J Chem Ecol (2013) 39:283–297 DOI 10.1007/s10886-013-0248-5

REVIEW ARTICLE

Flavonoids: Their Structure, Biosynthesis and Role in the Rhizosphere, Including Allelopathy

Leslie A. Weston & Ulrike Mathesius

Received: 29 December 2012 /Revised: 18 January 2013 /Accepted: 23 January 2013 /Published online: 9 February 2013 # Springer Science+Business Media New York 2013

Abstract are biologically active low molecular Keywords Plant interference . Roots . Exudation . weight secondary metabolites that are produced by plants, Rhizosphere . Secondary metabolites . Phenolics with over 10,000 structural variants now reported. Due to their physical and biochemical properties, they interact with many diverse targets in subcellular locations to elicit various Introduction activities in microbes, plants, and animals. In plants, flavo- noids play important roles in transport of auxin, root and Flavonoids are low molecular weight secondary metabolites shoot development, pollination, modulation of reactive ox- that are produced by plants, and generally are described as ygen species, and signalling of symbiotic bacteria in the non- essential for plant survival, unlike primary metabolites. legume Rhizobium symbiosis. In addition, they possess an- Secondary products are biologically active in many ways, tibacterial, antifungal, antiviral, and anticancer activities. In and over 10,000 structural variants of flavonoids have been the plant, flavonoids are transported within and between reported (Williams and Grayer, 2004; Ferrer et al., 2008); plant tissues and cells, and are specifically released into their synthesis appears to be ubiquitous in plants and the rhizosphere by roots where they are involved in plant/- evolved early during land plant evolution, aiding in plant plant interactions or allelopathy. Released by root exudation protection and signalling (Pollastri and Tattini, 2011; Delaux or tissue degradation over time, both aglycones and glyco- et al., 2012). Due to their physical and biochemical proper- sides of flavonoids are found in soil solutions and root ties, flavonoids also are able to interact with many diverse exudates. Although the relative role of flavonoids in allelo- targets in subcellular locations to elicit various activities in pathic interference has been less well-characterized than that microbes, plants and animals (Taylor and Grotewold, 2005; of some secondary metabolites, we present classic examples Buer et al., 2010). Although flavonoids have many roles in of their involvement in autotoxicity and allelopathy. We also plants, including their influence on the transport of auxin describe their activity and fate in the soil rhizosphere in (Brown et al., 2001; Wasson et al., 2006; Peer and Murphy, selected examples involving pasture legumes, cereal crops, 2007), they also play important roles in modulating the and ferns. Potential research directions for further elucida- levels of reactive oxygen species (ROS) in plant tissues tion of the specific role of flavonoids in soil rhizosphere (Taylor and Grotewold, 2005; Agati et al., 2012), and pro- interactions are considered. vide colouring to various tissues including flowers (Davies et al., 2012). In addition, they are required for signalling symbiotic bacteria in the legume rhizobium symbiosis (Djordjevic et al., 1987; Zhang et al., 2009), and are impor- * L. A. Weston ( ) tant in root and shoot development (Buer and Djordjevic, EH Graham Centre, Charles Sturt University, Wagga Wagga, NSW 2678, Australia 2009). e-mail: [email protected] In relation to their role in allelopathy and the inhibition of seedling root growth, the activity of flavonoids as regulators U. Mathesius of auxin transport and degradation is likely to be of partic- Division of Plant Science, Research School of Biology, Australian National University, Canberra, ACT 0200, Australia ular importance. Depending on their structure, flavonoids e-mail: [email protected] can impact the breakdown of auxin by IAA oxidases and 284 J Chem Ecol (2013) 39:283–297 peroxidases (Furuya et al., 1962; Stenlid, 1963; Mathesius, elucidated, compared to biosynthetic pathways of other 2001) and also affect polar auxin transport (Stenlid, 1976; secondary products (Dixon and Steele, 1999; Winkel- Jacobs and Rubery, 1988; Peer and Murphy, 2007), thereby Shirley, 2001). Flavonoids are synthesized through the impacting root growth of target species. Some or acetate-malonate metabolic pathway, phytoalexins act as cofactors to auxin in adventitious root which also is well-described in Arabidopsis. Interestingly, development, although the mode of action of these mole- unlike legumes, Arabidopsis lacks chalcone reductase and cules remains unknown (Yoshikawa et al., 1986). In addi- synthase enzymes, so therefore it cannot pro- tion, flavonoids show affinity for many enzymes and other duce one subset of flavonoids, the (Buer et proteins in plants and animals, including those required for al., 2007, 2010,). mitochondrial respiration. In this case, certain flavonoids Arabidopsis mutants (Peer et al., 2001) and transgenic contribute to inhibition of NADH oxidase and the balance legumes with modified branches of the pathway of reactive oxygen species (Hodnick et al., 1994, 1988), (Yu et al., 2003; Subramanian et al., 2005, 2006; Wasson et thereby impacting respiration. al., 2006) now are available and provide a unique tool for In animal systems, plant-produced flavonoids are impor- studying the role of flavonoids in rhizosphere interactions. tant dietary components, and are known to possess a broad Interestingly, flavonoids have similar precursors to those range of properties including antibacterial, antifungal, anti- utilized for lignin biosynthesis but exhibit a number of basal viral, and anticancer activity (Taylor and Grotewold, 2005; structures that result in generation of diverse structures Soto-Vaca et al., 2012). Many flavonoids also have served including , , -3-ols, , iso- as templates in the development of new pharmaceuticals flavanones, isoflavans, and pterocarpans (Fig. 1). (Cutler et al., 2007). Interestingly, flavonoids in planta can Substitution by glycosylation, malonylation, methylation, be transported within and between tissues and cells, and hydroxylation, acylation, prenylation, or polymerization often are released into the rhizosphere where they are in- leads to diversity in this family and has important impact volved in plant to plant interactions, specifically allelopathic upon function, solubility, and degradation (Dixon and interference (Hassan and Mathesius, 2012). They can be Steele, 1999; Winkel-Shirley, 2001; Zhang et al., 2009). released by root exudation or through tissue degradation In higher plants, flavonoid synthesis begins when en- over time, and although both aglycones and glycosides of zyme complexes form on the cytosolic side of the endoplas- flavonoids are found in root exudates, their relative role in mic reticulum (Jorgensen et al., 2005),whichthenmay allelopathic interference, specific activity and selectivity, localize to the tonoplast for subsequent glycosylation and and mode(s) of action remain less well-characterised storage in the vacuole (Winkel, 2004). In specific tissues, (Berhow and Vaughn, 1999;WestonandDuke,2003; flavonoid synthesis and accumulation often is located in Levizou et al., 2004; Hassan and Mathesius, 2012). This distinct cells (Fig. 2). Subcellularly, flavonoids have been review describes the diversity of flavonoids produced by found in the nucleus, the vacuole, cell wall, cell membranes higher plants, their biosynthesis and transport, their roles in and the cytoplasm (Hutzler et al., 1998; Erlejman et al., the rhizosphere, and gives particular emphasis to their re- 2004; Saslowsky et al., 2005; Naoumkina and Dixon, cently described roles in allelopathic interference with other 2008). While flavonoid glycosides stored in the vacuole plants. We also outline potential research directions for the probably do not generally have active roles, their released future to further elucidate the specific role of flavonoids in aglycone counterparts could have functions in the plant soil-rhizosphere interactions. cytoplasm, e.g., in regulation of enzyme activity, formation of reactive oxygen species, and auxin transport (Taylor and Grotewold, 2005; Naoumkina and Dixon, 2008). In some Flavonoid Structure, Function, and Biosynthesis studies, flavonoid glycosides also have been found to have in Plants active roles, e.g., in regulation of IAA oxidase, which could lead to changes in auxin accumulation (Furuya et al., 1962; Flavone ring structures are found in fruits, vegetables, Stenlid, 1968). Accumulation of flavanols () often grains, nuts, stems, leaves, flowers and roots and are ubiq- has been observed in nuclei, especially in gymnosperm uitous throughout nature, playing an integral role in plant species. Their roles could include the regulation of gene growth and development (Harborne, 1973). The term flavo- expression through chromatin remodelling and effects on noid generally is used to describe a broad collection of enzymes and protein complexes that regulate gene expres- natural products that possess a C6-C3-C6 skeleton, or more sion (Feucht et al., 2012). specifically a phenylbenzopyran function (Marais et al., In root tissues, flavonoids can accumulate at the root tip 2007). The typical flavone ring is the backbone of flavonoid and in root cap cells from where they can be exuded or structure, or the nucleus of diverse flavonoid molecules sloughed off into the soil (see below). Flavonoids also are (Fig. 1). The flavonoid biosynthetic pathway now is well localized in specific cell types of the root (Fig. 2), and can J Chem Ecol (2013) 39:283–297 285

