Synthesis,release, and transmission of alfalfa signals to rhizobial symbionts

In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa L.) releases two betaines, trigonelline and stachydrine, that induce nodulation (nod) genes inRhizobium meliloti. Experiments with¹⁴C-phenylalanine in the presence and absence of phenylalanine ammonia-lyase inhibitors show that exudation of flavonoidnod-gene inducers from alfalfa roots is linked closely to their concurrent synthesis. In contrast, flavonoid and betainenod-gene inducers are already present on mature seeds before they are released during germination. Alfalfa seeds and roots release structurally differentnod-gene-inducing signals in the absence of rhizobia. WhenR. meliloti is added to roots, medicarpin, a classical isoflavonoid phytoalexin normally elicited by pathogens, and anod-gene-inducing compound, formononetin-7-O-(6-O-malonylglycoside), are exuded. Carbon flow through the phenylpropanoid pathway and into the flavonoid pathway via chalcone synthase is controlled by complexcis-acting sequences andtrans-acting factors which are not completely understood. Even less information is available on molecular regulation of the two other biosynthetic pathways that produce trigonelline and stachydrine. Presumably the three separate pathways for producingnod-gene inducers in some way protect the plant against fluctuations in the production or transmission of the two classes of signals. Factors influencing transmission of alfalfanod-gene inducers through soil are poorly defined, but solubility differences between hydrophobic flavonoids and hydrophilic betaines suggest that the diffusional traits of these molecules are not similar. Knowledge derived from studies of how legumes regulate rhizobial symbionts with natural plant products offers a basis for defining new fundamental concepts of rhizosphere ecology.     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3′,4′,5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.     

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394–5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using ¹H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.     

Major flavonoids in uninoculated and inoculated roots of Vicia sativa subsp. nigra are four conjugates of the nodulation gene-inhibitor kaempferol

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841–852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.     

Release of Flavonoids by the Soybean Cultivars McCall and Peking and Their Perception as Signals by the Nitrogen-Fixing Symbiont Sinorhizobium fredii

Sinorhizobium fredii strain USDA191 forms N-fixing nodules on the soybean (Glycine max L. Merr.) cultivars (cvs) McCall and Peking, but S. fredii strain USDA257 nodulates only cv Peking. We wondered whether specificity in this system is conditioned by the release of unique flavonoid signals from one of the cultivars or by differential perception of signals by the strains. We isolated flavonoids and used nodC and nolX, which are nod-box-dependent and -independent nod genes, respectively, to determine how signals activate genes in the microsymbionts. Seeds of cv McCall and cv Peking contain the isoflavones daidzein, genistein, and glycitein, as well as their glucosyl and malonylglucosyl glycosides. Roots exude picomolar concentrations of daidzein, genistein, glycitein, and coumestrol. Amounts are generally higher in cv Peking than in cv McCall, and the presence of rhizobia markedly influences the level of specific signals. Nanomolar concentrations of daidzein, genistein, and coumestrol induce expression of nodC and nolX in strain USDA257, but the relative nolX-inducing activities of these signals differ in strain USDA191. Glycitein and the conjugates are inactive. Strain USDA257 deglycosylates daidzin and genistin into daidzein and genistein, respectively, thereby converting inactive precursors into active inducers. Although neither soybean cultivar contains unique nod-gene-inducing flavonoids, strain- and cultivar-specific interactions are characterized by distinct patterns of signal release and response.     

