root colonization, and infection, have been identied as avonoids.178 In studies with M. sativa, roots began to accumulate avonoids prior to the colonization by Glomus intraradix, indicating elicitation by an AM fungi-derived signal.190 It would be interesting to test whether this signal corresponds to the recently identied mycorrhizal derived lipochitooligosaccharides (LCOs), termed myc factors.191 In contrast to non-colonized roots, avonoid proles changed over time in M. sativa during colonization with G. mosseae and in M. truncatula colonized with G. versiforme.192 However, contradictory effects have been reported based on the type of avonoid molecule, the nutrient status, the developmental stage, the
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Fig. 3 The different in planta biological functions of the specific flavonoid classes, with representative compounds, are depicted on a Medicago arabica plant.
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degree of colonization, and the studied target organism.181 Hyperoside, the 3-O-galactoside of quercetin, is the dominant avone released by M. sativa seeds and stimulates spore germination in G. etunicatum and G. macrocarpum, whereas other avones were only active in the former, and the isoavone formononetin even inhibited spore germination in both.193 In addition, when adding formononetin and ononin to already colonized roots of M. sativa, only ononin was able to further stimulate root colonization.188 Isoavonoids have also been shown to promote AM. Coumestrol was identied as an active stimulator of hyphal growth in M. truncatula, and a coumestrol hyperaccumulating M. truncatula mutant was found to be hyperinfected by its mycorrhizal symbiont.194 In addition, AM of M. truncatula and M. sativa by G. versiforme was found to cause a transient increase in medicarpin levels.195 3.4.3. Defense. Of course, not all plant microbeinteractions are benecial and avonoids also play an important role in the defense against a wide range of pathogens, ranging from bacteria and fungi to insects and nematodes.196,197 Isoavonoids, such as daidzein, glycitein and formononetin glycosides, of which the aglycones restrict the growth of microbial pathogens, accumulate constitutively and, hence, behave as phytoanticipins.192 Isoavonoids represent a major class of phytoalexins in legume plant species. Gene expression proling has shown an elevation in the expression of avonoid biosynthesis genes and/ or higher metabolite levels in M. truncatula plants challenged by Phymatotrichopsis omnivora, the causative agent of root rot in alfalfa or infected with the necrotrophic pathogen Phoma medicaginis.196,198 Furthermore, accumulation of coumestrol correlated nicely with the level of disease aer P. medicaginis infection in several cultivars of annual Medicago species in the eld.199 Isoavonoid-derived pterocarpans, such as medicarpin, also have antimicrobial properties and are produced either constitutively or aer induction by pathogens or endogenous elicitors.192 Medicarpin from M. sativa protects the plant from the pathogenic fungus Rhizoctonia solani.104 Alfalfa seedlings challenged with the fungal pathogen Colletotrichum trifolii exhibited a defense response that was accompanied with an increased expression of avonoid biosynthesis genes and accumulation of medicarpin and sativan.200 The same was observed in a microarray analysis of M. truncatula infected with Erysiphe pisi, the causative agent of powdery mildew. Seven of the eleven medicarpin biosynthesis genes were strongly upregulated.201 Furthermore, the avanone naringenin was shown to interfere with the quorum sensing-controlled production of virulence factors from Pseudomonas aeruginosa PAO1.202 Besides isoavonoids and avanones, avonols, such as quercetin, also have strong antimicrobial properties.104 Biodegradation of avonoids is one mechanism by which ‘non-target’ bacteria may cope with the toxic concentrations of avonoids, however, many others have evolved an inducible resistance mechanism. For instance, Agrobacterium tumefaciens possesses an isoavonoid-inducible isoavonoid efflux pump which contributes signicantly to its colonization of M. sativa roots.203 Flavonoids are also known to be highly effective against insects and nematodes. Several insects are sensitive to
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avonoids and are deterred in feeding tests, since avonoids serve as anti-feedants, digestibility reducers, and toxins.197 Nematode-resistant cultivars of alfalfa contained increased amounts of isoavonoids. For instance, the phytoalexin medicarpin inhibited the motility of the nematode Pratylenchus penetrans in alfalfa,204 and accumulation of isoavonoids in response to infection with the stem nematode Ditylenchus dipsaci correlated with resistance and was even induced systemically.205 In vitro bioassays revealed that the avonols kaempferol, quercetin, and myricetin act as repellants for the root lesion nematode Radopholus similis and the root knot nematode Meloidogyne incognita, while the isoavones genistein and daidzein and the avone luteolin only acted on the former species.206 The former three avonols also inhibited the motility of M. incognita and kaempferol inhibited egg hatching of R. similis.206 3.4.4. Allelopathy. Besides their well-known role in plantmicrobe interactions, avonoids also play an important role as allelochemicals in plant-plant interactions. Allelopathy describes negative biochemical interactions between plants for the competition for natural resources, such as nutrients, light, and water.181 A few detailed studies have been performed on the direct inhibitory effects of avonoid exudates on the growth and development of neighboring plants in leguminous species. The isoavone formononetin and the pterocarpan medicarpin were found to inhibit the germination of Allium cepa.207 The avanone naringenin was found to have direct inhibitory effects on root growth of soybean (Glycine max) seedlings, likely due to premature lignication.208 Allelopathic interactions are not always caused by direct toxicity of the allelochemicals themselves, but can also be induced by biotic or abiotic structural modications caused by avonoids in the rhizosphere.181,209 Another phenomenon illustrating this is replant disease, also known as autotoxicity or soil sickness. It is an example of intraspecic allelopathy, where the plant produces allelochemicals when it starts decaying in the soil and these allelochemicals are detrimental to the establishment of new seedlings of the same species. In the case of alfalfa, it can reduce the development and productivity of the crop itself and cause permanent morphological reductions in root and shoot growth.99 Additionally, the observed damping-off of M. sativa seedlings by the fungal pathogens Pythium spp. and Rhizoctonia solani and causing the death or weakening if the seeds or seedlings before or aer they germinate, has been attributed to autotoxicity of undecomposed M. sativa plant residues.210 3.4.5. Developmental regulators. As mutants decient in the production of certain avonoids show a variety of developmental defects, ranging from lack of gravitropism,211 root looping, and aberrant root outgrowths212 to defects in whole plant growth,213 avonoids may serve as developmental regulators. Studies have shown that avonoids affect long-distance polar auxin transport (PAT) streams.214,215 For instance, RNAimediated silencing of avonoid biosynthesis in M. truncatula hairy roots led to increased auxin transport, indicating that avonoids act as auxin transport inhibitors.184,186 More specically, it has been shown that the isoavone formononetin, the avone dihydroxyavone, and the avonol kaempferol and not
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the chalcone isoliquiritigenin, nor the pterocarpan medicarpin were able to inhibit acropetal PAT in M. truncatula roots.216 Because they interfere with multiple aspects such as auxin transporters, kinases, and the trafficking machinery, avonoids are not considered to be specic regulators modulating auxin transport.214 Besides, CHS-silenced M. truncatula hairy roots, decient of avonoids, showed impaired nodulation, but no defect in lateral root formation.184,217 This suggests that avonoids are not required as general regulators of the organogenesis of secondary root organs. High sunlight induces the synthesis of both auxin and quercetin derivatives, and quercetin displays a great capacity for netuning auxin gradients as well as local auxin concentrations, which represent the actual determinants for different morphological responses.218 As a result, it was postulated that the highlight induced biosynthesis of antioxidant avonoids may have a role in regulating whole-plant and individual organ architecture.219 3.5. Pharmacological properties of Medicago avonoids Besides their important biological functions in plants, avonoids are signicant components of the human diet and have numerous pharmaceutical properties.220,221 In traditional medicine, many plant-derived infusions, balms, and spices, containing avonoids as active ingredients, have been used for centuries.222 M. sativa has long been used as traditional herbal medicine in many countries, such as China, India, and America.12,223 Nowadays, the amount of research on avonoids has tremendously increased, in part because they have been implicated in the prevention of numerous physiological disorders and diseases, within which chronic inammation is key to most. This potential resides in a number of biological activities, including antioxidant, anti-inammatory, anti-carcinogenic, and phyto-estrogenic abilities, though their interactions with intracellular signaling pathways and regulation of cell survival/ apoptotic genes and mitochondrial function. Although most of the studied bioactive, potentially valuable avonoids, such as coumestrol, daidzein, genistein, (iso)liquiritigenin, luteolin, quercetin, kaempferol, naringenin, apigenin, catechin, epicatechin, cyanidin, and delphinidin, can be encountered in Medicago, to our knowledge, none of these molecules are specic for Medicago species. Hence we will not discuss their pharmacological properties in detail but refer to excellent reviews published elsewhere.101,224–227 3.6. Determination of Medicago avonoids Many different techniques can be used to identify and/or quantify avonoids. An overview of the currently commonly used as well as new emerging separation and identication techniques has recently been published.228,229 These techniques range from simple bench methods to the use of sophisticated instrumentation, such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and laser-induced uorescence (LiF) detection, each having unique advantages and disadvantages.230 Coupling chromatography to MS offers an extra level of information to avonoid elucidation and is ideal for the analysis
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of complex plant tissue samples. Advances in sample extraction, chromatographic separation, detection and structural analysis of avonoids have dramatically inuenced the evolution of avonoid discovery and improved avonoid research in general.228 High-performance liquid chromatography (HPLC) coupled to UV detection (HPLC-UV) and to MS systems (HPLC-UV-MS) are well recognized methods to prole avonoid conjugates in plant tissue extracts. As such, various classes of avone, avonol, and isoavone glycoconjugates and aglycones have been identied in Medicago. However, these methods are insufficient to distinguish isomeric and isobaric compounds, which require tandem mass spectrometry with collision induced dissociation (CID MS/MS) for functional characterization. In one study, an integrated approach utilizing HPLC-UV coupled to electrospray ionization (ESI)-MS and gas chromatography (GC)-MS was used to elucidate the avonoid proles from M. truncatula root and cell cultures. A quadrupole time-of-ight (QTOF) MS device was used for all structural identications, but where the stereochemistry of sugar conjugates was uncertain, enzymatic hydrolysis, followed by GCMS to assign a correct sugar stereochemical conguration, was used.231 The same group also used reversed-phase highperformance liquid chromatography coupled to UV photodiode array detection and electrospray ionization ion-trap mass spectrometry (HPLC-PDA-ESI-ITMS) to analyze the intra- and extracellular phenylpropanoid and isoavonoid metabolome of M. truncatula cell cultures in response to YE or MeJA.115 More recently, this technique was validated in the same M. truncatula system to identify the biosynthetic mechanism for the aurone hispidol.136 With a more or less similar methodology, HPLC-UVMS was used in two studies to discover the changes of avonoid accumulation in M. truncatula leaves infected with P. medicaginis.162,196 In another comparable study, both a low resolution ion trap (IT) and a high resolution tandem QTOF LC-UV-MS system was used to evaluate the fragmentation pathways of M. truncatula avonoids aer CID experiments. It was concluded that although some decent fragmentations of minor compounds could be obtained using the low resolution IT, this technology does not always allow proper identication of all molecules in the sample. For that reason and because of the presence of equal nominal masses of some different substituents, a high resolution instrument could only be used to correctly analyze these derivatives.232 In addition, the same high resolution methodology was used to compare the avonoid proles from M. truncatula seedling roots, hairy roots, and suspension root cell cultures.233 In a more recent study, a new stop-and-go two-dimensional chromatography was used for the preparative separation of avonoids from M. sativa, combining counter-current chromatography and liquid chromatography (2D CCC LC). Moreover, two new avonoids were identied coupling this technique to ESI-MS, ESITOF-MS, and 1D and 2D NMR.234
4 The ‘omics’ toolkit for Medicago research Over the past two decades, M. truncatula has been established as a model system. Initially, it was chosen for its small diploid
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genome, short generation time, self-fertility and relative ease of transformation and regeneration. Since M. truncatula is also closely related to important forage and food crops, it has additional value as a model species for doing translational research.13,14,133 Furthermore, this model species also has the ability to engage in several plant-microbe interactions, both mutualistic and detrimental in nature, resulting in the evolution of a rich variety of natural product pathways.235 Due to a tremendous effort of the scientic Medicago community, numerous genetic, functional genomic, and molecular tools and resources have become available, making over the years signicant contributions to the elucidation and characterization of the secondary metabolism of Medicago species in particular, and legumes in general. Substantial efforts undertaken by the Medicago Genome Initiative that started over a decade ago, culminated in the publication of the rst dra genome in 2011.236 However, since relevant gene info was clearly still missing,237 e.g. several of the known (published) saponin biosynthesis genes are missing from the v3.5 version, a curated version (v4.0) has now been released. In addition, there are almost 270,000 expressed sequence tags (ESTs) deposited in Genbank (September 2013) for M. truncatula and more than 11,000 for M. sativa. Another important pillar of the Medicago’s functional genomic toolkit is the Medicago Gene Expression Atlas (MtGEA; http://mtgea.noble.org/v3/), which provides quantitative gene expression data for most M. truncatula genes in all major organs during plant development and in response to multiple environmental stimuli.238,239 It currently contains expression data from 670 Affymetrix GeneChip Medicago Genome Arrays (September 2012), each covering 50,900 probe sets. Besides evoking the ‘guilt by association’ principle, in which tightly coexpressed genes are likely involved in similar processes, some specic conditions, e.g. MeJA or YE elicitation, might allow for identication of specic proteins involved in Medicago secondary metabolism, including TFs or other regulatory proteins, transporters, and enzymes.49 Mining of the MtGEA allowed for instance to pinpoint HMGR as the putative target for the RING E3 Ubiquitin ligase MKB1.54 Improved functional genomic technologies such as transcriptomics, proteomics, and metabolomics provide opportunities for an in-depth understanding of secondary metabolism. For example, an integrated functional genomics approach was used to study natural product biosynthesis in M. truncatula and led to the reconstruction of the metabolic map of all pathways in MedicCyc, the rst legume pathway database for M. truncatula (http://www.noble.org/MedicCyc/).41,51,52,160,240,241 Of course, gene function discovery at a large scale cannot be accomplished without the availability of complementary substantial mutant collections to bridge the gap between genotype and phenotype. The generation of these Medicago collections has been pursued using different strategies, i.e. (i) chemical: ethyl methanesulphonate (EMS)242–245 for single-base substitutions; (ii) physical: fast neutron bombardment (FNB)246 and g-ray247 for deletion of DNA fragments of variable length; and (iii) biological: transferred DNA (T-DNA),248 transposons,249,250 the tobacco Tnt1 retrotransposon243,251–254 or the