Phenylalanine

4-coumaroyl CoA + 3 x malonyl CoA

Chalcone synthase

trihydroxy tetrahydroxy chalcone Phlobaphenes chalcone Chalcone Dihydroflavonol isomerase reductase

OH liquiritigenin Flavones OH OH Flavone HO O HO O synthase I + II Isoflavone synthase Isoflavone Luteolin synthase OH O isoflavone OH O

OH Isoflavone Vestitone reductase 3-OH-flavanones Flavonols OH reductase Flavonol HO O synthase

OH Isoflavonoids flavan-3,4-diols OH O HO O

O 3-OH- Condensed tannins

Medicarpin OCH3

Fig. 1 Simplified overview of the flavonoid biosynthetic pathway. Note that specific branches of the pathway might be plant species-specific. Major enzymes and end products are highlighted be readily studied by use of fluorescent imaging due to their transport is likely to be catalyzed by members of the ABC autofluorescence (Bayliss et al., 1997; Hutzler et al., 1998; transporter families because application of ABC transporter Mathesius et al., 1998). Roots typically produce many di- inhibitors reduced long distance auxin transport (Buer et al., verse flavonoids and these are stored as glycosides or agly- 2007). However, the specific molecular mechanisms of cones and released both by root exudation and tissue inter- and intracellular flavonoid transport require further decomposition or leaching (Rao, 1990). Root–produced fla- study. vonoids play roles in signalling to microbes and other Flavonoids have been shown to be readily exuded into plants, as well as in protection from soil pathogens, and the rhizosphere, although few studies have been able to their accumulation in roots is highly dependent on biotic quantify their exudation into soil. Flavonoid exudation has and abiotic environmental conditions (Rao, 1990). been shown to increase in response to various microbial signal molecules of symbionts and pathogens (Schmidt et al., 1994; Armero et al., 2001) and to abiotic stress including Flavonoid Transport, Exudation and Potential Roles nitrogen, temperature, and water stress (Coronado et al., in the Rhizosphere 1995; Dixon and Paiva, 1995; Juszczuk et al., 2004). Flavonoids also can be passively released from decompos- While flavonoid accumulation can be cell- and tissue- ing root cap and root border cells directly into the rhizo- specific, there is evidence for intra- and intercellular flavo- sphere (Hawes et al. 1998; Shaw et al. 2006). Although noid movement, in some cases through active transport. ABC transporters have been implicated in their exudation, Intracellular movement is most likely to occur via vesicle detailed mechanisms are not well understood. ABC trans- mediated transport or through membrane bound transporters porter mutants of Arabidopsis showed altered root exudate of the ABC (ATP binding cassette) or MATE (Multidrug profiles, which contained changes in flavonoids including And Toxic Extrusion compound) families (Zhao and Dixon, phytoalexins, as well as organic acids and sugars (Badri et 2009). In Arabidopsis, application of flavonoid aglycones to al., 2008, 2009, 2012) An ATP-dependent ABC transporter the root or the shoot leads to their transport across several also has been shown to be involved in the exudation of cell layers, suggesting that they can be easily transported genistein, an isoflavonoid, from soybean root plasma mem- between cells (Buer et al., 2007). This long distance brane vesicles (Sugiyama et al., 2007). Similarly, silencing 286 J Chem Ecol (2013) 39:283–297