Implications of long-distance flavonoid movement in Arabidopsis thaliana

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symbiotic interactions with other organisms. Recently we showed that flavonoids can move long distances in plants, which may facilitate these molecules reaching positions in the plant where these processes are regulated. The localised application of selective flavonoids to tt4 mutants such as naringenin, dihydrokaempferol and dihydroquercetin showed that they were taken up at the root tip, mid-root or cotyledons and travelled long distances via cell-to-cell movement to distal tissues and converted to quercetin and kaempferol. In contrast, kaempferol and quercetin do not move long distances. They were taken up only at the root tip and did not move from this position. Here we show the movement of endogenous flavonoids by using reciprocal grafting experiments between tt4 and wild-type seedlings. These results demonstrated that to understand the distribution of flavonoids in Arabidopsis, it is necessary to know where the flavonoid biosynthetic enzymes are made and to understand the mechanisms by which certain flavonoids move from their site of synthesis.Key words: flavonoid movement, reciprocal graft, quercetin, kaempferol, Arabidopsis thaliana, fluorescence, aglyconeFlavonoids are plant secondary metabolites made by the phenylpropanoid pathway. The central biosynthetic pathway is known and in Arabidopsis most of the enzymes in flavonoid synthesis are encoded by single copy genes.¹ The isolation of mutants with defects in the genes encoding these flavonoid biosynthetic enzymes has allowed researchers to understand the biochemical complexity of flavonoid synthesis and their biological roles. Flavonoid synthesis is more complex in other species, such as legumes, which produce a greater diversity of flavonoid molecules, and in which gene families encode the key enzymatic branch points of the pathway.^2,3The functions of flavonoids were demonstrated using genetic approaches that blocked flavonoid synthesis in Arabidopsis and other species. In Arabidopsis, flavonoids play important roles in UV protection⁴ and regulate auxin transport and dependent physiological processes, such as gravity responses,^5–7 and lateral root formation.⁸ In petunia, maize and tomato, pollen without flavonoids is infertile and this phenotype is reversed by flavonoid addition.^9–11 However, the enigma of why flavonoid-deficient Arabidopsis seedlings are fertile has not been resolved.¹² Flavonoids appear to interact with Multidrug resistance (MDR)/P-glycoproteins (PGP)/ABC-Type B proteins⁷ that transport auxin, regulate phosphatases and kinases, and may have regulatory roles as scavengers of reactive oxygen species (reviewed in ref. ¹³). These results are consistent with a diversity of important functions for flavonoids in plants that require careful control of flavonoid synthesis and localization.We have explored the possibility that flavonoid accumulation in specific locations is also modulated by movement of early intermediates of the flavonoid pathway. Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue.¹⁴ More recent grafting experiments showed that root synthesised metabolites, perhaps carotenoids, regulate shoot development,^15,16 flowering inducers travel long distances,¹⁷ and phytohormones are translocated (reviewed in ref. ¹⁸).We recently showed that flavonoids moved long distances in Arabidopsis using several approaches.¹⁹ The roots of Arabidopsis seedlings grown in complete darkness do not accumulate flavonoids⁵ since expression of early genes encoding enzymes of flavonoid biosynthesis are light dependent.²⁰ Yet, flavonoids accumulate in root tips of seedlings with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain (diphenyl boric acid 2-amino ethyl ester [DPBA]), and localised aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating that early intermediates in the flavonoid pathway can move long distances. This was confirmed by time-course fluorescence experiments and HPLC. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application mid-root or to cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol and dihydroquercetin were taken up at the root tip, mid-root or through cotyledons and travelled long distances via cell-to-cell movement to distal tissues followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Uptake of flavonoids at the root tip was inhibited by glybenclamide, a specific inhibitor of ABC type transporters²¹ suggesting a possible role for transporters of this class in the movement of flavonoids.To show that endogenous flavonoids are capable of long distance movement, we performed reciprocal butt grafting between tt4 and wild-type seedlings.²² In these experiments we asked whether flavonoids moved from wild-type tissues to flavonoid-deficient tissues of tt4. DPBA fluorescence detection was used to detect flavonoid movement into tt4 tissues.¹⁹ Seedlings were grafted and grown on filter paper in Petri dishes for 8 d. The seedlings were then transferred to equal parts sand, perlite and vermiculite to avoid the possibility of uptake of pre-existing flavonoids that may be natural soil components. After 14 d, the seedlings were stained with DPBA. When tt4 roots were grafted to tt4 shoots, the samples showed dim greenish autofluorescence in roots and only red chlorophyll fluorescence in the shoot. When either tt4 roots or shoots were grafted onto wild-type shoots or roots, respectively, the tt4 tissues showed bright yellow DPBA fluorescence (Fig. 1). The results of these experiments clearly showed that endogenous flavonoids moved across the graft to the reciprocal tissue. The flavonoid movement is specific to certain tissues, as flavonoids are not transported into the seeds developing on tt4 shoots grafted on wild-type roots, which retain the transparent testa phenotype. Although flavonoids clearly travelled from wild-type root tissue to mutant shoots, they were not capable of complementing the seed colour defect of tt4. In addition, adding naringenin to tt4 plants either to the media, or to the soil, also did not complement the seed colour phenotype in tt4 (data not shown). Recent research by Hsieh and Huang²³ may account for this inability to complement seed colouration, as flavonoids in the Brassicaceae end up in the pollen coat rather than the testa. The testa tissue derives from ovular tissue,²⁴ and thus is maternal in origin.Open in a separate windowFigure 1Grafting shows flavonoid movement occurs across grafts. Reciprocal grafting between wild type and tt4 indicated flavonoid movement from the flavonoid producing tissue to the chalcone synthase-deficient tissue. The order of the graft is indicated by the left legend as aerial tissue over root tissue in the graft. Micrographs are DPBA stained tissue excited with 488 nm wavelength. The tt4/tt4 control graft shows no flavonoids are present, even though a wound has occurred on the leaf which generally exacerbates flavonoid fluorescence. Scale bar = 100 µm. Green fluorescence is from kaempferol and gold from quercetin. The red fluorescence is from chlorophyll.The complexity of the flavonoid biosynthetic pathway and the large number of modified flavonoids that can be made through the complex series of glycosylation reactions suggests that distinct flavonoid molecules may have unique function. To fully understand these molecules, it is necessary to dissect the synthesis pathways for these glycosylated flavonoids. Two unnamed flavonoid glycoside mutants isolated in 1998,²⁵ have profound developmental phenotypes, supporting this hypothesis. These mutations resulted in whorled cauline leaves on inflorescences and double the number of rosette leaves. Our lab is in the process of determining if other phenotypes exist in flavonoid mutants.A critical feature of the observations of flavonoid movement is understanding the biological context of this movement. First, a number of recent studies reported physiological functions of flavonoids in roots, ranging from modulation of auxin transport and root gravitropism,^5–8 to nodulation³ and root branching,⁸ while it is clear that flavonoid synthesis is absent in dark-grown seedlings.⁵ Yet, for flavonoids to function in roots of plants grown in soil, the light signal and/or flavonoid precursors must travel to the roots. Additionally, transient flavonoid accumulation has been reported in roots reoriented relative to the gravity vector.⁶ For flavonoids to transiently accumulate at the root tip (at 2 hours after reorientation) and to return to lower levels (within 2 additional hours), suggests that more than flavonoid synthesis is regulated. Perhaps this transient flavonoid accumulation requires localized enzyme activation and transport mechanisms. As flavonoid transport is inhibited by a compound that blocks ABC transporters, which include the newly identified auxin transporters of the MDR/PGP class, perhaps there are connections between flavonoid and auxin transport that allow this transient accumulation. A more detailed understanding of this role of flavonoid movement in controlling plant development awaits additional experimentation.     