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endogenous MERE1 retrotransposon from M. truncatula255 for insertional mutagenesis. To investigate these mutant collections, both reverse and forward genetic approaches have been applied. Traditionally, map-based cloning was the method of choice in forward genetic screens, which required the identication of closely linked molecular markers to initiate chromosome walking and sequencing to identify the mutant locus.253 This tedious and difficult strategy was replaced by the targeting-induced local lesions in genomes (TILLING) approach, which is a PCR-based reverse genetics method that can detect mutations ranging from single nucleotide changes to deletions of several kilobases; the latter being a modied strategy termed deletion TILLING (De-TILLING).246 TILLING involves the detection of mismatches in heteroduplexes that are formed by the annealing between mutant and wild-type alleles for a target locus, using pooled DNA from a mutant collection.256 However, for Tnt1 mutant populations an alternative reverse genetic strategy can be used, because they are generated by a tractable genetic system in which the DNA sequence used to mutate the genome can also be used to identify the mutation. For this purpose a Tnt1 anking sequence tag (FST) database was generated by the Samuel Roberts Noble Foundation (http://Medicago-mutant. noble.org/mutant/index.php), which currently contains 72,159 FSTs (August, 2013).254 With the availability of all these screening tools, Tnt1 mutant populations have already proven to be extremely useful in screens for the discovery of regulators, transporters, and enzymes involved in avonoid production.146,164,165,175
5 Metabolic engineering Targeted metabolic engineering in Medicago species has mainly been focusing on the production of phenolic compounds such as avonoids and lignin. To the best of our knowledge, targeted metabolic engineering for triterpene saponin production has not been reported yet. 5.1. Engineering of avonoid biosynthesis in Medicago Because of the various functions of avonoids in plants, and their potential nutritional and pharmacological utility, engineering the plant’s ability to biosynthesize these bioactive natural compounds, either in the legumes where they are endogenously produced or heterologously in non-leguminous plants, such as Arabidopsis, tobacco, corn and tomato, or in microbial systems, has become a densely covered research area. Besides creating sustainable production sources for pure avonoids, legume avonoid engineering may encompass other goals, such as the improvement of the nutritional value (of e.g. soybean), or the modulation of plant-microbe interactions.159 Basic strategies that can be applied for the genetic modulation of avonoid biosynthesis in plants may involve: (i) increasing endogenous avonoid levels through the upregulation of structural or regulatory genes; (ii) overexpression of heterologous structural and regulatory genes that are not present in the gene pool of the target plant to open the
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endogenous Medicago pathways to new, non-Medicago metabolites; and (iii) blocking of specic steps in the avonoid biosynthetic pathway by RNA interference strategies to steer the metabolic ux toward the desired end-products.127,143 Effects of heterologous overexpression of Arabidopsis or maize genes that encode regulatory TFs of avonoid synthesis on accumulation of anthocyanins have already been investigated.255–259 The expression of the maize bHLH B-Peru and the MYB C1 (colourless) TF genes failed to increase anthocyanin levels in alfalfa, whereas transgenic alfalfa populations expressing the maize bHLH TF Lc (leaf color) gene showed a slight increase in anthocyanin production when the plants were exposed to abiotic stress.260 Similar to its maize C1 counterpart, overexpression of the Arabidopsis MYB TF Production of Anthocyanin Pigment 1 (PAP1) was not able to stimulate anthocyanin biosynthesis in M. truncatula or alfalfa. In contrast, overexpression of the M. truncatula C1/PAP1 homolog LAP1, increased anthocyanin production in transgenic M. truncatula, alfalfa, or white clover when ectopically overexpressed. Hence, LAP1 can be used as a tool for anthocyanin engineering in legumes.153 Ectopic expression of Arabidopsis TT2, a MYB factor involved in the regulation of PA biosynthesis, in M. trunculata hairy roots caused a massive accumulation of PAs as a result of the activation of genes involved in PA synthesis, transport, and oligomerization.144 Heterologous overexpression of TaMYB14, which encodes a MYB from Trifolium arvense, resulted in the synthesis and accumulation of PAs in M. sativa leaves.261 Likewise, the ectopic expression of the M. truncatula MYB TF MtPAR increased the expression of PA/anthocyanin biosynthesis and the accumulation of PAs in M. truncatula hairy roots.165 Overexpression of MtPAR also resulted in a higher accumulation of PAs in alfalfa shoots.165 In an attempt to create crops with improved diseaseresistance, constitutive overexpression of an isoavonoid biosynthesis gene, i.e. isoavone O-methyltransferase, in transgenic alfalfa caused a more rapid and increased production of medicarpin following infection by P. medicaginis, which ameliorated disease symptoms.262 Overexpression of another biosynthesis gene, IFS, from M. truncatula in alfalfa corroborated these results, with an additional accumulation of the isoavones formononetin and daidzein aer P. medicaginis infection.159 The expression of M. truncatula IFS1 in alfalfa also resulted in the accumulation of genistein glycosides in transgenic plants.159,263,264 Medicago genes were also used as tools for avonoid engineering in other plant species. Co-expression of MtBAN, encoding ANR, and Arabidopsis PAP1 in tobacco resulted in a signicant decrease in anthocyanin levels but a higher content of PA precursors.265 Overexpression of MtDFR1 in tobacco induced changes in ower anthocyanin proles, while overexpression of MtDFR2 did not induce such changes.140 In another study, an articial bifunctional enzyme was engineered, containing CHI from alfalfa and IFS from soybean that, upon overexpression, increased isoavone levels in transgenic tobacco as compared to plants transformed with IFS alone.266 Engineering micro-organisms by tweaking metabolic uxes and introducing novel plant-derived biosynthetic enzymes for a
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sustainable production of avonoids is also actively being pursued and has been reviewed elsewhere.267 Here we will only mention a few engineering studies in which Medicago genes were used. An Escherichia coli cell factory was developed to produce 7-O-methyl aromadendrin, a medicinally valuable avonoid, by introducing structural genes from several plants, among which CHI from M. sativa.268 A codon-optimized version of the same gene was also introduced in Streptomyces venezuelae to produce the avanones naringenin and pinocembrin.269 Finally, the production of 5-deoxyavanones in Saccharomyces cerevisiae was made possible by introducing CHS, CHI, and CHR from M. sativa.270 5.2. Engineering of lignin biosynthesis in Medicago Lignin is a complex biopolymer that is encountered in plant secondary cell walls and that plays crucial roles in mechanical support of the cell wall and the plant as a whole, in water conductance in plant stems and in defense against pathogens. The lignin polymer is tightly woven with cellulose and hemicellulose polymers in the lignocellulose matrix that provides strength to the plant cell wall. Lignin is the main hurdle in the processing of plant lignocellulosic biomass for liquid biofuels and negatively inuences the digestibility of forages.271–273 As the reduction of lignin content can improve the digestibility of forages,272 considerable efforts are undertaken to engineer lignin biosynthesis in the important forage crop alfalfa. Downregulation of nearly each of the monolignol biosynthesis genes leads to lower lignin concentrations and altered lignin composition (altered S/G ratio) in transgenic alfalfa,274–279 resulting in increased digestibility,274,277–280 especially when genes early in the pathway are silenced.272,281 The increased digestibility of the transgenic plant material is solely due to a decreased lignin content, since an altered S/G ratio does not seem to inuence digestibility.272,278,281 Though benecial for digestibility, large reductions in lignin content have always been accompanied with a signicant yield penalty.272,277–279,281–283 Dwarng of the engineered plants could be attributed to impaired water transport because of distorted xylem vessels279,283 or a constitutively activated defense response.284 Indeed, studies in Arabidopsis have shown that reduced lignin levels led to increased levels of the stress hormone salicylic acid (SA) but that the stunted growth phenotype could be reversed by keeping the SA levels low in the lignin-silenced Arabidopsis plants.285 This indicates that it should be feasible to engineer alfalfa plants with reduced lignin levels showing no yield penalty.
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Outlook
The occurrence of a wide range of natural products in the Medicago genus supports the intrinsic potential of this species as a platform for the development and sustainable production of (novel) bioactive compounds. Taking into account the benecial effects of Medicago secondary metabolites for the agricultural industry or for animal and human health, Medicago species seem to hold great capacity. Therefore, continued
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exploration of the elements involved in all aspects of secondary metabolite production in Medicago plants will be required to provide the necessary basis for the establishment of such a platform. The information currently available through studies focusing on the biology, genomics, biochemistry and metabolic engineering of Medicago species shows steady progress, in particular in the past decade and largely thanks to the substantial efforts of the Medicago Genome Initiative. Discovery of the genes corresponding to the structural pathway elements, i.e. the enzymes catalyzing the metabolic reactions, has greatly beneted from the booming number of sequence data that currently are available for Medicago and other plant species. Nonetheless, numerous molecular details remain largely unresolved, in particular with regard to the regulation and management of secondary metabolite synthesis, either during development or in response to environmental cues. The booming genomics, transcriptomics, proteomics, and metabolomics technologies have certainly made exploration of plant natural product biosynthesis pathways more accessible. The current advances in the omics area and the current strategies for gene discovery will undoubtedly continue to have a huge impact and to allow capturing some of the transcriptional regulators of Medicago natural product biosynthesis, which ultimately might be useful as master switches in plant metabolic engineering programs. However, identication of all structural elements, as well as all control mechanisms of bioactive natural production in the Medicago genus, may be mandatory to secure a sustainable, reliable and large-scale production of its compounds, whether it is through modulating the metabolic network of the Medicago species itself or through synthetic biology programs in heterologous hosts. As was and is the case for the enzyme-encoding genes, a substantial part of this information might be obtained by mining the available and ever-increasing number of gene sequences by current approaches such as looking for coexpression patterns in transcriptomes. This almost ‘classical’ strategy has proven its utility in practically every plant species studied for a particular metabolite, including Medicago species.28 More recently, another promising trend emerged, i.e. looking for physical metabolic gene clusters in plant genomes, an approach originally supposed to be applicable only in microbes where physical gene clustering is the rule rather than the exception. However, metabolic gene clusters are being discovered in an increasing number of plants,136,286 including e.g. a triterpene gene cluster in the legume Lotus japonicus.287 New or superior omics tools will need to be employed or developed though, to reach the demanded level of knowledge. Transcriptome analysis may benet from an increased spatiotemporal resolution, for instance allowing high-throughput single-cell proling, to address an important bottleneck in the study of plant secondary metabolism; the fact that it oen occurs in specic tissues or organs only consisting of a small number of specialized cells. Transcriptome or genome mining will also need increased computing power, in rst instance to allow building metabolic networks, either from genomes, such as MedicCyc, or transcriptomes, such as CathaCyc for Catharanthus roseus,288 and in second instance to create and improve
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databases, such as the Plant Metabolic Network (http:// www.plantcyc.org/), within which the respective networks of the different species can be compared. This will provide evolutionary information that might allow prioritizing genes for functional analysis. Omics should also go beyond the genome and transcriptome. The proteome and metabolome are the obvious next levels but crucial information is denitely hidden in the interface between these ‘omes’, i.e. at the interactome, for instance between DNA and proteins, such as transcription factors, or between different proteins, such as in transcription factor complexes or channels of metabolic enzymes (the so-called metabolons). Methods to study such interactomes gain performance and will gradually prove their value for plant metabolism as well.49,289–291 Another, yet virtually unexplored level of interactions is that between metabolites and proteins. Considering that metabolites comprise a large and important fraction of the cellular molecules, occur in an enormous range of physiological concentrations, and participate in a wide variety of biochemical and regulatory functions, for instance as ligands for receptors, and as substrates, products, cofactors, or allosteric regulators of enzymes, studying this interactome should deserve more attention. In this regard, a pioneering study in yeast, in which an assay for the large-scale identication of in vivo proteinmetabolite interactions was designed, provides sufficient proof-of-concept, since it allowed revealing numerous new metabolite interactions, both with enzymes from the sterol pathway as with regulatory proteins such as protein kinases.292 Finally, omics won’t be able to solve it all. One should keep in mind that only for a few 100 of the more than 5,000 metabolites that each plant species can synthesize, the structure has been unambiguously identied. ‘Classical’ metabolite purication and identication should therefore not be neglected in future research programs, because sooner or later any omics adept will face a metabolite of interest that is key to the studied network, but with a yet unknown structure.
7
Acknowledgements
We thank Annick Bleys for help in preparing the manuscript. N.D.G. and A.G. are indebted to the Agency for Innovation by Science and Technology (IWT) and the Iranian Ministry for Health and Medical Education, respectively, for predoctoral fellowships. J.P. is a postdoctoral fellow of the Research Foundation Flanders (FWO).
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270 Y. Yan, L. Huang and M. A. Koffas, Biotechnol J, 2007, 2, 1250–1262. 271 N. D. Bonawitz and C. Chapple, Annu. Rev. Genet., 2010, 44, 337–363. 272 H. J. Jung, D. A. Samac and G. Sarath, Plant Sci., 2012, 185– 186, 65–77. 273 X. Li, J. K. Weng and C. Chapple, The Plant Journal, 2008, 54, 569–581. 274 M. Baucher, M. A. Bernard-Vailh´ e, B. Chabbert, J. M. Besle, C. Opsomer, M. Van Montagu and J. Botterman, Plant Mol. Biol., 1999, 39, 437–447. 275 F. Chen, M. S. Srinivasa Reddy, S. Temple, L. Jackson, G. Shadle and R. A. Dixon, The Plant Journal, 2006, 48, 113–124. 276 D. Guo, F. Chen, K. Inoue, J. W. Blount and R. A. Dixon, The Plant Cell, 2001, 13, 73–88. 277 L. A. Jackson, G. L. Shadle, R. Zhou, J. Nakashima, F. Chen and R. A. Dixon, BioEnergy Res., 2008, 1, 180–192. 278 M. S. Reddy, F. Chen, G. Shadle, L. Jackson, H. Aljoe and R. A. Dixon, Proc. Natl. Acad. Sci. USA, 2005, 102, 16573– 16578. 279 G. Shadle, F. Chen, M. S. Srinivasa Reddy, L. Jackson, J. Nakashima and R. A. Dixon, Phytochemistry, 2007, 68, 1521–1529. 280 D. Guo, F. Chen, J. Wheeler, J. Winder, S. Selman, M. Peterson and R. A. Dixon, Transgenic Res., 2001, 10, 457–464. 281 F. Chen and R. A. Dixon, Nat. Biotechnol., 2007, 25, 759– 761. 282 N. D. Bonawitz and C. Chapple, Curr. Opin. Biotechnol., 2013, 24, 336–343. 283 J. Nakashima, F. Chen, L. Jackson, G. Shadle and R. A. Dixon, The New Phytologist, 2008, 179, 738–750. 284 L. Gallego-Giraldo, L. Escamilla-Trevino, L. A. Jackson and R. A. Dixon, Proc. Natl. Acad. Sci. USA, 2011, 108, 20814– 20819. 285 L. Gallego-Giraldo, Y. Jikumaru, Y. Kamiya, Y. Tang and R. A. Dixon, The New Phytologist, 2011, 190, 627–639. 286 H. Y. Chu, E. Wegel and A. Osbourn, The Plant journal: for cell and molecular biology, 2011, 66, 66–79. 287 A. Krokida, C. Delis, K. Geisler, C. Garagounis, D. Tsikou, L. M. Pena-Rodriguez, D. Katsarou, B. Field, A. E. Osbourn and K. K. Papadopoulou, New Phytol., 2013, 200, 675– 690. 288 A. Van Moerkercke, M. Fabris, J. Pollier, G. J. Baart, S. Rombauts, G. Hasnain, H. Rischer, J. Memelink, K. M. Oksman-Caldentey and A. Goossens, Plant & cell physiology, 2013, 54, 673–685. 289 J. E. Bassard, L. Richert, J. Geerinck, H. Renault, F. Duval, P. Ullmann, M. Schmitt, E. Meyer, J. Mutterer, W. Boerjan, G. De Jaeger, Y. Mely, A. Goossens and D. Werck-Reichhart, Plant Cell, 2012, 24, 4465–4482. 290 B. L. Møller, Science, 2010, 330, 1328–1329. 291 Q. Zhao and R. A. Dixon, Trends Plant Sci, 2011, 16, 227– 233. 292 X. Li, T. A. Gianoulis, K. Y. Yip, M. Gerstein and M. Snyder, Cell, 2010, 143, 639–650.