A B Hooker, 2008). Their mobility in soil varies greatly with

p chemical composition, e.g., glycosylation, which deter- mines their water solubility. In turn, the presence of flavo- noids in soil can alter soil composition and nutrient availability through their activity as antioxidants and metal chelators. Chelation and reduction of metals in the soil c impact nutrient availability, especially phosphorus and iron. For example, an isoflavonoid identified from Medicago sativa L. root exudates dissolved ferric phosphate, enhanc- C ing phosphate and iron availability (Masaoka et al., 1993). The flavonoids genistein, quercetin and alter iron availability by reducing Fe(III) to Fe(II) and by chelat- ing unavailable iron from iron oxides (Cesco et al., 2010). c Some of the more well-known biological roles of flavo- noids in the rhizosphere include the activation of nod genes from symbiotic rhizobia, chemo-attraction of rhizobia and nematodes, inhibition of pathogens, and activation of D mycorrhizal spore germination and hyphal branching (Table 1). These functions can indirectly affect the growth of conspecifics through the regulation of nitrogen fixation and mycorrhization, as well as their susceptibility to pathogens (Fig. 3). Legume root exudates contain species-specific flavo- noids, specifically flavones, that activate the nodulation genes of their respective symbionts by binding to the tran- Fig. 2 Flavonoid accumulation in roots is cell type specific. a Flavo- scriptional activator NodD (Redmond et al., 1986; Peters noid accumulation in root tips of Medicago truncatula.Blueand orange autofluorescence is due to the presence of flavonoids. Note and Long, 1988; Cooper, 2004; Peck et al., 2006). This the significant accumulation of flavonoids in the root tip and in root leads to Nod factor synthesis and subsequent infection and cap cells (orange, arrow). b Flavonoid accumulation in a mature white nodulation of the legume host. However, some flavonoids, clover (Trifolium repens L.) root. Note the accumulation of different especially isoflavonoids, also inhibit nod gene induction flavonoids exhibiting different emission wavelengths in different cell types, e.g. pericycle (p) and cortex (c). c Flavonoid accumulation in a (Zuanazzi et al., 1998). Some flavonoids that induce nod young but differentiated root section of white clover in cortex cells (c). genes, specifically luteolin and , have dual actions d Flavonoid accumulation in a section through the root tip of white as chemo-attractants, with different flavonoids attracting dif- clover showing flavonoid accumulation in nuclei of meristematic cells ferent Rhizobium species (Aguilar et al., 1988;Dharmatilake (light blue) and in the cytoplasm of epidermal and outer cortical cells (yellow, arrow). All images were taken using fluorescence microscopy and Bauer, 1992). Flavonoid exudation by the host changes with UV excitation. Magnification bars are 500μmina and 100μmin during different stages of the symbiosis, presumably fine- b, c and d tuning Nod factor synthesis during nodule development and colonisation (Dakora et al., 1993). Flavonoid composition in of the ABCG-type transporter MtABCG10 from Medicago the rhizosphere around legume roots also can be altered by truncatula Gaertn. recently was shown to significantly re- rhizobia, which metabolise and alter the structure of flavo- duce accumulation of several isoflavonoids in roots and root noids over time (Rao and Cooper, 1994, 1995). exudates, and this was accompanied by enhanced suscepti- Flavonoids and other phenolic compounds also specifi- bility of the roots to the pathogen Fusarium oxysporum cally repel soil-dwelling plant parasitic nematodes and affect (Banasiak et al., 2013). However, because ABC transporters hatching and migration. For example, the flavonols kaemp- are likely to have multiple substrates, future studies will ferol, quercetin, and myricetin acted as repellants for the have to demonstrate the specific role of (iso)flavonoids in root lesion nematode Radopholus similis and the root knot the altered exudates of these types of mutants.. nematode Meloidogyne incognita, whereas the isoflavo- Flavonoid persistence in the soil varies with environmen- noids genistein and and the flavone luteolin acted tal conditions and is influenced strongly by the presence of only on R. similis (Wuyts et al., 2006). Kaempferol, quer- soil microbes, some of which can metabolize or modify cetin, and myricetin also inhibited motility of M. incognita flavonoids (Hartwig and Phillips, 1991; Rao and Cooper, and kaempferol inhibited egg hatching of R. similis, whereas 1994,1995). Flavonoids also can become unavailable due to other nematodes were not affected by any of these com- absorption to soil particles and organic matter (Shaw and pounds. It was demonstrated that nematode-resistant plant J Chem Ecol (2013) 39:283–297 287

Table 1 Examples of flavonoids of different biosynthetic branches of the pathway and their roles in the rhizosphere

