Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont.     

Microbial oligosaccharides differentially induce volatiles and signalling components in Medicago truncatula

Plants perceive biotic stimuli by recognising a multitude of different signalling compounds originating from the interacting organisms. Some of these substances represent pathogen-associated molecular patterns, which act as general elicitors of defence reactions. But also beneficial microorganisms like rhizobia take advantage of compounds structurally related to certain elicitors, i.e. Nod-factors, to communicate their presence to the host plant. In a bioassay-based study we aimed to determine to what extent distinct oligosaccharidic signals are able to elicit overlapping responses, including the emission of volatile organic compounds which is mainly considered a typical mode of inducible indirect defence against herbivores. The model legume Medicago truncatula Gaertn. was challenged with pathogen elicitors (beta-(1,3)-beta-(1,6)-glucans and N,N',N',N'-tetraacetylchitotetraose) and two Nod-factors, with one of them being able to induce a nodulation response in M. truncatula. Single oligosaccharidic elicitors caused the emission of volatile organic compounds, mainly sesquiterpenoids. The volatile blends detected were quite characteristic for the applied compounds, which could be pinpointed by multivariate statistical methods. As potential mediators of this response, the levels of jasmonic acid and salicylic acid were determined. Strikingly, neither of these phytohormones exhibited changing levels correlating with enhanced volatile emission. All stimuli tested caused an overproduction of reactive oxygen species, whereas nitric oxide accumulation was only effected by elicitors that were equally able to induce volatile emission. Thus, all signalling compounds tested elicited distinct reaction patterns. However, similarities between defence reactions induced by herbivory and pathogen-derived elicitors could be ascertained; but also Nod-factors were able to trigger defence-related reactions.     

Soil nematodes mediate positive interactions between legume plants and rhizobium bacteria

Symbiosis between legume species and rhizobia results in the sequestration of atmospheric nitrogen into ammonium, and the early mechanisms involved in this symbiosis have become a model for plant-microbe interactions and thus highly amenable for agricultural applications. The working model for this interaction states that the symbiosis is the outcome of a chemical/molecular dialogue initiated by flavonoids produced by the roots of legumes and released into the soil as exudates, which specifically induce the synthesis of nodulation factors in rhizobia that initiate the nodulation process. Here, we argue that other organisms, such as the soil nematode Caenorhabditis elegans, also mediate the interaction between roots and rhizobia in a positive way, leading to nodulation. We report that C. elegans transfers the rhizobium species Sinorhizobium meliloti to the roots of the legume Medicago truncatula in response to plant-released volatiles that attract the nematode. These findings reveal a biologically-relevant and largely unknown interaction in the rhizosphere that is multitrophic and may control the initiation of the symbiosis.     

Genealogy of legume-Rhizobium symbioses.

Accumulating evidence suggests that lateral transfer of nodulation capacity is an important driving force in symbiotic evolution. As a consequence, many distantly related soil bacteria have acquired the capacity to invade plants and fix nitrogen within them. In addition to these proteins required for bacteroid development and nitrogen fixation, core symbiotic competence seems to require flavonoids, NodD proteins, lipochitooligosaccharidic Nod-factors, extra-cellular polysaccharides, as well as various exported proteins. Plants respond to different levels and combinations of these substances in species specific ways. After contact has been initiated by flavonoids and NodD proteins, constant signal exchange fine-tunes these symbiotic demands, especially to overcome defence reactions.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Role of rhizobial lipo-oligosacharides in root nodule formation on leguminous plants

During recent years signals leading to the early stages of nodulation of legumes by rhizobia have been identified. Plant flavonoids induce rhizobialnod genes that are essential for nodulation. Most of thenod gene products are involved in the biosynthesis of lipo-oligosaccharide molecules. The commonnodABC genes are minimally required for the synthesis of all lipo-oligosaccharides. Host-specificnod gene products in a givenRhizobium species are responsible for synthesis or addition of various moieties to those basic lipo-oligosaccharide molecules. For example, inR. leguminosarum, thenodFEL operon is involved in the production of lipo-oligosaccharide signals that mediate host specificity. AnodFE-determined highly unsaturated fatty acid (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic acid) is essential for inducing nodule meristems and pre-infection thread structures on the host plantVicia sativa. Lipo-oligosaccharides also trigger autoregulation of nodulation in pea and, if applied in excessive amounts to a legume, can prevent nodulation and thereby might play a role in competition. During our studies on the biosynthesis of lipo-oligosaccharides, we discovered that, besides the lipo-oligosaccharides, other metabolites are synthesizedde novo after induction of thenod genes. These novel metabolites appeared to be phospholipids, containing either one of the three fatty acids which are made by the action of NodFE inR. leguminosarum.     