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Natural product biosynthesis in Medicago species Cite this: DOI: 10.1039/c3np70104b
Azra Gholami,†ab Nathan De Geyter,†ab Jacob Pollier,†ab Sofie Goormachtigab and Alain Goossens*ab
Covering: up to the end of 2013 The genus Medicago, a member of the legume (Fabaceae) family, comprises 87 species of flowering plants, including the forage crop M. sativa (alfalfa) and the model legume M. truncatula (barrel medic). Medicago species synthesize a variety of bioactive natural products that are used to engage into symbiotic interactions but also serve to deter pathogens and herbivores. For humans, these bioactive natural products often possess promising pharmaceutical properties. In this review, we focus on the two most interesting and well characterized secondary metabolite classes found in Medicago species, the triterpene saponins and the flavonoids, with a detailed overview of their biosynthesis, regulation, and profiling methods. Furthermore, their biological role within the plant as well as their potential utility for Received 26th September 2013 Accepted 15th January 2014
human health or other applications is discussed. Finally, we give an overview of the advances made in metabolic engineering in Medicago species and how the development of novel molecular and omics toolkits can influence a better understanding of this genus in terms of specialized metabolism and
DOI: 10.1039/c3np70104b
chemistry. Throughout, we critically analyze the current bottlenecks and speculate on future directions
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and opportunities for research and exploitation of Medicago metabolism.
1 2 2.1. 2.2. 2.3. 2.4. 2.5. 3 3.1. 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.1.7. 3.1.8. 3.2. 3.3. 3.4.
Introduction Saponins Saponin biosynthesis in M. truncatula Regulation of saponin biosynthesis Biological role of Medicago saponins Commercial potential of Medicago saponins Determination of Medicago saponins Flavonoids Flavonoid biosynthesis in M. truncatula Early steps of avonoid biosynthesis Isoavonoid biosynthesis Flavonol biosynthesis Flavone biosynthesis Aurone biosynthesis Anthocyanin biosynthesis Proanthocyanidin biosynthesis Structural diversication and decoration of avonoids Regulation of avonoid biosynthesis Flavonoid localization Biological function of Medicago avonoids in planta
a
Department of Plant Systems Biology, VIB, Ghent University, Technologiepark 927, B-9052 Gent, Belgium. E-mail: [email protected]; Fax: +32 9 3313809; Tel: +32 9 3313800
b
Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, Belgium † These authors contributed equally to this work
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3.4.1. 3.4.2. 3.4.3. 3.4.4. 3.4.5. 3.5. 3.6. 4 5 5.1. 5.2. 6 7 8
1
Nutrient acquisition Benecial plant-microbe interactions Defense Allelopathy Developmental regulators Pharmacological properties of Medicago avonoids Determination of Medicago avonoids The ‘omics’ toolkit for Medicago research Metabolic engineering Engineering of avonoid biosynthesis in Medicago Engineering of lignin biosynthesis in Medicago Outlook Acknowledgements References
Introduction
Legumes (Fabaceae) constitute a highly diverse plant family that encompasses economically important crops, such as pea and soybean, and that provides about one-third of humankind’s protein intake, fodder and forage for livestock, and raw materials for industries. Legumes are particular among cultivated plants because of their ability to establish symbiotic interactions such as with nitrogen-xing bacteria.1–3 In addition, leguminous plant species are rich sources of health promoting phytochemicals. In particular, the legume subfamily of
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Papilionoideae contains the predominant producers of the very benecial isoavonoids.4–8 Azra Gholami (born 1981) received her Pharm. D degree in Pharmacy from Shiraz University of Medical Sciences and Health Services (Iran, 2006). Aerwards she worked as a researcher in the department of pharmacognosy and medicinal plants of Shiraz University of Medical Science and Health services for two years. In 2009 she started as a predoctoral researcher in the lab of Alain Goossens (VIB-Ghent University, Belgium) to obtain her PhD degree in 2013. Her PhD research focused on the identication of potential regulators of jasmonatemodulated secondary metabolism in Medicago truncatula. Nathan De Geyter (born 1986) graduated as a Master in Sciences, Biology at Ghent University in 2009. In 2010, he started his PhD research in the lab of Alain Goossens and Soe Goormachtig aer he obtained a scholarship from the Agency for Innovation by Science and Technology in Flanders. His work focuses on the Tandem Affinity Purication (TAP) technology in the model legume Medicago truncatula. Using the TAP technique one can identify all unknown protein interactors from a known ‘bait’ protein. The baits that are further investigated are involved in MeJA-dependent regulation of secondary metabolism in M. truncatula. Jacob Pollier (born 1980) obtained his PhD in Biotechnology under the guidance of Alain Goossens at Ghent University (Belgium, 2011). His PhD research focused on the development of a combinatorial biosynthesis platform in plants using triterpene saponins as target compounds. Presently he works as a postdoctoral fellow of the Research Foundation-Flanders in the lab of Alain Goossens. His main research interests are jasmonate signaling and the regulation of triterpene saponin biosynthesis in Medicago truncatula, gene discovery in plant specialized metabolism and combinatorial biosynthesis of triterpenoids in plants and engineered yeast.
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The genus Medicago belongs to this legume subfamily and comprises 87 species of owering plants, including the widely cultivated crop species M. sativa (alfalfa or lucerne).9–11 Alfalfa is the fourth economically most valuable crop in North America (aer corn, soybean, and wheat, respectively) and the temperate world’s most important forage crop. It is a valuable feed source for livestock and alfalfa sprouts are eaten by humans.12 Besides alfalfa, several other Medicago species are used as medicine, human food, green manure, and source of industrial enzymes in biotechnology.10,13 Alfalfa is an obligate outcrossing and tetraploid species, which makes genetic and genomic studies difficult. Therefore, the closely related M. truncatula (barrel medic) was developed as a model legume.14,15 M. truncatula is an annual, diploid and autogamous legume with a moderate genome size (500–550
Soe Goormachtig (born 1969) obtained her PhD in Plant Biotechnology in Marc Van Montagu’s lab at Ghent University (Belgium 1997), studying the symbiotic interaction between the tropical legume Sesbania rostrata and its microsymbiont Azorhizobium caulinodans. Aer a post-doc in the Lab of Prof. Ingo Potrykus (ETH Z¨ urich, Switzerland), she returned to Ghent University where she was appointed as a Professor in functional biology in 2005. She is now also appointed as principal investigator within the VIB Department of Plant Systems Biology at Ghent University. Her research focusses on rhizosphere interactions more specically on metabolites that inuence root organogenesis as well as nodule development in Medicago truncatula. Alain Goossens (born 1971) obtained his PhD in Plant Biotechnology in Marc Van Montagu’s lab at Ghent University (Belgium, 1998), studying plant seed storage protein synthesis. Subsequently, he performed postdoctoral studies at the IBMCP in Valencia (Spain) with Ram´ on Serrano, working on yeast salt tolerance. He returned to Ghent, where he was appointed as a Principle Investigator within the VIB Department of Plant Systems Biology at Ghent University (2003). His research program focuses on jasmonate signaling, gene discovery in plant metabolism and synthetic biology. He has been appointed as a Professor at Ghent University (2010) and is teaching ‘Metabolic Engineering’.
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Review
Mbp). The availability of a wide range of genomic and genetic resources makes it an invaluable model for studying legume secondary metabolism at the molecular genetic level.15 Among the secondary metabolite classes produced by Medicago species, the triterpene saponins and isoavonoids are of the highest interest and have been explored more thoroughly than the others.4 Lignins are other phenolic compounds that are found in all higher plants and are an important factor affecting cell wall digestibility in forage legumes, including alfalfa.4 In addition, the occurrence of alkaloids like stachydrine and trigonelline,16 cyanogenic glycosides (cyanogens) and nonprotein amino acids (NPAAS) such as canavanine17 have also been reported in Medicago species. This review provides a comprehensive overview of the structural variability, biosynthesis, regulation, physiological role in planta and biological activity of the triterpene saponins and the avonoids found in Medicago. For the other classes of Medicago specialized metabolites, we refer to previous reviews on Medicago and plant metabolism.4,17 Finally, the advances made in metabolic engineering of Medicago species are discussed, and the growing molecular and omics toolkits available for the Medicago community are highlighted.
2 Saponins Saponins are a structurally diverse class of amphipathic glycosides with a lipophilic steroid, steroidal alkaloid, or triterpenoid aglycone backbone (also called sapogenin) that is covalently linked to one (monodesmosidic) or more (di- or tridesmosidic) hydrophilic sugar chains via a glycosidic bond. The name saponin is derived from the Latin word for soap, sapo, and points to an important physicochemical property of the compounds, i.e. their ability to form a colloidal solution in water that forms a stable foam when shaken. This makes them useful as emulsiers and foaming agents in food and beverage industries. Saponins also possess various pharmacological properties, making them commonly used in phytotherapy and cosmetics.18–20 The saponins that are present in the various Medicago species all have pentacyclic oleanane-type sapogenins that are characterized by the presence of a C-12–C-13 double bond, and an oxygen atom at the 3b-position.21 The sapogenins are derived from b-amyrin through various oxidative modications, and based on the oxidation pattern, two distinct types of aglycones can be distinguished in Medicago (Fig. 1). A rst class consists of aglycones that are oxidized at the C-28 position, which is frequently accompanied with oxidation at the C-23 position. Aglycones of this class in Medicago are oleanolic acid, queretaroic acid, hederagenin, caulophyllogenin, bayogenin, medicagenic acid, and zanhic acid (Fig. 1). A second class of sapogenins possesses a hydroxy group at the C-24 position, which excludes oxidation at the C-28 position.21 In Medicago, sapogenins of this class include the soyasapogenols A, B, and E (Fig. 1). A comprehensive overview of the various sapogenins and saponins present in the genus Medicago can be found elsewhere.21,22
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2.1. Saponin biosynthesis in M. truncatula The triterpene saponins present in Medicago share a common biogenic origin with the sterols. 2,3-oxidosqualene, the last common precursor molecule between the primary sterol metabolism and the secondary triterpenoid metabolism, is synthesized in the cytosol from isopentenyl pyrophosphate (IPP) derived from the mevalonate (MVA) pathway.23–26 In M. truncatula, ve isoforms of the key MVA pathway enzyme HMGR have been characterized.27 For the remaining steps of the MVA pathway, candidate genes were identied via a functional genomics approach.28 The end-product of the MVA pathway, IPP, is isomerised by IPP isomerase (IPPI) to yield the allylic isomer dimethylallyl pyrophosphate (DMAPP).29 Subsequently, the prenyl transferase farnesyl pyrophosphate synthase (FPS) catalyzes the sequential condensation reactions of DMAPP with two units of IPP to form farnesyl pyrophosphate (FPP).30 Neither IPPI nor FPS have been functionally characterized in M. truncatula, but candidate genes can be found in the genome.28 In the next step of the biosynthetic pathway leading to 2,3-oxidosqualene, two molecules of FPP are coupled head-to-head to form squalene. This reaction is catalyzed by squalene synthase (SQS), for which a single gene copy can be found in the M. truncatula genome.31 Finally, squalene is oxidized by squalene epoxidase (SQE) to 2,3-oxidosqualene. In M. truncatula, at least three SQE genes are present,28 two of which have been characterized.31 The cyclization of 2,3-oxidosqualene forms the branch-point between the primary sterol and the secondary triterpene saponin metabolism. Cycloartenol (Fig. 1), the tetracyclic plant sterol precursor, is synthesized through cyclization of 2,3-oxidosqualene by cycloartenol synthase (CAS),32,33 the ancestral enzyme of all plant oxidosqualene cyclases (OSCs) involved in secondary metabolism.23,34 For the biosynthesis of triterpene saponins in Medicago, 2,3-oxidosqualene is cyclized to the pentacyclic oleanane-type triterpene backbone b-amyrin by the OSC b-amyrin synthase (BAS) (Fig. 1).31,35 Besides this characterized BAS gene, several other OSCs can be identied in the M. truncatula genome, among which genes homologous to OSCs that catalyze the formation of other types of triterpene backbones, such as lupeol. However, lupeol or derivatives thereof have not yet been detected in M. truncatula.28 Aer the synthesis of b-amyrin, the competitive action of two enzymes causes another branching of the saponin biosynthetic pathway. The P450 enzyme CYP716A12 catalyzes the carboxylation of b-amyrin at the C-28 position, which seems to exclude hydroxylation of b-amyrin at the C-24 position catalyzed by the P450 enzyme CYP93E2, or vice versa (Fig. 1).21,36,37 Carboxylation of b-amyrin at the C-28 position leads to oleanolic acid, which is further modied to yield haemolytic saponins, whereas hydroxylation at the C-24 position is essential for the production of the non-haemolytic soyasapogenol glycosides.21,36 Aer the oxidative modications at the C-24 and C-28 positions, several other positions of the b-amyrin backbone are oxidized in Medicago. In the non-haemolytic branch, CYP72A61v2 catalyzes the oxidation at the C-22 position to yield the soyasapogenol B aglycone (Fig. 1).38 In the haemolytic branch, the C-23 position
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Fig. 1 Sapogenins detected in Medicago species according to Tava, et al.21 and their theoretical biosynthetic pathway in M. truncatula. Cycloartenol and the biosynthetic intermediates in grey have not been detected as saponin aglycone in Medicago.
of oleanolic acid is oxidized by CYP72A68v2 (Fig. 1).38 Another M. truncatula P450, CYP72A63, catalyzes the oxidation of the C30 position of b-amyrin,39 leading to queretaroic acid (Fig. 1).