Flavonoid name Flavonoid class Function Reference

Luteolin Flavone Nod gene and chemotaxis inducer in rhizobia Cooper (2004); Peters and Long (1988) Nematode repellant Wuyts et al. (2006) 7,4′dihydroxy flavone Flavone Nod gene and chemotaxis inducer in rhizobia Cooper (2004); Djordjevic et al. (1987); Hyphal branching stimulator Redmond et al. (1986); Tsai and Phillips (1991) 5,7,4′-trihydroxy- Flavone Allelopathic inhibitor of seedling growth Kong et al. (2004, 2007) ‘,5’dimethoxyflavone Quercetin Flavonol Nod gene inducer Cooper (2004) Iron chelator Cesco et al. (2010) Nematode repellant and motility inhibitor Wuyts et al. (2006) Antimicrobial Naoumkina et al. (2010) Hyphal branching stimulator Tsai and Phillips (1991) Allelopathic inhibitor of seedling growth Rice (1984) Kaempferol Flavonol Nod gene and chemotaxis inducer in rhizobia Cooper (2004) Iron chelator Cesco et al. (2010) Nematode repellant, egg hatching and motility Wuyts et al. (2006) inhibitor Allelopathic inhibitor of seedling growth Levizou et al. (2004); Rice (1984) Myricetin Flavonol Nod gene inducer Cooper (2004) Nematode repellant Wuyts et al. (2006) Formonenetin Isoflavonoid Nod gene inhibitor Djordjevic et al (1987) Hyphal branching inhibitor Tsai and Phillips (1991) Genistein Isoflavonoid Nod gene inducer Cooper (2004) Iron chelator Cesco et al. (2010) Nematode repellant Wuyts et al. (2006) Daidzein Isoflavonoid Nod gene inducer Cooper (2004) Nematode repellant Wuyts et al. (2006) Coumestrol Isoflavonoid Nod gene inducer Cooper (2004) Hyphal branching stimulator Morandi et al. (2009) Medicarpin Isoflavonoid-derived Antimicrobial phytoalexin Naoumkina et al. (2010) pterocarpan Maackiain Isoflavonoid-derived Antimicrobial phytoalexin Naoumkina et al. (2010) pterocarpan

cultivars contained increased amounts of flavonoids, spe- pisatin from pea provided protection from pathogenic fungi cifically the isoflavonoids and the pterocarpan medicarpin, and oomycetes (Pueppke and Vanetten, 1974). The likely in alfalfa (M. sativa L.) (Baldridge et al., 1998; Edwards mechanism of action against fungi is through inhibition of et al., 1995) elongation of fungal germ tubes and mycelial hyphae Flavonoids also inhibit a range of root pathogens, espe- (Blount et al., 1992; Higgins, 1978). cially fungi (Makoi and Ndakidemi, 2007). Generally, iso- During pathogen attack in a resistant plant species, phy- flavonoids, , or flavanones have been found as the toalexins are thought to become oxidized, leading to forma- most potent antimicrobials. These defense compounds can tion of toxic free radicals that can stimulate cell death during either be induced upon pathogen attack (phytoalexins) or be a hypersensitive response (Heath, 2000). Flavonols also preformed (phytoanticipins), while others are exuded into contribute to resistance against pathogens. For example, the soil (Armero et al., 2001). In this role, flavonoids have quercetin is antimicrobial and inhibits the ATPase activity been shown to act as antimicrobial toxins (Cushnie and of DNA gyrase in bacteria (Plaper et al., 2003; Naoumkina Lamb, 2011) and anti- or pro-oxidants (Jia et al., 2010). et al., 2010). Carnation (Dianthus caryophyllus) also has Pterocarpans, end products of the isoflavonoid pathway, been shown to mount a significant defense against Fusarium including medicarpin, pisatin and maackiain also have anti- oxysporum by formation of the fungitoxic flavonol triglyco- microbial properties (Naoumkina et al., 2010). For example, side of kaempferide (Curir et al., 2005). 288 J Chem Ecol (2013) 39:283–297

OH OH

HO O

OH O Luteolin (flavone): Nod gene inducer p-coumaric acid and other simple phenolics: OH Phytoinhibitors OH OH HO O OH

OH HO O Coumestrol OH O Quercetin (flavonol): OH (coumestan, Maackiain Antioxidant, chelator OH O isoflavonoid): (pterocarpan, Kaempferol (flavonol) of iron in soil, Stimulator of VAM isoflavonoid): Phytoinhibitor, nematode repellant, hyphal branching, antimicrobial auxin transport inhibitor, phytoinhibitor Nod gene inhibitor phytoalexin nematode repellant

Fig. 3 Overview of flavonoid functions in the rhizosphere

Other known roles of flavonoids in the rhizosphere in- Role of Flavonoids in Allelopathy and Plant Defense clude effects on arbuscular mycorrhizal fungi, which form a beneficial symbiosis with the majority of land plants under Allelopathy is defined as the direct or indirect effect of conditions of phosphorus deficiency (Harrison, 2005). secondary products produced by a donor plant upon a re- Hyphae of the mycorrhizal fungi are attracted to root exu- ceptor plant; these interactions are most often classified as dates, and in some cases this has been attributed to the detrimental or harmful, but allelopathic interactions also can presence of flavonoids, which stimulate hyphal branching be described as beneficial (Rice, 1984). The importance of and presymbiotic growth towards the host (Siqueira et al., allelopathy in agriculture is increasingly recognized(e.g., 1991; Scervino et al., 2005a, b, 2006, 2007; Steinkellner et see papers in this issue by; Kato-Noguchi and Peters, al., 2007). In Medicago truncatula Gaertn., the hyper- 2013; Shulz et al., 2013; Weston et al., 2013; Wothington accumulation of coumestrol, a potent hyphal stimulator, is and Reberg-Horton, 2013; Zhang et al., 2013), and allelo- correlated with hyperinfection by the symbiont (Morandi et pathic interactions are often used for weed suppression or al., 2009). However, both hosts and non-hosts also have additional management of weeds in diverse cropping sys- been reported to exude flavonoids that inhibit hyphal tems (Weston, 1996; Xuan and Tsuzuki, 2002; Weston and branching (Tsai and Phillips, 1991; Akiyama et al., 2010). Duke, 2003;). Exudation of flavonoids from the host also is phosphorus- Flavonoids have been reported in the literature for over regulated (Akiyama et al., 2002), similar to the dependence 50 years as chemical mediators involved in allelopathic of flavonoid accumulation on nitrogen availability in interactions in the soil rhizosphere. Many plants produce legumes forming nitrogen-fixing symbioses (Coronado et copious quantities of diverse flavonoids from their living al., 1995). As one can see from review of the plant roots, and our capacity to identify these metabolites in trace literature, the role of flavonoids in plant defense in the quantities has improved dramatically in recent years, hence rhizosphere and other physiological processes is complex many recent publications have focused on the role of flavo- and will no doubt be the subject of additional study as noids in allelopathy. As a result, flavonoids are more fre- we continue to evaluate regulation of these processes quently implicated in allelopathic interactions in the soil from a molecular perspective. rhizosphere as they have been identified in significant J Chem Ecol (2013) 39:283–297 289 concentrations in many bioactive root exudates. Both simple and more complex phenolics, including flavonoids, are re- leased from decomposing plant tissues as leachates and through the process of microbial degradation and transfor- mation in soil. In this review, we examine several case studies of allelopathy in both natural and managed eco- systems, including plants of diverse taxonomic origin, to demonstrate how allelopathic interference is potentially modulated by the presence of bioactive flavonoids in the rhizosphere.