The Symbiotic Biofilm of Sinorhizobium fredii SMH12, Necessary for Successful Colonization and Symbiosis of Glycine max cv Osumi,Is Regulated by Quorum Sensing Systems and Inducing Flavonoids via NodD1

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.     

Localized Changes in Flavonoid Biosynthesis in Roots of Lotus pedunculatus after Infection by Rhizobium loti

Two-dimensional paper chromatography in four solvent systems, high-sensitivity spray reagents, and UV absorption spectroscopy were used to separate and characterize flavonoids and isoflavonoids in roots and root nodules of 20-d-old Lotus pedunculatus Cav. Seedlings were grown either under sterile conditions or after inoculation with Fix⁺ or Fix⁻ strains of Rhizobium loti. Flavonoids rather than isoflavonoids predominated in all tissues. Flavonoid profiles in sterile and denodulated root tissues were remarkably similar, both qualitatively and quantitatively. At least 14 partially purified flavonoid aglycones and conjugates were found in root extracts; denodulated root tissues contained no compounds that were not also present in sterile roots. Fix⁺ rhizobia were responsible for major postinfection shifts in plant flavonoid biosynthesis at the sites of nodule morphogenesis. Polymeric flavolans were absent from Fix⁺ nodules but present in all root tissues and in Fix⁻ nodules. Catechin was detected only in Fix⁺ nodules.     

Active extrusion of protons into deionized water by roots of intact maize plants

The investigations were focussed on the question as to whether roots of intact maize plants (Zea mays L. cv Blizzard) release protons into deionized H₂O. Plants in the six to seven leaf stage depressed the pH of deionized H₂O from 6 to about 4.8 during an experimental period of 4 hours. Only one-third of the protons released could be ascribed to the solvation of CO₂ in H₂O. The main counter anions released were Cl⁻, NO₃⁻, and SO₄²⁻. At low temperature (2°C), the H⁺ release was virtually blocked while a relatively high amount of K⁺ was released. The presence of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the external solution increased the H⁺ secretion significantly. Addition of vanadate to the outer medium inhibited the H⁺ release while fusicoccin had a stimulating effect. Substituting the nutrient solution of deionized H₂O resulted in a substantial increase of the membrane potential difference from −120 to −190 millivolts. The experimental results support the conclusion that the H⁺ release by roots of intact maize plants is an active process driven by a plasmalemmalocated ATPase. Since the net H⁺ release was not associated with a net uptake of K⁺, it is unlikely to originate from a K⁺/H⁺ antiport.