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Aglycones with this type of modication have been detected in M. Arabica,40 however, none have been reported yet in M. truncatula, and thus this enzyme may be involved in the
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biosynthesis of minor saponins and/or may be restricted to specic tissues or organs of M. truncatula plants.39 For the remaining oxidative modications, no enzymes have been characterized yet, but likely they are also catalyzed by P450s. In the haemolytic branch, two enzymes are still missing: an oxidase that catalyzes the 16-a hydroxylation of hederagenin and medicagenic acid, leading to caulophyllogenin and zanhic acid, respectively, and an oxidase that catalyzes the 2-b hydroxylation of the oleanane backbone. In the non-haemolytic branch, the enzymes catalyzing the C-21 oxidation of soyasapogenol B to soyasapogenol A and the C-22 oxidation of soyasapogenol B to soyasapogenol E remain to be discovered. Aer the various oxidative modications of the b-amyrin backbone, the resulting sapogenins are glycosylated at different positions. In Medicago, glycosylation occurs mainly at the C-3 hydroxy and the C-28 carboxy groups of the aglycone. Rarely, additional glycosylation is observed at the C-23 position.22 In M. truncatula, several glucosyltransferases capable of catalyzing the transfer of glucosyl residues to the sapogenins have been identied. UGT71G1 and UGT73K1 were found to transfer glucosyl residues to different sapogenins in vitro, however, the specic position to which the glucosyl residues were transferred was not determined.41 UGT71G1 also recognizes isoavones and the avonol quercetin as substrates. UGT71G1 glucosylates these compounds with higher efficiencies than triterpenes,41,42 suggesting that it might not correspond to a saponin specic biosynthetic enzyme. Another glucosyltransferase, UGT73F3, was found to catalyze the glucosylation of sapogenins at the C-28 carboxy group in vitro. This effect was conrmed in vivo by genetic loss-of-function studies.28 Besides glucose, several other sugars, including glucuronic acid, galactose, rhamnose, arabinose and xylose are found to be part of the sugar chains of Medicago saponins.22 To date however, no enzymes catalyzing the transfer of these sugar residues to the aglycones have been discovered in Medicago. Other types of modications occurring on the Medicago saponins include addition of malonyl and methyl functionalities on the sugar residues. In M. sativa, the occurrence of soyasaponin VI, containing a C-22 maltol functionality has been reported.19,22,43 No specic enzymes for these modications have been reported yet.
2.2. Regulation of saponin biosynthesis In Medicago plants, a tissue-specic accumulation of saponins is oen observed. Soyasapogenol glycosides are the main accumulating saponins in M. truncatula roots, whereas none of the major medicagenic acid and zanhic acid glycosides that accumulate in the aerial parts, are present in roots.19,44 This tissue-specic accumulation is likely the result of their tissuespecic biosynthesis, and points to a role for saponins in specic defense mechanisms in different Medicago tissues.19,45 The differential accumulation of specic saponin mixtures in different tissues also suggests the existence of specic regulatory mechanisms that steer the biosynthesis toward the desired biosynthetic end-products. To date, however, no regulators responsible for this tissue-specic saponin compendium have been discovered in Medicago.
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Besides the tissue-specic, constitutive accumulation of saponins, induced saponin biosynthesis is oen observed in response to herbivore feeding or pathogen attack.46 For instance, seven days aer Spodoptera littoralis larvae fed on M. sativa leaves, the total saponin content was increased 84% in the foliage of the damaged plants,47 with doubled concentrations of 3-GlcA,28-AraRhaXyl medicagenic acid and soyasaponin I. Furthermore, the damaged leaves had a deterrent effect on the herbivorous larvae, and when fed on damaged leaves, the larval performance was reduced.48 The increased saponin accumulation is mediated by increased steady-state transcript levels of the corresponding saponin biosynthetic genes. This transcriptional response to pathogen or herbivore attack is controlled by a complex signaling cascade in which jasmonates (JAs) play a crucial role.49,50 As such, treatment of Medicago plants or plant cell cultures with JAs can mimic pathogen or herbivore attack. Accordingly, exposure of M. truncatula cell suspension cultures to methyl jasmonate (MeJA) leads to the transcriptional activation of saponin biosynthetic genes and as a consequence to an increased saponin accumulation.28,31,41,51,52 The JA perception and initial signaling cascades leading to the concerted transcriptional activation of entire secondary metabolic pathways is conserved across the plant kingdom. However, the downstream transcriptional machineries that regulate the specic biosynthetic genes are mostly speciesspecic and remain largely unknown.26,49,50,53 So far, no specic transcription factors that regulate the saponin biosynthesis have been characterized in Medicago. However, another regulatory mechanism, operating at a posttranscriptional level, has recently been discovered. To secure plant development and integrity, the unrestrained production of bioactive saponins upon JA perception needs to be prevented. In M. truncatula, the RING E3 ubiquitin ligase MAKIBISHI1 (MKB1), a functional homolog of the mammalian RMA1 proteins involved in the endoplasmic reticulumassociated degradation (ERAD) protein quality control system, is co-expressed with the saponin biosynthesis enzymes.54 By recruiting the ERAD machinery, MKB1 controls the levels and activity of HMGR, the rate-limiting enzyme in the MVA pathway. M. truncatula roots, in which MKB1 is silenced, overaccumulate bioactive monoglycosylated saponins and lose their integrity, demonstrating the need of this regulatory system to manage saponin biosynthesis.
2.3. Biological role of Medicago saponins The green parts of plants are the main target for herbivores, so the saponins present in the aerial parts of Medicago may exhibit antiherbivory activities by several different mechanisms, thereby inuencing animal metabolism from feed intake to excretion.22,55 First, the accumulating saponins may turn the plants unpalatable for herbivores, for instance due to the bitter avor, as was illustrated with a sensory test performed with human volunteers that showed that the main saponin present in the aerial parts of alfalfa, a tridesmoside of zanhic acid, was the most bitter, astringent and throat-irritating of all tested
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compounds.56 Alfalfa saponins also deter insects from eating, as was shown by larvae of Colorado potato beetles that died in four to six days, mainly because of fasting, when they were fed potato leaves treated with a 0.5% solution of total saponins of alfalfa.57 Second, when herbivores are not deterred and consume Medicago saponins, they may suffer from the various toxic or detrimental effects of the compounds. For instance, intraruminal administration of partially hydrolyzed alfalfa saponins led to reduced microbial fermentation and nutrient degradation in the rumen of sheep.58 Furthermore, the total protozoal count in the rumen was signicantly reduced by the alfalfa saponins, likely due to the disruption of protozoal cell membranes by the formation of water-insoluble addition products of medicagenic acid with membrane sterols.58,59 In addition, alfalfa saponins inhibit rumen motility in sheep and cattle,59 and they appear to be responsible for some cases of rumen bloat.55 Next to ruminants, alfalfa saponins also exhibit toxic effects on monogastric animals55,60 and herbivorous insects.22,61–65 Saponins that accumulate in roots or that are released in the rhizosphere may protect Medicago plants against parasitic nematodes. In vitro assays have shown that saponins from M. arborea, M. arabica, and M. sativa exert toxicity toward the phytoparasitic nematode Xiphinema index.66 Addition of dry plant material from M. arborea and M. sativa roots and tops was shown to suppress root and soil population densities of the rootknot nematode Meloidogyne incognita and the yellow potato cyst nematode Globodera rostochiensis in potting mixes and in eld trials, with saponins being held at least partially responsible for the nematicidal activity.67,68 Saponins released in the rhizosphere may also be involved in negative allelopathic interactions of Medicago plants with surrounding plants.69 It was observed that germination, growth, and yield of cotton was much lower when sown in soil aer alfalfa cultivation, than when sown in soil aer wheat cultivation or in bare soil, likely due to the release of saponins by alfalfa roots.70 The addition of powdered alfalfa roots to sand was shown to inhibit wheat seedling growth.71 Later, a structure-activity relationship study with puried alfalfa saponins showed that medicagenic acid and the monoglycosides 3-O-Glc-medicagenic acid and 3-O-Glc-hederagenin exerted the highest growth inhibitory effects on wheat seedlings. These compounds accumulate in considerable amounts in alfalfa seedlings and roots, whereas they are absent in mature alfalfa tops.72 This underscores that the tissue-specic accumulation of Medicago saponins is correlated with their biological roles. In seeds, saponins may be involved in the protection against insects. For instance, M. truncatula seed our has a strong toxic effect on the rice weevil Sitophilus oryzae, a major pest of stored cereals. Purication and identication of the entomotoxic compound revealed that 3-GlcA,28-AraRhaXyl medicagenic acid protects M. truncatula seeds against the weevil.73 The saponins accumulating in Medicago may also be involved in the protection of the plants against infections by phytopathogenic fungi and microbes. The anti-fungal activity of the saponins present in Medicago is well established, with the medicagenic acid aglycone being the major contributor.74 Besides defense, the saponins present in Medicago roots may also play a role in the interaction of the plant with symbiotic
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partners, such as nitrogen-xing rhizobia and mycorrhizal fungi. For instance, colonization of M. truncatula with the mycorrhizal fungus Glomus intraradices does not lead to quantitative changes in the total saponin content but to a reduced ratio between malonylated and corresponding non-malonylated saponins.46,75 This might hint to a specic, but yet unknown, role of saponins in the mycorrhization process. Intriguingly also the receptor-like kinase NORK, required for Nod factor signaling and root nodule development in M. truncatula, was found to interact with HMGR. Chemical inhibition of HMGR activities or silencing of HMGR expression in M. truncatula roots decreased nodulation, indicating that HMGR and the MVA pathway are essential for nodule development.27 It was postulated that recruitment of HMGR by NORK might modulate the production of specic primary terpenoid compounds, such as particular hormones or sterols, but it is equally possible that it rather affects synthesis of secondary terpenoids such as specic bioactive saponins that might play important roles in the plantmicrobe mutualism.
2.4. Commercial potential of Medicago saponins The combined deterrent and toxic effects on insects make Medicago saponins suitable for use against pest insects in agriculture and horticulture.62 One way to use Medicago saponins against herbivorous insects is by selecting cultivars that accumulate high amounts of saponins.62 For instance, the development, survival, and reproduction of pea aphids fed on an alfalfa line with a high saponin content was reduced as compared to aphids fed on an alfalfa cultivar with a low saponin content. Moreover, it was shown that zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid accumulating in the high saponin cultivar were the main compounds contributing to the resistance of alfalfa against the pea aphid.65,76,77 Besides selecting for Medicago cultivars with a high saponin content, extracted Medicago saponins could be used to spray other plants, as was illustrated with larvae of Colorado potato beetles that died aer feeding on potato leaves treated with a 0.5% solution of total saponins of M. sativa.57 The nematicidal activity of the accumulating saponins allows Medicago biomass to be used as a biological agent to control phytoparasitic nematodes. A eld trial in which soil was amended with pelleted M. sativa reduced the soil population density of the root-knot nematode M. incognita, and tomato plants growing on the amended soil developed less root galls and delivered increased tomato crop yields.67,68 The antifungal activity of alfalfa saponins may also reduce the presence of phytopathogenic fungi in the amended soil. Incorporation of alfalfa plant material in soil inoculated with Phytophthora capsici reduced Phytophthora blight of capsicum in pots and eld trials.78 Total saponins and individual compounds from different Medicago species were shown to prevent fusariosis on tulip bulbs.79,80 Hence, the activity of Medicago saponins on phytopathogenic fungi and nematodes, in combination with the high nitrogen content of the plant material, make Medicago biomass especially useful as agent against soil-born plant pathogens and biological fertilizer.
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Because of the antimicrobial activity, Medicago saponins may also nd applications as antibacterial and antifungal agents. Saponin extracts from M. sativa, M. murex, M. arabica, and M. hybrida inhibit the growth of dermatophytic fungi and monodesmosidic glycosides of medicagenic acid were shown to be the most active compounds.81 Similarly, testing of saponins from M. sativa, M. arborea, and M. arabica against a selection of medically important yeasts and bacteria revealed a high activity against Gram-positive bacteria like Bacillus cereus, B. subtilis, Staphylococcus aureus, and Enterococcus faecalis. M. arabica saponins, in which hederagenin is the dominant aglycone, showed a broad spectrum of action, evidencing the possible application of those compounds as antibacterial agents.82 The ability to rupture erythrocyte membranes (haemolysis) is another important property of several of the saponins present in Medicago. Saponins bind to the sterols present in the erythrocyte membrane, which causes irreversible damage to the lipid bilayer and results in increased membrane permeability and haemoglobin loss.83,84 Recently, it was reported that the M. truncatula mutants lacking hemolytic activity (lha) were devoid of haemolytic activity, because they were unable to produce the aglycones oleanolic acid, hederagenin, bayogenin, medicagenic acid, and zanhic acid. This was caused by mutations that render the CYP716A12 enzyme, which catalyzes the carboxylation of bamyrin at the C-28 position, non-functional,36 and conrms previous reports about the importance of C-28 carboxylation in addition to C-3 glycosylation for haemolytic activity.85 The haemolytic activity has even been used as a detection and quantication method for saponins.55,84,86 Like many triterpenoid saponins, Medicago saponins also exert cytotoxic effects,22 and a toxicity screening of saponins from 12 different Medicago species with the brine shrimp Artemia salina pointed toward hederagenin and bayogenin glycosides as the main cytotoxic compounds.87
2.5. Determination of Medicago saponins The saponins present in Medicago plants occur in complex mixtures of structurally related high-molecular weight compounds and are oen only present in low concentrations.19,22,88,89 This complicates the detection and quantication of saponins and hinders purication of individual compounds, which is necessary for structure elucidation and biological activity testing. In addition, Medicago saponins lack chromophores that allow detection in UV, limiting the choice of detection methods that can be employed for analytical purposes.89,90 The screening of crude extracts using hyphenated techniques, such as LC-MS, provides information about the composition of the saponin mixture present in the investigated plant material. In a pioneering study, a large number of saponins in root extracts of M. sativa and M. truncatula were separated with HPLC, and 15 and 27 saponins of M. sativa and M. truncatula, respectively, were tentatively identied based on fragmentation data under negative ionization.88 More recently, by coupling HPLC to the highly accurate Fourier transform ion cyclotron resonance MS, reliable prediction of the molecular
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formula of the detected saponins was possible. This has led, in combination with the fragmentation spectra under negative ionization, to the tentative identication of 79 saponins in M. truncatula hairy roots,19,54 and further underscores the complexity of the saponin mixture present in Medicago. Such qualitative proling of the saponin mixture, however, does not provide conclusive evidence for the absolute chemical structures of the saponins present in Medicago, which can only be obtained by subjecting puried compounds to a combination of analytical methods, including MS and NMR.22 The purication of saponins from the aerial parts of M. truncatula has led to the chemical characterization of 15 individual compounds.91 Several other saponin molecules have been puried and structurally resolved from M. arborea, M. hybrida, M. arabica, and M. polymorpha.40,92–95 A detailed overview of the different saponin structures reported in various Medicago species can be found elsewhere.22 So far however, the purication of compounds has only led to the identication of the dominant compounds in the saponin mixture; the purication of low abundant compounds remains a serious challenge. The puried and characterized compounds can be used as standards to generate response curves that can be used for the absolute quantication of saponins with HPLC/MS. Differential accumulation of saponins in the various organs of M. truncatula was reported, with higher levels of medicagenic acid conjugates present in leaves and seeds, and higher levels of soyasapogenol conjugates present in the roots.45 In a similar approach, saponins in the aerial parts of three different M. truncatula cultivars were quantied, showing very similar saponin mixtures in the three cultivars, with medicagenic acid, zanhic acid, and soyasapogenol glycosides being the dominant compounds. The observed total saponin concentration in M. truncatula is also very similar to that in M. sativa. However, M. truncatula has a higher concentration of zanhic acid glycosides, and a lower concentration of soyasapogenol glycosides as compared to M. sativa.44 The high levels of zanhic acid glycosides (>40% of the total saponin content) observed in this study are in disagreement with the study by Huhman, et al.,45 which reports that zanhic acid glycosides constitute only 0.6% of the total saponin content. This difference may be attributed to the lack of appropriate standards in the latter study, in which no zanhic acid glycosides were available as standards for absolute quantication.45,91 Besides HPLC/MS, GC/MS can also be used for the quantication of Medicago saponins. However, being high-molecular weight compounds, saponins are not volatile and, consequently, GC/MS analysis can solely be performed on hydrolyzed saponins, thus only providing information on the aglycone structures. Furthermore, hydrolysis is oen not complete or can lead to artefacts or decomposition of aglycones, thereby negatively inuencing the nal result.36,90,96
3 Flavonoids Flavonoids constitute a diverse class of metabolites that are ubiquitously present in the plant kingdom and contains more than 10,000 different structures. The name avonoid is derived
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from the Latin word for yellow, avus, and points to the color of many avonoids. Flavonoids are responsible for much of the yellow, red, blue, and purple plant pigmentation. They play important biochemical and physiological roles in planta and are involved in interactions of the plant with its environment, both for benecial symbioses and for biotic and abiotic stress responses.97–99 Flavonoids also possess various pharmacological properties and are thought to have health-promoting effects.100,101 Flavonoids are characterized by a polyphenolic structure with 15 carbon atoms arranged in a basic C6-C3-C6 structural skeleton, in which two benzene rings (A & B) are linked by a linear three carbon bridge (in the case of chalcones) or by a pyran or pyrone ring (C). According to the different alterations and modications of ring C, avonoids have been classied into different subclasses. All main subclasses of plant avonoids, i.e. anthocyanidins, aurones, chalcones, avanols (proanthocyanidins), avanones, avonols, avones and isoavones are present in the genus Medicago, yielding a complex and branched synthetic network (Fig. 2). In contrast to the triterpene saponins, Medicago avonoids are less genus-specic and generally encountered in many legumes. Nonetheless, bearing the role of Medicago as a model system for legume and avonoid research in mind, we considered it appropriate to provide a comprehensive overview of Medicago avonoid synthesis, by describing the Medicago metabolites, genes and enzymes encountered already and listing the missing gaps in the pathway knowledge.