Alfalfa and Clover Autotoxicity The perennial legumes al- falfa (Medicago sativa L.) and red or white clover (Trifolium pratense L. or Trifolium repens L.) are widely used in Fig. 4 Established stand of alfalfa (Medicago sativa L.) in Australian temperate regions as high quality pastures and fodder plants paddock. Note the concentric space around each alfalfa plant, with containing substantial levels of protein (Oleszek and little to no other vegetation in this concentric ring. Older alfalfa stands often exhibit autotoxicity and allelopathy over time and individual Jurzysta, 1987; Hancock, 2005). These crops also are im- plant growth becomes limited by presence of adjacent plants. Photo portant for their contributions of large quantities of organic taken by P. A. Weston, Australia matter to the soil, improvement of soil structure, and en- hanced water infiltration following establishment. Alfalfa generally contributes about two fold higher levels of organic numerous phytoinhibitors including that were sol- dry matter in comparison to the forage crops of red or white uble in water or alcohol, the presence of medicagenic acid clover. Most alfalfa and certain clovers typically are estab- along with other unidentified water- soluble inhibitors was lished as perennials, and as such they tend to be fairly associated with inhibition. resistant to weed infestation over time. However, as pastures Other investigators also have found that alfalfa plants age, both established alfalfa and clover pasture stands often released water-soluble allelochemicals from fresh leaves, exhibit significant reductions in plant counts and productiv- stems and crown tissues as well as dry hay, older roots, ity. This phenomenon, known as autotoxicity, severely lim- and seeds (Klein and Miller, 1980; Hedge and Miller, its the ability of producers to renovate declining pastures 1992; Tsuzuki et al., 1999; Xuan and Tsuzuki, 2002). In a (Tesar, 1993; Hancock, 2005). When renovating these pas- cultivar study, the Japanese cultivar Lucerne was deter- tures, the planting of successive crops also frequently leads mined to be most inhibitory to seedling germination and to poor stands in crops immediately following established growth when compared to other cultivars in laboratory and clover or alfalfa. In the past, the cause of this phenomenon greenhouse experimentation (Xuan and Tsuzuki, 2002). was thought to be depletion of soil moisture and nutrients or Miller (1996) and Chung and Miller (1995) also observed build-up of soil pathogens during perennial crop growth, but significant cultivar differences in phytotoxicity of alfalfa now there is evidence that these crops also exhibit phyto- extracts upon seedling growth, with Pioneer 5472 the most toxicity or allelopathy, through production and release of suppressive when compared with other American alfalfa toxic secondary metabolites (see also this issue Huang et al., cultivars. 2013). In the case of alfalfa or lucerne, autotoxicity can limit Hancock has speculated on the evolutionary role or pur- the development and productivity of the crop itself pose of autotoxicity in alfalfa (also known as lucerne) or (Cosgrove and Undersander, 2003; Hancock, 2005)and clover. Alfalfa, along with other perennial pasture legumes, result in permanent morphological reductions in root devel- is believed to have developed and evolved in the northern opment and shoot growth (Jennings, 2001; Jennings and and eastern coastal regions of the Mediterranean. During Nelson, 2002) (Fig. 4.) the period in which evolution was thought to have oc- Since these perennial legumes are known to be both curred, these areas likely experienced hot dry conditions autotoxic and allelopathic (Hedge and Miller, 1992), numer- and resource limitations (Hancock, 2005). Under condi- ous investigators have attempted to identify the allelochem- tions such as these, Hancock and others postulated that a icals responsible for phytotoxicity, with limited success. competitive advantage would arise if other plants, includ- (Oleszek and Jurzysta, 1987) reported the release of water- ing alfalfa seedlings, could be prevented from establish- soluble allelochemicals from alfalfa and red clover root ing near mature plants, specifically through autotoxicity extracts, which inhibited fungal and seedling growth in a (Jennings, 2001)(Figs. 4, 5 and 6). variety of soils with different textural properties over time. In many legumes it has been well established that phe- They concluded that although the extracts contained nolics, including flavonoids, are routinely produced and 290 J Chem Ecol (2013) 39:283–297