Early interactions between legumes and rhizobia: disclosing complexity in a molecular dialogue

The exchange of chemical signals between soil bacteria (rhizobia) and legumes has been termed a molecular dialogue. As initially conceived in the early 1990s, it involved two main groups of molecules: nod gene-inducing flavonoids from plants and the mitogenic lipochito-oligosaccharide Nod factors of rhizobia. This review considers how subsequent research revealed the existence of a more complex set of interactions, featuring expanded roles for the original participants and contributions from additional plant and bacterial metabolites. Rhizobia respond to chemoattractants and growth-enhancing compounds in root exudates, and several plant nonflavonoids possess nod gene-inducing properties. Expression of non-nod genes is induced by flavonoids; these include encoders of a type I secreted protein and the entire type III, and possibly also type IV, secretion systems. Many other genes and proteins in rhizobia are flavonoid-inducible but their functions are largely unknown. Rhizobia produce far more Nod factor variants than was previously envisaged and their structures can be influenced by the pH of the environment. Other symbiotically active compounds or systems of rhizobia, some of them universally present, are: the surface polysaccharides, quorum-sensing N-acyl homoserine lactones, plant growth-promoting lumichrome and two-component regulatory systems.     

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.     

The Rhizobium, Bradyrhizobium, and Azorhizobium NodC proteins are homologous to yeast chitin synthases.

The nodABC genes of rhizobia are essential for the synthesis of lipo-oligosaccharidic (N-acylated chitin oligomers) nodulation signals. nodC gene products from Rhizobium, Bradyrhizobium, and Azorhizobium exhibit extensive homology with chitin synthases, suggesting that the NodC proteins are involved in the synthesis of the chitin oligomer backbone by catalyzing the beta-1,4-linkage between N-acetyl-D-glucosamine residues.     

Molecular mechanisms of Nod factor diversity

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.     

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.     

Chemotaxis,induced gene expression and competitiveness in the rhizosphere

Rhizobia are soil bacteria which symbiotically infect legume roots and generate nodules in which they fix atmospheric nitrogen for the plant in exchange for photosynthetically fixed carbon. A crucial aspect of signal exchange between these symbionts is the secretion of phenolic compounds by the host root which induce nodulation gene expression in the bacteria. Stimulation of nod gene expression by host phenolics is required for nodule formation, is biochemically specific at 10^-6 M, and is mediated by nodD. We and others have shown that rhizobia display chemotaxis to 10^-9 M of the same phenolic compounds. Chemotaxis to inducer phenolics is selectively reduced or abolished by mutations in certain nod genes governing nodulation efficiency or host specificity. Conversely, mutations in rhizobia that affect general motility or chemotaxis have substantial effects on nodulation efficiency and competitiveness. These findings suggest that microbes entering the rhizosphere environment may utilize minor, non-nutrient components in root exudates as signals to guide their movement towards the root surface and elicit changes in gene expression appropriate to this environment.     

Chitin recognition in rice and legumes

This review focuses on a comparison of plant reception of chitin oligosaccharides by legumes and rice. Chitin oligosaccharides (dp=6-8) released from fungal pathogens induce plant defense reactions in rice, while lipo-chitin oligosaccharides (dp=4-5) induce the development of a new plant organ, the nodule, in legumes during infection by rhizobia. The former situation is pathogenic and the latter situation beneficial to the plant. However, these two systems do share some common features. We hypothesize that rice and legumes, as well as other plants, may possess members of an evolutionarily conserved family of chitin binding proteins. These proteins may play an important role in chitin reception and subsequent signal transduction. However, data support the idea that legumes may possess a second chitin binding receptor that shows a greater specificity for the lipo-chitin nodulation signals. The presence of this second receptor may be one of the key factors that distinguishes plants capable of nodulation by rhizobia (e.g., soybean) from those that cannot be nodulated (e.g., rice).     

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.     

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.     

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.     

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.