3.1. Flavonoid biosynthesis in M. truncatula 3.1.1. Early steps of avonoid biosynthesis. The biosynthetic pathway leading to the production of avonoids is derived from the phenylpropanoid pathway (Fig. 2). It starts with the formation of p-coumaric acid and p-coumaroyl-CoA from phenylalanine, catalyzed by the sequential activities of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) on the one hand, and the cytosolic formation of malonyl-CoA from acetyl-CoA by acetyl-CoA carboxylase (ACC) on the other hand. Then, one molecule of p-coumaroyl-CoA and three molecules of malonylCoA serve as substrates for chalcone synthase (CHS) to run a series of sequential decarboxylation and condensation reactions, leading to the formation of a polyketide intermediate. This intermediate undergoes cyclization and aromatization reactions to form the A-ring and the resultant chalcone (naringenin chalcone). In Medicago species., CHS is encoded by multiple (oen 10 or more) gene copies.102–104 Chalcone reductase (CHR), of which the corresponding gene is found only in leguminous plants, removes the hydroxyl group of the second malonyl-CoA during chalcone biosynthesis, which together with the CHS leads to the biosynthesis of isoliquiritigenin, the precursor of the 5-deoxyavonoids.105 In dicotyledonous plants like Medicago, p-coumaric acid and p-coumaroyl-CoA are also the precursors of the monolignols, the monomer building blocks of the lignin heteropolymer. Although engineering of lignin synthesis in Medicago species
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has been pursued (see section 5.2), we will not further discuss this branch of the phenylpropanoid pathway here but instead refer to the literature.106–108 The two avanones, naringenin and liquiritigenin, the precursors of 5-hydroxy- and 5-deoxy-avonoids, respectively, are produced via the enzymatic activity of chalcone isomerase (CHI). Two classes of CHIs have been described. Type I CHIs are found in both legumes and nonlegumes, and convert naringenin chalcone into naringenin. Type II CHIs are legumespecic enzymes that can isomerize both naringenin chalcone and isoliquiritigenin to naringenin and liquiritigenin, respectively (Fig. 2).109,110 Genes encoding enzymes of both classes of CHIs have been identied in Medicago.111 The resulting avanones, naringenin and liquiritigenin, serve as substrates for isoavone synthase (IFS), avone synthase (FNS) and avanone 3-hydroxylase (F3H) to produce isoavonoids, avones and (dihydro)avonols, respectively (Fig. 2). 3.1.2. Isoavonoid biosynthesis. Isoavonoids have the Bring attached to the C-ring via the C-3 instead of the C-2 position (Fig. 2). The rst committed step of isoavonoid biosynthesis begins with an oxidative aryl migration from C-2 to C-3, catalyzed by the cytochrome P450 (P450) enzyme 2-hydroxyisoavanone synthase (2HIS, also known as isoavone synthase, IFS).112 Subsequently, dehydration of the 2-hydroxyisoavanone intermediates, i.e. 2,5,7,40 -tetrahydroxyisoavanone and 2,7,40 -trihydroxyisoavanone, by 2-hydroxyisoavanone dehydratase (2HID) forms the “undecorated” isoavones genistein and daidzein, respectively. The 40 -O-methylated forms of genistein and daidzein, biochanin A and formononetin, respectively, which are the most abundant isoavonoid aglycones in M. truncatula roots,113 are biosynthesized by catalytic action of 2,7,40 -trihydroxyisoavanone-40 -O-methyltransferase (HI40 OMT) on the 2-hydroxyisoavanone intermediates prior to 2HID activity. The isoavonoid aglycones can undergo further modications, such as C-30 hydroxylation of the B-ring. M. truncatula I30 H (CYP81E9) was shown to catalyze the hydroxylation of both biochanin A and formononetin to pratensein and calycosin, respectively.114 In addition, Medicago accumulates isoavonoid aglycones with various modications of the A-ring, such as afrormosin, alfalone, irisolidone, and irilone (Fig. 2). Labeling studies indicated that the rst two are derived from formononetin,115 but the specic enzymes involved in the biosynthesis of these compounds have not been discovered yet. Pterocarpans form a class of isoavonoid derivatives in which the B-ring is coupled to the C-ring via a furan ring. The biosynthesis of medicarpin, the pterocarpan accumulating in Medicago, starts with the conversion of formononetin to 20 hydroxyformononetin, catalyzed by the P450 enzyme I20 H (CYP81E7).114 Next, 20 -hydroxyformononetin is converted to the 20 -hydroxylated isoavanone vestitone by the NADPHdependent isoavone reductase (IFR). Subsequently, vestitone is converted into medicarpin via a 2-step enzymatic conversion catalyzed by vestitone reductase, that reduces vestitone to 7,20 dihydroxy-40 -methoxy-isoavanol (DMI), and by DMI dehydratase, that dehydrates DMI, thereby forming the ether linkage of the furan ring of the pterocarpan skeleton (Fig 1).116,117
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Fig. 2 General phenylpropanoid and (iso)flavonoid biosynthetic pathway in M. truncatula. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; CHR, chalcone reductase IFS, isoflavone synthase; 2HID, 2-hydroxyisoflavanone dehydratase; FNS, flavone synthase; F3H, flavanone 3-hydroxylase; IFR, isoflavone reductase; I20 H, isoflavone 20 -hydroxylase; VR, vestitone reductase; DMID, dihydroxy-40 -methoxy-isoflavanol dehydratase; FLS, flavonol synthase; PRX, peroxidases; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; PTR, pterocarpan reductase.
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Pterocarpans can be further reduced by pterocarpan reductase,118 leading to isoavans, of which vestitol and sativan are compounds accumulating in Medicago. Coumestans, oxidation products of pterocarpans, constitute another class of isoavonoid derivatives. Coumestrol is the main coumestan accumulating in Medicago and is synthesized from the isoavonoid daidzein, likely through dihydrodaidzein and a pterocarpan intermediate.119–121 Up until now, however, the genes and enzymes involved in its biosynthesis have not been elucidated yet. 3.1.3. Flavonol biosynthesis. Flavonols are the most widespread subclass of avonoids in higher plants. They are derived from the dihydroavonols, which are the product of the F3H enzyme, of which the corresponding genes have been identied and characterized both in M. truncatula and M. sativa.122,123 Flavonol synthase (FLS) catalyzes the key step in avonol biosynthesis, converting dihydroavonols, such as dihydrokaempferol, dihydroquercetin, and dihydromyricetin, to the corresponding avonols, kaempferol, quercetin, and myricetin, respectively (Fig. 2). FLS has been characterized in several plant species, including A. thaliana,124,125 Citrus unshiu Marc.,126 parsley,127 soybean,128 and strawberry,129 but not yet in Medicago species. 3.1.4. Flavone biosynthesis. The biosynthesis of avones from avanones occurs by introducing a double bond between C-2 and C-3 that is catalyzed by avone synthase (FNS) (Fig. 2). Two different FNS (FNSI and FNSII) have been characterized in plants. FNSI is mainly found in members of the Apiaceae family as well as monocotyledonous plants,130,131 while FNSII is found widespread among all higher plants.132 All known FNSII proteins belong to the plant P450 subfamily CYP93B and two different mechanisms for their catalytic activity have been reported, i.e. some convert avanone substrates to avones via a 2-hydroxyavanone intermediate, whereas others directly convert avanones to avones.133,134 Two FNSII genes, MtFNSII-1 (CYP93B10) and MtFNSII-2 (CYP93B11), have been characterized in M. truncatula. Both convert avanones to 2-hydroxyavanones and have a distinct tissue-specic expression pattern. MtFNSII-1 is mostly expressed in roots and seeds where the major accumulating avones are 7,40 -dihydroxyavone and apigenin/luteolin, respectively, while MtFNSII-2 is highly expressed in owers and pods.135 Medicago enzyme(s) that catalyze the conversion of the intermediate 2-hydroxyavanones into the accumulating avones have currently not been identied. 3.1.5. Aurone biosynthesis. Structurally, aurones are isomers of avones (Fig. 2). Aurones are widely distributed in fruits and owers, in which they are responsible for yellow pigmentation. Accumulation of the aurone hispidol and its glycoside derivative, hispidol-40 -O-glucoside, is reported in yeast elicitor (YE)-induced cell cultures of M. truncatula. Three peroxidase genes, MtPRX1, MtPRX2 and MtPRX3, were suggested to be involved in hispidol biosynthesis from isoliquiritigenin, based on the observation that their expression proles were correlated with hispidol accumulation. Subsequently, the aurone synthase activity of recombinant MtPRX1 and MtPRX2 proteins was demonstrated in vitro.136
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3.1.6. Anthocyanin biosynthesis. The presence of a positive charge in the anthocyanin structure at acidic pH, called a avylium cation (2-phenylbenzopyrylium), distinguishes anthocyanins from other subgroups of avonoids (Fig. 2). The number and position of hydroxy and methoxy groups on the anthocyanidin skeleton; the identity and number of sugars, and the positions at which they are attached; the extent of sugar acylation and the identity of the acylating agent are all responsible for the variation in the anthocyanin structures.137 Enzymes controlling the B-ring hydroxylation of e.g. naringenin in the early steps of avonoid biosynthesis, such as avonoid 30 hydroxylase (F30 H) and avonoid 30 ,50 -hydroxylase (F30 50 H), are key determinants for the structural fate of the resulting anthocyanins as well as 2,3-cis-avan-3-ols.138 The rst dedicated step toward the biosynthesis of anthocyanins (and proanthocyanidins, see section 3.1.7) is controlled by dihydroavonol-4-reductase (DFR) that uses the same substrates as FLS. Dihydrokaempferol, dihydroquercetin, and dihydromyricetin, which differ only in the hydroxylation pattern of their B-ring, are common substrates of DFR and are converted into leucopelargonidin, leucocyanidin, and leucodelphinidin, respectively.139 In M. truncatula, two DFRs, MtDFR1 and MtDFR2, have been characterized that have distinct substrate preferences. Although the expression pattern of the two characterized MtDFRs is very similar in most tissues tested, it is proposed that MtDFR1 has a more pronounced role than MtDRF2 in anthocyanin (cyanidin glucoside) biosynthesis in leaves.140 The next step in anthocyanin biosynthesis is catalyzed by anthocyanidin synthase (ANS) that converts the leucoanthocyanidins leucocyanidin, leucopelargonin, and leucodelphinidin to the corresponding anthocyanidins cyanidin, pelargonin, and delphinidin, respectively.141 In vitro assays characterized the single MtANS as a bifunctional enzyme involved in the conversion of leucocyanidin to cyanidin during (pro)anthocyanidin biosynthesis, and, more efficiently, dihydroquercetin to quercetin during avonol biosynthesis.142 MtANS is mainly expressed in the seed coat of Medicago species, but also in other tissues to play a role in (pro)anthocyanidin biosynthesis. In addition, downregulation of MtANS resulted in reduced levels of anthocyanins in leaves and of soluble and insoluble proanthocyanidins in seeds of Medicago species.142 3.1.7. Proanthocyanidin biosynthesis. Proanthocyanidins (PAs), also called avanols or condensed tannins, comprise mixtures of oligomers or polymers of avan-3-ol units that share the upstream biosynthetic pathway with the anthocyanins (Fig. 2). The structural diversity of PAs depends on the stereochemistry and hydroxylation pattern of the avan-3-ol units, the position and stereochemistry of the interavanyl linkage between the monomeric units, the extent of polymerization, and the type of modications, like various methyl-, acyl- or glycosyl- substitutions of the monomeric units.103,143 Hydroxylation on the A-ring leads to stereochemistry at C-4, which has great importance in the biosynthesis of PAs, since all chiral intermediates of PA biosynthesis up to (+)-catechin possess the 2,3-trans stereochemistry. Conversely, ()-epicatechin with
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2,3-cis stereochemistry arises from achiral anthocyanidin precursors that provide another starter unit for PA biosynthesis.103,143 Although the only difference between the two PA starter units 2,3-cis-2R,3R-()-epicatechin and 2,3-trans-2R,3S(+)-catechin is the cis- or trans- stereochemical conguration, they are synthesized by two distinct biosynthetic pathway branches (Fig. 2).143 In M. truncatula the structural genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), both involved in PA biosynthesis, have been characterized.142,144–146 LAR is encoded by a member of the plant reductase-epimerase-dehydrogenase (RED) supergene family and is closely related to IFR.147 It uses the same substrates as ANS, i.e. avan-3,4-diols, to convert them to their corresponding PA “starter units”, the 2,3-trans-avan-3-ols catechin, afzelechin and gallocatechin, respectively, in an NADPH-dependent manner. This rst committed step in PA biosynthesis diverges from the pathway common with that of the anthocyanins.145 In M. truncatula, the MtLAR gene is expressed in owers, pods, and seed coats.142 ANR catalyzes the conversion of the anthocyanidins formed by ANS into 2,3-cis-avan-3-ols (()-epicatechin, ()-epiafzelechin and ()-epigallocatechin, respectively). The ANR enzyme is encoded by the BANYULS (BAN) gene, identied rst through characterization of the banyuls locus in Arabidopsis148 and later described in detail in M. truncatula.140,145 Similar to LAR, ANR also belongs to the IFR-like group of the plant RED superfamily. 3.1.8. Structural diversication and decoration of avonoids. Flavonoids oen accumulate as malonylated or acetylated glycoconjugates. For instance, almost all anthocyanidin aglycones are conjugated with one or more sugar moieties, mainly at the C-3 position. Glycosylation enhances the watersolubility of anthocyanidins and is critical for the transport and sequestration of these compounds in the vacuole. Of the more than 100 identied UDP-glycosyltransferases (UGTs) in the M. truncatula genome, UGT71G1, UGT85H2, UGT78G1, and UGT72L1 have been shown to possess activity on avonoid aglycones.144,41,149–151 UGT71G1 was shown to be capable of glycosylating avonols (quercetin), isoavonoids (genistein), and saponins.41,42 UGT85H2 is a multifunctional avonoid glycosyltransferase with the ability to glycosylate various avonoids, such as isoavones (biochanin A), avonols (kaempferol), and chalcones (isoliquiritigenin).150 Also UGT78G1 has a broad substrate range, i.e. on formononetin, kaempferol, and the anthocyanidins pelargonidin and cyanidin.151 Although it had been shown that isoavones were the preferred in vitro substrates, yielding the corresponding 7-Oglucosides,152 the phenotypes observed by UGT78G1 overexpression in alfalfa, as well as in M. truncatula retrotransposon insertion lines, rather support a role in anthocyanidin glycosylation. Expression of UGT78G1 is strongly upregulated by Legume Anthocyanin Production 1 (LAP1), a MYB-family transcription factor (TF) involved in anthocyanin biosynthesis but not isoavone biosynthesis.153 Correspondingly, UGT78G1 is expressed in anthocyanin accumulating sites, i.e. owers, leaves, and buds, rather than in roots, which are the major sites for isoavone accumulation in Medicago.152 Since PAs are