interactions with fungal pathogens suggest that they also may be important for protection in plants against soil- borne pathogens while stimulating VAM fungi (Hartwig et al., 1991). Somewhat surprisingly, we have found that no systematic soil-based studies have attempted to characterize the bioactive constituents produced by exudation or released by older established Medicago sativa roots into the rhizo- sphere, nor has anyone attempted a practical determination of their concurrent impacts on seedling establishment, path- ogen suppression, VAM fungi or rhizobacterial growth and development. These organisms all are commonly found in temperate soils, and are likely to be impacted by secondary products such as flavonoids in the rhizosphere. Given the likelihood that a complex mixture of water-soluble com- pounds is released by mature alfalfa roots, secondary prod- ucts in a complex mixture could play either stimulatory or inhibitory roles in the rhizosphere, and these roles may change depending on available concentrations in the soil water solution. As we currently have the ability to accurately detect trace quantities of bioactive secondary products and sensitively perform metabolomic profiling by using both gas or liquid chromatography (GC or LC) coupled to mass spec- trometry, it is now possible to fully characterize production and turnover of flavonoids and other secondary products in the rhizosphere of mature alfalfa stand. Future studies could be designed to investigate alfalfa autoxicity under field con- Fig. 5 and 6 In contrast to an established alfalfa (Medicago sativa L.) ditions, in both young and mature alfalfa stands to further stand with limited weed infestation present (right), an adjoining determine the role of specific flavonoids and phenolics in this paddock in the absence of alfalfa shows significant infestation of both detrimental phenomenon. grass and broadleaf weeds (left). Photos taken by P. A. Weston, Flavonoid production in roots of perennial legumes also Australia has been shown to be strongly regulated by nitrogen supply. Under nitrogen limiting conditions, flavonoid biosynthesis released from developing roots during seed germination and genes such as and isoflavone reductase seedling establishment (Mandal et al., 2010) . As described, are upregulated and show enhanced expression, indicating these compounds play various critical roles in mediating the nitrogen nutrition status of the plant plays a role in legume Rhizobium symbiosis, but also play important roles impacting secondary product production (Coronado et al., in stimulation or inhibition of other soil organisms or prop- 1995). This also is the case for other secondary plant prod- agules (Buer et al., 2010; Mandal et al., 2010; Hassan and ucts such as the hydroxamic acids BOA and DIMBOA Mathesius, 2012). Interestingly, the same phenolic acids that produced by Secale cereale L. (Mwaja et al., 1995). The stimulate rhizobial defense and IAA production also function bioavailability of soil nitrogen could thus also play a critical as potent inhibitors of seed germination and seedling growth, role in the regulation of allelopathy or autoallelopathic and include p-coumaric acid, vanillic acid, ferulic acid, gallic interactions in established legume stands. acid, p-hydroxybenzoic acid, and aldehyde and other cinnamic In perhaps the most interesting field study outlining the acid derivatives (Rice, 1984; Weston and Duke, 2003; Batish fate of flavonoids over time, Fomsgaard and colleagues used et al., 2006). Flavonoids typically produced by alfalfa seed- sensitive LC-MS/MS techniques to profile a diverse group lings also promote spore germination of Glomus spp., impor- of over 20 flavonoids released from living and decomposing tant AM fungi that beneficially infect plants. Quercetin, 4’–7- white clover stands in Denmark, in situ and after soil incor- dihydroxyflavone and 4′–7-dihydroxyflavanone were shown poration of the clover as a green manure (Carlsen et al., to be especially stimulatory to spore germination in AM fungi 2012). As the authors report, numerous studies have impli- (Tsai and Phillips, 1991). cated allelochemicals produced by white clover with weed Quercetin, luteolin, and other substituted flavones and suppression, as well as negative interactions associated with flavanones are released by germinating seeds and living allelopathy or replant/pathogenesis problems following roots of alfalfa and related legume crops over time, and their white clover establishment. This ground-breaking study J Chem Ecol (2013) 39:283–297 291 evaluated the pattern of flavonoid release from living clover induced strong ageotropic responses. The loss of normal grown under field conditions and also from leachates fol- gravitropic orientation could result from the activity of some lowing incorporation of green cover crops into field soil. flavonoids as polar auxin transport inhibitors, and resulting Their findings help to explain the potential for allelopa- perturbations in growth could impact ability to acquire thy and autoallelopathic interactions associated with estab- resources and competitive interference. As mode of action lished white clover stands. Specifically, the flavonoid studies are limited, additional studies with specific flavo- aglycones formononetin, medicarpin, and kaempferol pre- noids and mixtures are needed to further define their impact dominated in soil analyses, with glycosides of kaempferol on root growth and morphology. However, as Levizou et al. and quercetin also present at relatively high concentrations. (2004) suggest, the lack of dose dependent inhibition Kaempferol persisted for days in field soil surrounding responses with many flavonoids indicates that activity is living or incorporated clover stands. These aglycones highly concentration dependent; some of these compounds and related constituents have specifically been noted to could be inhibitory or stimulatory depending on available possess substantial phytoinhibitory activity (Rice, 1984). concentration in the soil/water solution in the rhizosphere. Kaempferol and kaempferol-3-O-L-arabinofuranoside stim- Legume root exudates also contain allelochemicals active ulated seed germination at low concentrations but inhibited against the parasitic weed Striga, which constitutes a major seedling growth at higher concentrations (Hai et al., 2008); constraint to African agriculture with yield losses up to these compounds also are present in walnut (Juglans regia 100 % in large parts of sub Saharan Africa (Gressel et al., L.) leaf extracts. The Carlsen study (2012) also noted that 2004). The forage legume Desmodium uncinatum was iden- highest concentrations of flavonoids in clover crops were tified as an effective intercropping plant because it inhibits associated with presence of clover flowers, in comparison to post-germination and attachment of Striga, and this inhibi- leaves, stems or roots in soil degradation studies. Several of tion was mimicked by several (iso)flavonoid glycosides the flavonoids identified are also known inhibitors of fungal identified from its root exudates (Hooper et al., 2010; growth, while others are associated with stimulation of Khan et al., 2010). Interestingly, some of these microbial growth in the rhizosphere (Mandal et al., 2010). stimulated Striga germination, and this could later be po- In a recent study, Sosa and colleagues (2010) noted that tentially exploited to cause ‘suicidal’ germination of Striga. flavonoid aglycones derived from Cictus landanifer also In Africa, the use of Desmodium as an intercrop plant has persisted for very long periods of time in soil before degra- been a cheap and successful strategy for smallholder farmers dation, even with no further addition of plant leachates. to control Striga infestations in their fields, with added Based on these interesting studies, we suggest that addi- advantage that some of the isoflavonoids are also active tional experimentation is required to determine 1) mobility against stem borers affecting the crops (Khan et al., 2006). of these compounds in various soil types and profiles, 2) location of maximal concentrations (likely to be nearest The Role of Flavonoids in Barley and Rice Allelopathy living roots, for example), and 3) half-life of major flavo- Barley (Hordeum vulgare L.) is an important and ancient noids and their glycosides in living soils. The application of cereal crop grown as a source of protein for human and comprehensive metabolic and proteomic profiling per- animal consumption in temperate regions of the world. formed from similarly designed experimentation with Barley was reported to exhibit weed suppression over legumes growing in a field setting will most certainly aid 2,000 years ago (Bertholdsson, 2004; Kremer and Ben- in further defining the roles of flavonoids, phenolics, and Hammouda, 2009). More recently, barley has been noted their related degradation products in the rhizosphere. to exhibit allelopathic activity when used as a rotational crop Although many flavonoids have been implicated in allelo- with succeeding small-seeded crops (Bertholdsson, 2004). It pathic inhibition of seedling growth and radicle elongation was also noted to suppress successive crops of bread and such as kaempferol and 6-methoxy-kaempferol, and rham- durum wheat (Triticum aestivum L. and T. durum L., netin and isorhamnetin (Levizou et al., 2004), the mode of respectively) (Kremer and Ben-Hammouda, 2009). Certain action of these inhibitors has not often been studied cultivars of barley also exhibit strong autotoxic tenden- (Berhow and Vaughn, 1999). cies, especially when grown under droughty conditions In studies that evaluate the activity of selected flavonoids in the field (Lovett and Hoult, 1995;KremerandBen- implicated in allelopathic interactions, some of the flavo- Hammouda, 2009). Repeated plantings of barley have prov- noid mixtures tested caused inhibition of root growth, re- en to be high risk for the development of autoallelopathy, duction in frequency of cell division in root meristematic particularly in droughty areas, especially with certain culti- regions, and suppression in the formation of root hairs and vars. Barley residues also were weed suppressive in trials statocytes in root cap cells (Levizou et al., 2004). Further conducted in the US, Canada and Europe. Spring barley evaluation of primary root formation in lettuce indicated that residues were particularly inhibitory to newly germinated eriodictyol, naringenin, and quercetin 3,3-dimethylether weed seedlings (Putnam and DeFrank, 1983). Despite the 292 J Chem Ecol (2013) 39:283–297 considerable literature pointing to the fact that barley is weed seeds, including barnyardgrass (Echinochloa crus- more allelopathic under certain field conditions, very limited galli L.), several Cyperus spp., and also to spore germina- research has yet been performed to identify the phytotoxins tion of several soil-borne fungal pathogens. The combina- associated with interference. Kremer and Ben-Hammouda tion of both compounds resulted in greater inhibitory (2009) compiled the results of numerous field and green- activity to weed propagules. Both compounds were shown house experiments involving barley and found that 44 dif- to be released into the soil rhizosphere, after degradation or ferent allelochemicals were potentially associated with leaching of rice residues, and infestation of barnyardgrass toxicity in these studies. These were primarily identified around living rice resulted in greater release of these sec- from leaf, stem and seed extracts, but a number of com- ondary products in soil compared to weed free plots (Kong pounds also were identified from root exudates or extracts. et al., 2004, 2007). Silencing the PAL pathways in rice by Potential allelochemicals included alkaloids, phenolics, fla- RNA interference (RNAi) leads to decreased phenolics pro- vonoids, cyanoglucosides, polyamines and hydroxamic duction and root exudation and subsequent effects on soil acids, indicating that barley possesses a diversity of bioac- microbial populations (see this issue, Fang et al., 2013). tive secondary products capable of eliciting numerous rhi- Interestingly, a review of the literature related to flavo- zosphere interactions. The alkaloids hordenine and gramine noid activity shows that many of the same compounds were exceptionally disruptive of cellular processes in affected associated with reduction in seed germination or seedling plants, and five phenolic acids were also implicated in barley growth also are active on soil pathogens, indicating some autoxicity studies. broad spectrum activity of both commonly reported and rare Barley was noted to produce several unique flavonoids in plant flavonoids. In additional research performed by the its tissues, with most studies reporting existence of these Kong laboratory (Kong et al., 2007), two flavone glycosides compounds in shoot residues, as often only shoot tissues were identified in extracts obtained from the allelopathic were assayed in reported experiments. Unusual flavonoids rice cultivar. Glycosides could not be detected in rice soils identified included lutonarin, saponarin, and isovitexin, as as they degraded, but both aglycones were more stable, with well as and . Potential modes of action half-lives of up to 24 h in living rice soils. The flavonoid include inhibition of cell growth, disruption of ATP forma- aglycones were less mobile in the soil profile as compared to tion, and interference with auxin function (Berhow and glycosides, indicating the aglycones are likely