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typically sequestered in vacuoles in malonylated and glycosylated forms, glycosylation has been proposed to be critical for the transport and storage of these compounds at their nal destination in the vacuole. UGT72L1 is a Medicago glycosyltransferase responsible for the glycosylation of the PA precursor ()-epicatechin, leading to epicatechin 30 -O-glucoside. The expression pattern of UGT72L1 in developing seeds correlates with the presence of epicatechin 30 -O-glucoside and PA accumulation.144 However, since it is not proven that UGT72L1 by itself is essential for PA biosynthesis, redundant pathways for glycosylation and subsequent PA polymerization may exist.146 Malonylated and acetylated isoavonoids are considered as ‘storage forms’ that accumulate in the vacuoles, serving as a pool of biosynthetic precursors or inactive forms of phytoalexins.154,155 The malonyl residues substituted on the sugar moiety protect the isoavonoid glycoconjugates from enzymatic degradation, change their lipophilicity, and act as molecular tags promoting efficient vacuolar uptake of the conjugates.156,157 Three malonyltransferases from M. truncatula, named MtMaT1, MtMaT2, and MtMaT3, have been shown to catalyze the malonylation of a range of isoavone 7-O-glucosides in vitro and MtMaT1 and/or MtMaT2 were suggested to function as malonyl CoA:isoavone 7-O-malonyltransferases in vivo.158 3.2. Regulation of avonoid biosynthesis Flavonoids play a signicant role in responses to several environmental factors, including both biotic, e.g. pathogen attack and herbivory, and abiotic stresses, e.g. ultraviolet (UV)-light, salt stress, and nutrient deciencies.13,15,97,98,159 In Medicago, isoavonoid phytoalexins and related compounds accumulate in response to fungal or yeast elicitors (YE).51,52,110,115,155,160 In agreement, fungal pathogen infections are capable of massively changing the expression of phenylpropanoid biosynthesis genes, such as Phymatotrichopsis omnivore, responsible for destructive root rot disease in many dicot species, in M. sativa.161 In M. truncatula, increased levels of the medicarpin precursors formononetin 7-O-glucoside and malonylated formononetin 7-O-glucoside were observed aer infection with the fungal pathogen Phoma medicaginis.162 Intriguingly, in M. truncatula cell cultures, genes involved in the early steps of phenylpropanoid/isoavonoid biosynthesis are induced by YE treatment but not by MeJA elicitation, while downstream pathway genes specic for medicarpin formation were induced by both.155 The difference in outcome is due to the different triggers applied. Indeed, YE mimics pathogen attack in the plant cell, while MeJA induces wound signaling cascades. Two different strategies have been proposed for the induction of medicarpin in response to pathogen (YE) or wound signals (MeJA) in M. truncatula. In non-stress conditions, glycosylated and malonylated formononetin conjugates are sequestered in the vacuoles. When MeJA or wound stresses are applied, formononetin conjugates are converted to free formononetin isoavones and transferred to the cytosol. Concomitantly, the downstream enzymes are induced by MeJA and formononetin is converted to medicarpin. Elicitation with YE on the other hand leads to the elevation of medicarpin levels via de novo
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biosynthesis.155 Also the phytoalexin accumulation pattern during fungal infection reect the existence of different regulatory systems that orchestrate metabolic uxes through specic metabolic pathways.51,52,115,155,160 In M. truncatula, four YE-induced WRKY TFs (W100577, W100630, W108715, and W109669) were identied, of which the expression was correlated with that of the genes involved in the central phenylpropanoid pathway and in the downstream steps of medicarpin biosynthesis. Ectopic expression of these WRKY TFs in tobacco (Nicotiana tabacum) led to higher levels of avonoids and other phenolic compounds.160 In addition, studies about the regulation of anthocyanin and PA biosynthesis revealed additional ternary protein complexes composed of three families of TFs, i.e. the R2R3-MYB domain, the bHLH (basic helix–loop–helix) domain, and the WD40 repeat proteins.163 So far in Medicago, only the LAP1 R2R3-MYB from M. truncatula was described to be involved in the orchestration of anthocyanin biosynthesis.153 Overexpression of LAP1 in M. truncatula and several closely related species, including alfalfa and white clover, led to the accumulation of anthocyanin pigments, which was reective of the upregulation of a large number of genes associated with anthocyanin biosynthesis, including the glucosyltransferase UGT78G1.153 Also two major components of the regulatory complex involved in the regulation of PA biosynthesis have been found in M. truncatula, namely M. truncatula WD40-1 (MtWD40-1), which is a singlecopy WD40-repeat protein orthologous to Arabidopsis Transparent Testa Glabra 1 (TTG1) and designated as a positive regulator of PA biosynthesis in M. truncatula seeds,164 and MtPAR (M. truncatula proanthocyanidin regulator), which is an R2R3 MYB-type TF that plays a positive regulatory role on genes involved in the PA pathway.165 The gene encoding MtPAR is only expressed in the seed coat where PAs accumulate. In addition, ectopic expression of MtPAR in M. sativa was shown to cause accumulation of PAs in the shoots.165
3.3. Flavonoid localization Flavonoids can be transported within and between cells and even between tissue layers, oen leading to active or passive release at the plant surface to carry out their role in regulating interactions of the plant with its environment. In specic tissues, avonoid synthesis and accumulation is oen located in distinct cells. In M. truncatula roots, avonoids accumulate in specic cell types, i.e. at the root tip and in root cap cells, from where they can be exuded or sloughed off into the soil.99 With regard to the active intercellular transport of avonoids, ABC (ATP binding cassette) transporters are likely involved, since the M. truncatula ABC transporter MtABCG10 is located in the plasma membrane and active in the vascular tissue of most organs. Silencing MtABCG10 in M. truncatula hairy roots resulted in a lower accumulation and exudation of medicarpin and its isoavone precursors.166 At the cellular level, avonoids are found in the extracellular space and in most plant cell compartments, i.e. the cytosol, vacuole, endoplasmic reticulum, chloroplast, nucleus and small vesicles.167 However, most conjugated avonoids are
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sequestered in the vacuole, where for instance the vacuolar acidic environment and co-pigments determine anthocyaninmediated oral pigmentation.168 In addition, PA subunits are polymerized and subsequently converted to brown oxidation products in the vacuoles. Isoavonoids, such as formononetin, were also found to be sequestrated in the vacuoles in glycosylated and malonylated forms that were only converted to free formononetin and translocated to the cytosol for conversion to medicarpin in response to certain stresses.155 Two mechanisms have been proposed for intracellular avonoid movement: (i) vesicle trafficking-mediated transport and (ii) membrane transporter-mediated transport. Although most studies have been performed on anthocyanins only, this likely holds true for other avonoid classes as well.167,169 Via the rst mechanism, anthocyanins rst accumulate in anthocyanoplasts, which are vesicle-like structures, or in membrane-less proteinaceous matrices, called anthocyanic vacuolar inclusions (AVIs).156,170 Then, anthocyanoplasts and AVIs are covered by prevacuolar compartments (PVCs), i.e. endocytic multi-vesicle compartments involved in ER-Golgivacuole vesicle trafficking, and subsequently imported into the central vacuole.171 In addition, a direct trans-Golgi networkindependent trafficking pathway from the ER to PVCs exists that enables the transport of anthocyanins by the interaction of protein storage vacuoles (PSVs) and anthocyanin-containing vesicle-like structures.167 Membrane transporter-mediated transport of anthocyanins can happen in two ways: (i) primary transport mediated by multidrug resistance-associated protein (MRP)-type ABC transporters172,173 and (ii) proton gradient-dependent secondary transport that is mainly driven by V-ATPase and vacuolar H+pyrophosphatase.167 Furthermore, vacuole-localized multidrug and toxic extrusion (MATE) transporters have been identied as a transporter involved in avonoid/H+ exchange.174 In M. truncatula two MATE-type transporters, MATE1 and MATE2, have been identied as H+-gradient-dependent transporters of PAs and anthocyanin/avonols, respectively.158,175 MATE1 was shown to transport epicatechin 30 -O-glucoside and its expression is conned to the seed coat of M. truncatula. It is an ortholog of Arabidopsis Transparent Testa (TT12) and can complement the seed PA deciency phenotype of the tt12 mutation in Arabidopsis.158 MATE2 is produced primarily in leaves and owers and is involved in vacuolar sequestration of anthocyanins and other avonoids in owers and leaves. It has a higher transport capacity for anthocyanins than for other avonoid glycosides and in spite of its high similarity to MATE1 it cannot effectively transport PA precursors.175 Instead, MATE2 transports glycosylated and malonyl-glycosylated avonoids.175 Malonylation increases both the affinity and transport efficiency of MATE2 for glycosylated avonoid compounds.175 Coregulation of MATE2 with UGT78G1 and MaT4 by LAP1 in M. truncatula152,153 further supports the role of MATE2 in the transport of malonylated anthocyanin glycosides into the vacuole.175 Glutathione S-transferases (GSTs) are involved in both vesicle trafficking and membrane transporter-mediated transport of avonoids. Although their exact role in avonoid This journal is © The Royal Society of Chemistry 2014
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transport has not been determined yet, it has been suggested that they can bind to PAs, anthocyanins or avonols to form a GST-anthocyanin or GST-avonol complex, protecting them from oxidation and/or guiding them to the central vacuole.167,176
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3.4. Biological function of Medicago avonoids in planta Phenolic compounds were already present in the most early plant lineages and contributed to the successful evolutionary adaptation of plants from an aquatic environment to the land. Phenolics have acquired miscellaneous and sometimes crucial functions in plants. In particular in legumes, such as Medicago, avonoids perform many functions (Fig. 3).99,113 Flavonoids, especially avonols, play an important role as energy-escape valves, UV screens, and antioxidants in protecting the plant against abiotic stresses, e.g. oxidative damage associated with exposure to highly energetic, short wavelength solar light. Furthermore, avonoids, and especially anthocyanins, are involved in reproduction and seed dispersal via ower and fruit pigmentation, which attracts pollinators and herbivores. The above-mentioned roles are not truly specic for Medicago species though, and will not be reviewed here. We will rather focus on other roles, in nutrient acquisition, development, allelopathy, and response to biotic stress, on which specic studies on Medicago avonoids have been reported. 3.4.1. Nutrient acquisition. Flavonoids can affect plant nutrient acquisition through chemical changes of the soil, either via direct or indirect contributions to the availability of nitrogen (N), phosphorous (P) and iron (Fe).177 Conversely, N, P, and Fe supply in the soil can affect avonoid biosynthesis.178,179 It has been shown that avonoid exudation from the host is Pregulated and that avonoid accumulation is affected upon Nlimitation in nodulating legumes.99 Since avonoids can act as metal chelators, a putative function in the chemical mobilization of scarcely soluble soil Pforms has been postulated.177 For example, an isoavonoid identied in root exudates of M. sativa was able to dissolve ferric phosphate, thus making both phosphate and Fe available to the plant.180 Flavonoids, including the isoavone genistein and the avonols quercetin and kaempferol, can alter Fe availability by reducing Fe(III) to Fe(II) and by chelating otherwise unavailable iron into iron oxides and/or (poorly soluble) iron minerals.177 Although the role of avonoids in nutrient acquisition has been established already longtime, a lot of questions remain. Indeed, detailed information on the dynamic composition, concentrations, microbial modications, and persistence of the avonoid proles released in the rhizosphere, is oen lacking or contradicting.181 3.4.2. Benecial plant-microbe interactions. Flavonoids also serve an ‘indirect’, signaling-based role in enhancing the uptake of two of the most important macronutrients, N and P, by stimulating benecial plant-microbe symbioses, via the processes of nodulation and arbuscular mycorrhization (AM), respectively. Nodulation is a symbiosis that occurs predominantly between legumes and Gram-negative a/b-proteobacteria, collectively referred to as rhizobia. In this intimate interaction,
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bacteria x atmospheric nitrogen for the plant in exchange for organic sugars. The bidirectional communication during nodulation involves two main groups of molecules: the plant produces nodulation (nod) gene-inducing avonoids, and in response the bacteria secrete mitogenic lipo-chitooligosaccharide (LCO) Nod factors (NFs) that trigger early plant responses.1,177,182 About 30 nod gene-inducing avonoids have been isolated from nine legume genera. One of the rst avonoids discovered to possess nod gene-inducing activities was luteolin, isolated from M. sativa.177,182,183 In general, avones were shown to be the most potent inducers of nod genes in Sinorhizobium meliloti, which colonizes M. truncatula184 and hydroxylation at the C-4 and the C-7 positions is important for this activity.185,186 Mixtures can be more effective than single compounds though, and some avonoids act as inducers for certain rhizobia species and as anti-inducers for others.178,182 Previously, it was shown that some isoavonoids from M. sativa, such as medicarpin and coumestrol, even repress NF production.187 Besides their nod gene-inducing activity, most of these avonoids, especially luteolin and apigenin, also act as chemoattractants, thereby concentrating compatible rhizobia at the root surface.99,182 Flavonoid-decient transgenic M. truncatula roots, in which CHS was silenced, exhibited a near complete loss of nodulation, whereas avone-depleted roots had reduced nodulation and isoavone-decient roots nodulated normally.113,184 These differences could point to an important role of avonols, in particular kaempferol, during nodulation, more particular to cause an inhibition in auxin transport, resulting in local auxin accumulation, which precedes the initiation of cell division and is thus needed for correct nodule primordium development.113,186 Also the M. sativa isoavone formononetin and its 7-O-glucoside ononin were shown to be involved in nodulation, since they were able to counteract the autoregulation of nodulation, the process in which more extensive nodulation is suppressed when the plant is already fully engaged.188 The other important type of plant-microbe mutualism is the process of AM, which occurs between almost all land plants and fungi of the phylum Glomeromycota. In AM, the fungal partner extends the underground root system of the host, thereby greatly enhancing the uptake of water and nutrients, such as P and N, whereas the plant invests up to 20% of its xed carbon into the fungus.189 Some of the host plant exudates that stimulate processes promoting AM, e.g. spore germination, hyphal growth, hyphal branching in the soil, root colonization, and infection, have been identied as avonoids.178 In studies with M. sativa, roots began to accumulate avonoids prior to the colonization by Glomus intraradix, indicating elicitation by an AM fungi-derived signal.190 It would be interesting to test whether this signal corresponds to the recently identied mycorrhizal derived lipochitooligosaccharides (LCOs), termed myc factors.191 In contrast to non-colonized roots, avonoid proles changed over time in M. sativa during colonization with G. mosseae and in M. truncatula colonized with G. versiforme.192 However, contradictory effects have been reported based on the type of avonoid molecule, the nutrient status, the developmental stage, the
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Fig. 3 The different in planta biological functions of the specific flavonoid classes, with representative compounds, are depicted on a Medicago arabica plant.