to play a Vaughn, 1999). Traditional breeding and selection may be more important role in suppression of plant growth or soil useful for enhancement of allelopathic activity; Bertholdsson pathogens over time. (2005) showed that enhanced early vigor and weed suppres- sive tendencies could be selected for as useful traits in Flavonoid Production in Ferns is Associated With barley germplasm (Bertholdsson, 2005). Older land races Allelopathy Ferns often have been observed to form dense were likely to possess greater suppressive or allelopathic monocultural stands in conditions of low light in forest tendencies than newly developed cultivars (Bertholdsson, understories. Ferns are the oldest plant taxa and exhibit 2004) and could be a source of future germplasm for considerable morphological diversity, but despite taxonomic selection of enhanced weed suppression. interest in their origin and spread, few studies have been Research on rice (Oryza sativa L.) allelopathy has been conducted from a chemotaxonomic perspective. Ferns ex- underway for many years (Weston, 1996, 2005; Weston and hibit a rich array of secondary metabolites, and many exhibit Duke, 2003; see also this issue Kato-Noguchi and Peters, strong allelopathic tendencies, forming distinct concentric 2013; Gealy et al., 2013; Fang et al., 2013; Worthington and spheres of inhibition around mature plants. In an interesting Reberg-Horton, 2013). This tropical or semi-tropical cereal study performed with Pityrogramma ferns, bioactive flavo- has been known to exhibit weed suppressive tendencies that noids were identified in fern foliage, including high concen- are cultivar dependent. Currently, rice cultivars have been trations of chalcones and dihydrochalcones (Star, 1980). selected for enhanced allelopathic activity in both the US Some ferns produce flavonoids in their roots, and others and China (Weston and Duke, 2003; Kong et al., 2004). The produce high concentrations in powders, called farinas, of presence of momilolactone A and B in rice roots and white, yellow, or gold on the abaxial sides of their fronds. In shoots, as well as root exudates has been considered critical high concentrations, these powders flake off fern fronds and to allelopathic activity in certain rice cultivars (Kato- fall to the soil surface, thus interacting with germinating weed Noguchietal.,2002). However, a bioactive flavone seeds, and potentially contributing to the sphere of inhibition (5,7,4′-trihydroxy-3′,5′-dimethoxyflavone), a cyclohexe- surrounding established ferns (Cooper-Driver, 1980). none, and a mixture of long-chain and cyclic hydrocarbons In past literature, angiosperms were characterized on the were isolated from allelopathic rice residues, accession basis of their flavonoids; specifically, primitive woody PI312777 by the Kong laboratory in China. Both the fla- plants containing flavonols and proanthocyanaidins, herba- vones and cyclohexenone were particularly inhibitory to ceous advanced plants containing a mixture of flavonols and J Chem Ecol (2013) 39:283–297 293 flavones, and highly advanced herbaceous plants containing generation of mutants with high throughput sequencing only flavones (Cooper-Driver, 1980). Flavonoid distribution techniques may prove useful (see also this issue Duke et in ferns showed a similar pattern with the most primitive al., 2013). herbaceous fern containing flavones and biflavones but not Large knowledge gaps also remain in our understand- flavonols and . The mixture of flavonoids ing of how flavonoids act as allelopathic compounds. encountered in many ferns have proven to be effective This review has indicated that it will be important to antimicrobial agents, preventing disease in ferns and also identify molecular targets of flavonoids in plant species playing unknown roles in the rhizosphere as defense com- that are inhibited, thereby unravelling mechanisms of pounds. The role of flavonoids in fern ecology requires how allelopathic plants that produce flavonoids are pro- further investigation, in light of new findings regarding their tected from autotoxicity. So far it is unclear whether role in chemical signalling and allelopathic interactions. It flavonoids are taken up by target species, as suggested would be useful to revisit the chemotaxonomic surveys by experiments in Arabidopsis in vitro (Buer et al., performed prior to 1980, given our increased sensitivity 2007), whether the uptake varies among species, whether and capacity to detect and identify secondary metabolites plants utilize transporter proteins for flavonoid uptake, in planta and in surrounding soils, and the information we and whether flavonoids have different molecular targets have now garnered about flavonoids diversity in legumes in various species. It also is possible that flavonoids and other higher plant species. exuded into the rhizosphere are first modified by soil microorganisms (Rao and Cooper, 1994) before becoming bioactive as allelochemicals. Flavonoids could be taken Future Research Directions up into sensitive plants by traditional uptake and trans- port processes as well as by fungal networks involving Major gaps in our knowledge of flavonoid exudation and mycorrhizae (Barto et al., 2012). function in the rhizosphere, particularly these involving allelopathic interactions, include the detailed mechanisms Acknowledgements The authors are grateful to the Australian of flavonoid exudation and the identification of transport Research Council for funding for a Future Fellowship to UM (FT100100669) and New South Wales Office of Medical and Science mechanisms and transporter proteins specific to flavonoid Research for funding a Biofirst Life Sciences Research Fellowship to exudation. Future study of the regulation of flavonoid trans- LAW. The authors also acknowledge the helpful reviews received porters by abiotic and biotic rhizosphere signals will be during the review process. important to gain information on flavonoid release rates/flux over time. Additionally, measurements of actual flavonoid concentrations in real soil environments are largely lacking. This potentially could be be accomplished by solid phase References root zone extraction using micro-extraction techniques in specific rhizosphere locations to determine spatial and tem- AGATI, G., AZZARELLO, E., POLLASTRI, S., and TATTINI, M. 2012. poral changes in flavonoid exudation (Mohney et al., 2009; Flavonoids as antioxidants in plants: Location and functional significance. Plant. Sci. 196:67–76. Weidenhamer et al., 2009). Such an approach might allow AGUILAR, J. M. M., ASHBY, A. M., RICHARDS, A. J. M., LOAKE, G. J., more precise estimations of flavonoid breakdown and WATSON,M.D.,andSHAW, C. H. 1988. Chemotaxis of movement in the soil. 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