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degree of colonization, and the studied target organism.181 Hyperoside, the 3-O-galactoside of quercetin, is the dominant avone released by M. sativa seeds and stimulates spore germination in G. etunicatum and G. macrocarpum, whereas other avones were only active in the former, and the isoavone formononetin even inhibited spore germination in both.193 In addition, when adding formononetin and ononin to already colonized roots of M. sativa, only ononin was able to further stimulate root colonization.188 Isoavonoids have also been shown to promote AM. Coumestrol was identied as an active stimulator of hyphal growth in M. truncatula, and a coumestrol hyperaccumulating M. truncatula mutant was found to be hyperinfected by its mycorrhizal symbiont.194 In addition, AM of M. truncatula and M. sativa by G. versiforme was found to cause a transient increase in medicarpin levels.195 3.4.3. Defense. Of course, not all plant microbeinteractions are benecial and avonoids also play an important role in the defense against a wide range of pathogens, ranging from bacteria and fungi to insects and nematodes.196,197 Isoavonoids, such as daidzein, glycitein and formononetin glycosides, of which the aglycones restrict the growth of microbial pathogens, accumulate constitutively and, hence, behave as phytoanticipins.192 Isoavonoids represent a major class of phytoalexins in legume plant species. Gene expression proling has shown an elevation in the expression of avonoid biosynthesis genes and/ or higher metabolite levels in M. truncatula plants challenged by Phymatotrichopsis omnivora, the causative agent of root rot in alfalfa or infected with the necrotrophic pathogen Phoma medicaginis.196,198 Furthermore, accumulation of coumestrol correlated nicely with the level of disease aer P. medicaginis infection in several cultivars of annual Medicago species in the eld.199 Isoavonoid-derived pterocarpans, such as medicarpin, also have antimicrobial properties and are produced either constitutively or aer induction by pathogens or endogenous elicitors.192 Medicarpin from M. sativa protects the plant from the pathogenic fungus Rhizoctonia solani.104 Alfalfa seedlings challenged with the fungal pathogen Colletotrichum trifolii exhibited a defense response that was accompanied with an increased expression of avonoid biosynthesis genes and accumulation of medicarpin and sativan.200 The same was observed in a microarray analysis of M. truncatula infected with Erysiphe pisi, the causative agent of powdery mildew. Seven of the eleven medicarpin biosynthesis genes were strongly upregulated.201 Furthermore, the avanone naringenin was shown to interfere with the quorum sensing-controlled production of virulence factors from Pseudomonas aeruginosa PAO1.202 Besides isoavonoids and avanones, avonols, such as quercetin, also have strong antimicrobial properties.104 Biodegradation of avonoids is one mechanism by which ‘non-target’ bacteria may cope with the toxic concentrations of avonoids, however, many others have evolved an inducible resistance mechanism. For instance, Agrobacterium tumefaciens possesses an isoavonoid-inducible isoavonoid efflux pump which contributes signicantly to its colonization of M. sativa roots.203 Flavonoids are also known to be highly effective against insects and nematodes. Several insects are sensitive to
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avonoids and are deterred in feeding tests, since avonoids serve as anti-feedants, digestibility reducers, and toxins.197 Nematode-resistant cultivars of alfalfa contained increased amounts of isoavonoids. For instance, the phytoalexin medicarpin inhibited the motility of the nematode Pratylenchus penetrans in alfalfa,204 and accumulation of isoavonoids in response to infection with the stem nematode Ditylenchus dipsaci correlated with resistance and was even induced systemically.205 In vitro bioassays revealed that the avonols kaempferol, quercetin, and myricetin act as repellants for the root lesion nematode Radopholus similis and the root knot nematode Meloidogyne incognita, while the isoavones genistein and daidzein and the avone luteolin only acted on the former species.206 The former three avonols also inhibited the motility of M. incognita and kaempferol inhibited egg hatching of R. similis.206 3.4.4. Allelopathy. Besides their well-known role in plantmicrobe interactions, avonoids also play an important role as allelochemicals in plant-plant interactions. Allelopathy describes negative biochemical interactions between plants for the competition for natural resources, such as nutrients, light, and water.181 A few detailed studies have been performed on the direct inhibitory effects of avonoid exudates on the growth and development of neighboring plants in leguminous species. The isoavone formononetin and the pterocarpan medicarpin were found to inhibit the germination of Allium cepa.207 The avanone naringenin was found to have direct inhibitory effects on root growth of soybean (Glycine max) seedlings, likely due to premature lignication.208 Allelopathic interactions are not always caused by direct toxicity of the allelochemicals themselves, but can also be induced by biotic or abiotic structural modications caused by avonoids in the rhizosphere.181,209 Another phenomenon illustrating this is replant disease, also known as autotoxicity or soil sickness. It is an example of intraspecic allelopathy, where the plant produces allelochemicals when it starts decaying in the soil and these allelochemicals are detrimental to the establishment of new seedlings of the same species. In the case of alfalfa, it can reduce the development and productivity of the crop itself and cause permanent morphological reductions in root and shoot growth.99 Additionally, the observed damping-off of M. sativa seedlings by the fungal pathogens Pythium spp. and Rhizoctonia solani and causing the death or weakening if the seeds or seedlings before or aer they germinate, has been attributed to autotoxicity of undecomposed M. sativa plant residues.210 3.4.5. Developmental regulators. As mutants decient in the production of certain avonoids show a variety of developmental defects, ranging from lack of gravitropism,211 root looping, and aberrant root outgrowths212 to defects in whole plant growth,213 avonoids may serve as developmental regulators. Studies have shown that avonoids affect long-distance polar auxin transport (PAT) streams.214,215 For instance, RNAimediated silencing of avonoid biosynthesis in M. truncatula hairy roots led to increased auxin transport, indicating that avonoids act as auxin transport inhibitors.184,186 More specically, it has been shown that the isoavone formononetin, the avone dihydroxyavone, and the avonol kaempferol and not
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the chalcone isoliquiritigenin, nor the pterocarpan medicarpin were able to inhibit acropetal PAT in M. truncatula roots.216 Because they interfere with multiple aspects such as auxin transporters, kinases, and the trafficking machinery, avonoids are not considered to be specic regulators modulating auxin transport.214 Besides, CHS-silenced M. truncatula hairy roots, decient of avonoids, showed impaired nodulation, but no defect in lateral root formation.184,217 This suggests that avonoids are not required as general regulators of the organogenesis of secondary root organs. High sunlight induces the synthesis of both auxin and quercetin derivatives, and quercetin displays a great capacity for netuning auxin gradients as well as local auxin concentrations, which represent the actual determinants for different morphological responses.218 As a result, it was postulated that the highlight induced biosynthesis of antioxidant avonoids may have a role in regulating whole-plant and individual organ architecture.219 3.5. Pharmacological properties of Medicago avonoids Besides their important biological functions in plants, avonoids are signicant components of the human diet and have numerous pharmaceutical properties.220,221 In traditional medicine, many plant-derived infusions, balms, and spices, containing avonoids as active ingredients, have been used for centuries.222 M. sativa has long been used as traditional herbal medicine in many countries, such as China, India, and America.12,223 Nowadays, the amount of research on avonoids has tremendously increased, in part because they have been implicated in the prevention of numerous physiological disorders and diseases, within which chronic inammation is key to most. This potential resides in a number of biological activities, including antioxidant, anti-inammatory, anti-carcinogenic, and phyto-estrogenic abilities, though their interactions with intracellular signaling pathways and regulation of cell survival/ apoptotic genes and mitochondrial function. Although most of the studied bioactive, potentially valuable avonoids, such as coumestrol, daidzein, genistein, (iso)liquiritigenin, luteolin, quercetin, kaempferol, naringenin, apigenin, catechin, epicatechin, cyanidin, and delphinidin, can be encountered in Medicago, to our knowledge, none of these molecules are specic for Medicago species. Hence we will not discuss their pharmacological properties in detail but refer to excellent reviews published elsewhere.101,224–227 3.6. Determination of Medicago avonoids Many different techniques can be used to identify and/or quantify avonoids. An overview of the currently commonly used as well as new emerging separation and identication techniques has recently been published.228,229 These techniques range from simple bench methods to the use of sophisticated instrumentation, such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and laser-induced uorescence (LiF) detection, each having unique advantages and disadvantages.230 Coupling chromatography to MS offers an extra level of information to avonoid elucidation and is ideal for the analysis
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of complex plant tissue samples. Advances in sample extraction, chromatographic separation, detection and structural analysis of avonoids have dramatically inuenced the evolution of avonoid discovery and improved avonoid research in general.228 High-performance liquid chromatography (HPLC) coupled to UV detection (HPLC-UV) and to MS systems (HPLC-UV-MS) are well recognized methods to prole avonoid conjugates in plant tissue extracts. As such, various classes of avone, avonol, and isoavone glycoconjugates and aglycones have been identied in Medicago. However, these methods are insufficient to distinguish isomeric and isobaric compounds, which require tandem mass spectrometry with collision induced dissociation (CID MS/MS) for functional characterization. In one study, an integrated approach utilizing HPLC-UV coupled to electrospray ionization (ESI)-MS and gas chromatography (GC)-MS was used to elucidate the avonoid proles from M. truncatula root and cell cultures. A quadrupole time-of-ight (QTOF) MS device was used for all structural identications, but where the stereochemistry of sugar conjugates was uncertain, enzymatic hydrolysis, followed by GCMS to assign a correct sugar stereochemical conguration, was used.231 The same group also used reversed-phase highperformance liquid chromatography coupled to UV photodiode array detection and electrospray ionization ion-trap mass spectrometry (HPLC-PDA-ESI-ITMS) to analyze the intra- and extracellular phenylpropanoid and isoavonoid metabolome of M. truncatula cell cultures in response to YE or MeJA.115 More recently, this technique was validated in the same M. truncatula system to identify the biosynthetic mechanism for the aurone hispidol.136 With a more or less similar methodology, HPLC-UVMS was used in two studies to discover the changes of avonoid accumulation in M. truncatula leaves infected with P. medicaginis.162,196 In another comparable study, both a low resolution ion trap (IT) and a high resolution tandem QTOF LC-UV-MS system was used to evaluate the fragmentation pathways of M. truncatula avonoids aer CID experiments. It was concluded that although some decent fragmentations of minor compounds could be obtained using the low resolution IT, this technology does not always allow proper identication of all molecules in the sample. For that reason and because of the presence of equal nominal masses of some different substituents, a high resolution instrument could only be used to correctly analyze these derivatives.232 In addition, the same high resolution methodology was used to compare the avonoid proles from M. truncatula seedling roots, hairy roots, and suspension root cell cultures.233 In a more recent study, a new stop-and-go two-dimensional chromatography was used for the preparative separation of avonoids from M. sativa, combining counter-current chromatography and liquid chromatography (2D CCC LC). Moreover, two new avonoids were identied coupling this technique to ESI-MS, ESITOF-MS, and 1D and 2D NMR.234
4 The ‘omics’ toolkit for Medicago research Over the past two decades, M. truncatula has been established as a model system. Initially, it was chosen for its small diploid
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genome, short generation time, self-fertility and relative ease of transformation and regeneration. Since M. truncatula is also closely related to important forage and food crops, it has additional value as a model species for doing translational research.13,14,133 Furthermore, this model species also has the ability to engage in several plant-microbe interactions, both mutualistic and detrimental in nature, resulting in the evolution of a rich variety of natural product pathways.235 Due to a tremendous effort of the scientic Medicago community, numerous genetic, functional genomic, and molecular tools and resources have become available, making over the years signicant contributions to the elucidation and characterization of the secondary metabolism of Medicago species in particular, and legumes in general. Substantial efforts undertaken by the Medicago Genome Initiative that started over a decade ago, culminated in the publication of the rst dra genome in 2011.236 However, since relevant gene info was clearly still missing,237 e.g. several of the known (published) saponin biosynthesis genes are missing from the v3.5 version, a curated version (v4.0) has now been released. In addition, there are almost 270,000 expressed sequence tags (ESTs) deposited in Genbank (September 2013) for M. truncatula and more than 11,000 for M. sativa. Another important pillar of the Medicago’s functional genomic toolkit is the Medicago Gene Expression Atlas (MtGEA; http://mtgea.noble.org/v3/), which provides quantitative gene expression data for most M. truncatula genes in all major organs during plant development and in response to multiple environmental stimuli.238,239 It currently contains expression data from 670 Affymetrix GeneChip Medicago Genome Arrays (September 2012), each covering 50,900 probe sets. Besides evoking the ‘guilt by association’ principle, in which tightly coexpressed genes are likely involved in similar processes, some specic conditions, e.g. MeJA or YE elicitation, might allow for identication of specic proteins involved in Medicago secondary metabolism, including TFs or other regulatory proteins, transporters, and enzymes.49 Mining of the MtGEA allowed for instance to pinpoint HMGR as the putative target for the RING E3 Ubiquitin ligase MKB1.54 Improved functional genomic technologies such as transcriptomics, proteomics, and metabolomics provide opportunities for an in-depth understanding of secondary metabolism. For example, an integrated functional genomics approach was used to study natural product biosynthesis in M. truncatula and led to the reconstruction of the metabolic map of all pathways in MedicCyc, the rst legume pathway database for M. truncatula (http://www.noble.org/MedicCyc/).41,51,52,160,240,241 Of course, gene function discovery at a large scale cannot be accomplished without the availability of complementary substantial mutant collections to bridge the gap between genotype and phenotype. The generation of these Medicago collections has been pursued using different strategies, i.e. (i) chemical: ethyl methanesulphonate (EMS)242–245 for single-base substitutions; (ii) physical: fast neutron bombardment (FNB)246 and g-ray247 for deletion of DNA fragments of variable length; and (iii) biological: transferred DNA (T-DNA),248 transposons,249,250 the tobacco Tnt1 retrotransposon243,251–254 or the
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endogenous MERE1 retrotransposon from M. truncatula255 for insertional mutagenesis. To investigate these mutant collections, both reverse and forward genetic approaches have been applied. Traditionally, map-based cloning was the method of choice in forward genetic screens, which required the identication of closely linked molecular markers to initiate chromosome walking and sequencing to identify the mutant locus.253 This tedious and difficult strategy was replaced by the targeting-induced local lesions in genomes (TILLING) approach, which is a PCR-based reverse genetics method that can detect mutations ranging from single nucleotide changes to deletions of several kilobases; the latter being a modied strategy termed deletion TILLING (De-TILLING).246 TILLING involves the detection of mismatches in heteroduplexes that are formed by the annealing between mutant and wild-type alleles for a target locus, using pooled DNA from a mutant collection.256 However, for Tnt1 mutant populations an alternative reverse genetic strategy can be used, because they are generated by a tractable genetic system in which the DNA sequence used to mutate the genome can also be used to identify the mutation. For this purpose a Tnt1 anking sequence tag (FST) database was generated by the Samuel Roberts Noble Foundation (http://Medicago-mutant. noble.org/mutant/index.php), which currently contains 72,159 FSTs (August, 2013).254 With the availability of all these screening tools, Tnt1 mutant populations have already proven to be extremely useful in screens for the discovery of regulators, transporters, and enzymes involved in avonoid production.146,164,165,175
5 Metabolic engineering Targeted metabolic engineering in Medicago species has mainly been focusing on the production of phenolic compounds such as avonoids and lignin. To the best of our knowledge, targeted metabolic engineering for triterpene saponin production has not been reported yet. 5.1. Engineering of avonoid biosynthesis in Medicago Because of the various functions of avonoids in plants, and their potential nutritional and pharmacological utility, engineering the plant’s ability to biosynthesize these bioactive natural compounds, either in the legumes where they are endogenously produced or heterologously in non-leguminous plants, such as Arabidopsis, tobacco, corn and tomato, or in microbial systems, has become a densely covered research area. Besides creating sustainable production sources for pure avonoids, legume avonoid engineering may encompass other goals, such as the improvement of the nutritional value (of e.g. soybean), or the modulation of plant-microbe interactions.159 Basic strategies that can be applied for the genetic modulation of avonoid biosynthesis in plants may involve: (i) increasing endogenous avonoid levels through the upregulation of structural or regulatory genes; (ii) overexpression of heterologous structural and regulatory genes that are not present in the gene pool of the target plant to open the
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endogenous Medicago pathways to new, non-Medicago metabolites; and (iii) blocking of specic steps in the avonoid biosynthetic pathway by RNA interference strategies to steer the metabolic ux toward the desired end-products.127,143 Effects of heterologous overexpression of Arabidopsis or maize genes that encode regulatory TFs of avonoid synthesis on accumulation of anthocyanins have already been investigated.255–259 The expression of the maize bHLH B-Peru and the MYB C1 (colourless) TF genes failed to increase anthocyanin levels in alfalfa, whereas transgenic alfalfa populations expressing the maize bHLH TF Lc (leaf color) gene showed a slight increase in anthocyanin production when the plants were exposed to abiotic stress.260 Similar to its maize C1 counterpart, overexpression of the Arabidopsis MYB TF Production of Anthocyanin Pigment 1 (PAP1) was not able to stimulate anthocyanin biosynthesis in M. truncatula or alfalfa. In contrast, overexpression of the M. truncatula C1/PAP1 homolog LAP1, increased anthocyanin production in transgenic M. truncatula, alfalfa, or white clover when ectopically overexpressed. Hence, LAP1 can be used as a tool for anthocyanin engineering in legumes.153 Ectopic expression of Arabidopsis TT2, a MYB factor involved in the regulation of PA biosynthesis, in M. trunculata hairy roots caused a massive accumulation of PAs as a result of the activation of genes involved in PA synthesis, transport, and oligomerization.144 Heterologous overexpression of TaMYB14, which encodes a MYB from Trifolium arvense, resulted in the synthesis and accumulation of PAs in M. sativa leaves.261 Likewise, the ectopic expression of the M. truncatula MYB TF MtPAR increased the expression of PA/anthocyanin biosynthesis and the accumulation of PAs in M. truncatula hairy roots.165 Overexpression of MtPAR also resulted in a higher accumulation of PAs in alfalfa shoots.165 In an attempt to create crops with improved diseaseresistance, constitutive overexpression of an isoavonoid biosynthesis gene, i.e. isoavone O-methyltransferase, in transgenic alfalfa caused a more rapid and increased production of medicarpin following infection by P. medicaginis, which ameliorated disease symptoms.262 Overexpression of another biosynthesis gene, IFS, from M. truncatula in alfalfa corroborated these results, with an additional accumulation of the isoavones formononetin and daidzein aer P. medicaginis infection.159 The expression of M. truncatula IFS1 in alfalfa also resulted in the accumulation of genistein glycosides in transgenic plants.159,263,264 Medicago genes were also used as tools for avonoid engineering in other plant species. Co-expression of MtBAN, encoding ANR, and Arabidopsis PAP1 in tobacco resulted in a signicant decrease in anthocyanin levels but a higher content of PA precursors.265 Overexpression of MtDFR1 in tobacco induced changes in ower anthocyanin proles, while overexpression of MtDFR2 did not induce such changes.140 In another study, an articial bifunctional enzyme was engineered, containing CHI from alfalfa and IFS from soybean that, upon overexpression, increased isoavone levels in transgenic tobacco as compared to plants transformed with IFS alone.266 Engineering micro-organisms by tweaking metabolic uxes and introducing novel plant-derived biosynthetic enzymes for a
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sustainable production of avonoids is also actively being pursued and has been reviewed elsewhere.267 Here we will only mention a few engineering studies in which Medicago genes were used. An Escherichia coli cell factory was developed to produce 7-O-methyl aromadendrin, a medicinally valuable avonoid, by introducing structural genes from several plants, among which CHI from M. sativa.268 A codon-optimized version of the same gene was also introduced in Streptomyces venezuelae to produce the avanones naringenin and pinocembrin.269 Finally, the production of 5-deoxyavanones in Saccharomyces cerevisiae was made possible by introducing CHS, CHI, and CHR from M. sativa.270 5.2. Engineering of lignin biosynthesis in Medicago Lignin is a complex biopolymer that is encountered in plant secondary cell walls and that plays crucial roles in mechanical support of the cell wall and the plant as a whole, in water conductance in plant stems and in defense against pathogens. The lignin polymer is tightly woven with cellulose and hemicellulose polymers in the lignocellulose matrix that provides strength to the plant cell wall. Lignin is the main hurdle in the processing of plant lignocellulosic biomass for liquid biofuels and negatively inuences the digestibility of forages.271–273 As the reduction of lignin content can improve the digestibility of forages,272 considerable efforts are undertaken to engineer lignin biosynthesis in the important forage crop alfalfa. Downregulation of nearly each of the monolignol biosynthesis genes leads to lower lignin concentrations and altered lignin composition (altered S/G ratio) in transgenic alfalfa,274–279 resulting in increased digestibility,274,277–280 especially when genes early in the pathway are silenced.272,281 The increased digestibility of the transgenic plant material is solely due to a decreased lignin content, since an altered S/G ratio does not seem to inuence digestibility.272,278,281 Though benecial for digestibility, large reductions in lignin content have always been accompanied with a signicant yield penalty.272,277–279,281–283 Dwarng of the engineered plants could be attributed to impaired water transport because of distorted xylem vessels279,283 or a constitutively activated defense response.284 Indeed, studies in Arabidopsis have shown that reduced lignin levels led to increased levels of the stress hormone salicylic acid (SA) but that the stunted growth phenotype could be reversed by keeping the SA levels low in the lignin-silenced Arabidopsis plants.285 This indicates that it should be feasible to engineer alfalfa plants with reduced lignin levels showing no yield penalty.
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Outlook
The occurrence of a wide range of natural products in the Medicago genus supports the intrinsic potential of this species as a platform for the development and sustainable production of (novel) bioactive compounds. Taking into account the benecial effects of Medicago secondary metabolites for the agricultural industry or for animal and human health, Medicago species seem to hold great capacity. Therefore, continued
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exploration of the elements involved in all aspects of secondary metabolite production in Medicago plants will be required to provide the necessary basis for the establishment of such a platform. The information currently available through studies focusing on the biology, genomics, biochemistry and metabolic engineering of Medicago species shows steady progress, in particular in the past decade and largely thanks to the substantial efforts of the Medicago Genome Initiative. Discovery of the genes corresponding to the structural pathway elements, i.e. the enzymes catalyzing the metabolic reactions, has greatly beneted from the booming number of sequence data that currently are available for Medicago and other plant species. Nonetheless, numerous molecular details remain largely unresolved, in particular with regard to the regulation and management of secondary metabolite synthesis, either during development or in response to environmental cues. The booming genomics, transcriptomics, proteomics, and metabolomics technologies have certainly made exploration of plant natural product biosynthesis pathways more accessible. The current advances in the omics area and the current strategies for gene discovery will undoubtedly continue to have a huge impact and to allow capturing some of the transcriptional regulators of Medicago natural product biosynthesis, which ultimately might be useful as master switches in plant metabolic engineering programs. However, identication of all structural elements, as well as all control mechanisms of bioactive natural production in the Medicago genus, may be mandatory to secure a sustainable, reliable and large-scale production of its compounds, whether it is through modulating the metabolic network of the Medicago species itself or through synthetic biology programs in heterologous hosts. As was and is the case for the enzyme-encoding genes, a substantial part of this information might be obtained by mining the available and ever-increasing number of gene sequences by current approaches such as looking for coexpression patterns in transcriptomes. This almost ‘classical’ strategy has proven its utility in practically every plant species studied for a particular metabolite, including Medicago species.28 More recently, another promising trend emerged, i.e. looking for physical metabolic gene clusters in plant genomes, an approach originally supposed to be applicable only in microbes where physical gene clustering is the rule rather than the exception. However, metabolic gene clusters are being discovered in an increasing number of plants,136,286 including e.g. a triterpene gene cluster in the legume Lotus japonicus.287 New or superior omics tools will need to be employed or developed though, to reach the demanded level of knowledge. Transcriptome analysis may benet from an increased spatiotemporal resolution, for instance allowing high-throughput single-cell proling, to address an important bottleneck in the study of plant secondary metabolism; the fact that it oen occurs in specic tissues or organs only consisting of a small number of specialized cells. Transcriptome or genome mining will also need increased computing power, in rst instance to allow building metabolic networks, either from genomes, such as MedicCyc, or transcriptomes, such as CathaCyc for Catharanthus roseus,288 and in second instance to create and improve
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databases, such as the Plant Metabolic Network (http:// www.plantcyc.org/), within which the respective networks of the different species can be compared. This will provide evolutionary information that might allow prioritizing genes for functional analysis. Omics should also go beyond the genome and transcriptome. The proteome and metabolome are the obvious next levels but crucial information is denitely hidden in the interface between these ‘omes’, i.e. at the interactome, for instance between DNA and proteins, such as transcription factors, or between different proteins, such as in transcription factor complexes or channels of metabolic enzymes (the so-called metabolons). Methods to study such interactomes gain performance and will gradually prove their value for plant metabolism as well.49,289–291 Another, yet virtually unexplored level of interactions is that between metabolites and proteins. Considering that metabolites comprise a large and important fraction of the cellular molecules, occur in an enormous range of physiological concentrations, and participate in a wide variety of biochemical and regulatory functions, for instance as ligands for receptors, and as substrates, products, cofactors, or allosteric regulators of enzymes, studying this interactome should deserve more attention. In this regard, a pioneering study in yeast, in which an assay for the large-scale identication of in vivo proteinmetabolite interactions was designed, provides sufficient proof-of-concept, since it allowed revealing numerous new metabolite interactions, both with enzymes from the sterol pathway as with regulatory proteins such as protein kinases.292 Finally, omics won’t be able to solve it all. One should keep in mind that only for a few 100 of the more than 5,000 metabolites that each plant species can synthesize, the structure has been unambiguously identied. ‘Classical’ metabolite purication and identication should therefore not be neglected in future research programs, because sooner or later any omics adept will face a metabolite of interest that is key to the studied network, but with a yet unknown structure.
7
Acknowledgements
We thank Annick Bleys for help in preparing the manuscript. N.D.G. and A.G. are indebted to the Agency for Innovation by Science and Technology (IWT) and the Iranian Ministry for Health and Medical Education, respectively, for predoctoral fellowships. J.P. is a postdoctoral fellow of the Research Foundation Flanders (FWO).
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