Long-distance control of nodulation: Molecules and models

Legume plants develop root nodules to recruit nitrogen-fixing bacteria called rhizobia. This symbiotic relationship allows the host plants to grow even under nitrogen limiting environment. Since nodule development is an energetically expensive process, the number of nodules should be tightly controlled by the host plants. For this purpose, legume plants utilize a long-distance signaling known as autoregulation of nodulation (AON). AON signaling in legumes has been extensively studied over decades but the underlying molecular mechanism had been largely unclear until recently. With the advent of the model legumes, L. japonicus and M. truncatula, we have been seeing a great progress including isolation of the AON-associated receptor kinase. Here, we summarize recent studies on AON and discuss an updated view of the long-distance control of nodulation.     

Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation

Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot.     

Autoregulation and Nitrate Inhibition of Nodule Formation in Soybean cv. Enrei and its Nodulation Mutants

The regulation and nitrate inhibition of nodule formation insoybean, Glycine max (L.) Merr., was further examined usingthe nodulation mutants of cv. Enrei. The non-nodulating mutantsEn115, Enl282, and En1314 produced extremely few markedly-curledroot hairs which were all devoid of infection threads, and invariablyfailed to initiate sub-epidermal cell divisions (SCDs) in theroot cortex. A considerable number of arrested SCDs was foundbefore nodule emergence in Enrei, but not in En6500 which hadsignificantly more SCDs that progressively increased at moreadvanced stages of nodule ontogeny. These observations indicatethat autoregulation acts by blocking the developmental stagebefore nodule emergence. In both Enrei and En65OO, the maturationof emerged nodules was restricted by a late-acting nodulationcontrol mechanism that is apparently unrelated to autoregulation.Reciprocal wedge-grafts of plants inoculated at sowing showedthat the control of the supernodulating phenotype resides inthe shoot, while the non-nodulating phenotype is strictly root-controlled.The nodulation phenotype of the current non-nodulating mutantsresults not from an alteration of the autoregulatory mechanism,but from mutation that exerts a root-localized effect that blocksSCDs which trigger the autoregulatory mechanism. Reciprocalgrafting experiments on Enrei and En6500 seedlings grown undervarious nitrate levels suggest that nitrate inhibition of nodulation,like autoregulation, is shoot-controlled. Since these two processesare invariably expressed together, they are probably causallyrelated, acting synergistically to regulate nodule formationin soybean. These results indicate that the regulation and nitrateinhibition of nodulation in the nodulation mutants of cv. Enreiare similar to those of cv. Bragg nodulation mutants. Key words: Autoregulation, nitrate-tolerant symbosis, non-nodulating mutants, soybean, supernodulating mutant     

Molecular mechanisms controlling legume autoregulation of nodulation

Background

High input costs and environmental pressures to reduce nitrogen use in agriculture have increased the competitive advantage of legume crops. The symbiotic relationship that legumes form with nitrogen-fixing soil bacteria in root nodules is central to this advantage.

Scope

Understanding how legume plants maintain control of nodulation to balance the nitrogen gains with their energy needs and developmental costs will assist in increasing their productivity and relative advantage. For this reason, the regulation of nodulation has been extensively studied since the first mutants exhibiting increased nodulation were isolated almost three decades ago.

Conclusions

Nodulation is regulated primarily via a systemic mechanism known as the autoregulation of nodulation (AON), which is controlled by a CLAVATA1-like receptor kinase. Multiple components sharing homology with the CLAVATA signalling pathway that maintains control of the shoot apical meristem in arabidopsis have now been identified in AON. This includes the recent identification of several CLE peptides capable of activating nodule inhibition responses, a low molecular weight shoot signal and a role for CLAVATA2 in AON. Efforts are now being focused on directly identifying the interactions of these components and to identify the form that long-distance transport molecules take.     

60Ma of legume nodulation. What's new? What's changing?

Current evidence suggests that legumes evolved about 60 million years ago. Genetic material for nodulation was recruited from existing DNA, often following gene duplication. The initial process of infection probably did not involve either root hairs or infection threads. From this initial event, two branched pathways of nodule developmental processes evolved, one involving and one not involving the development of infection threads to 'escort' bacteria to young nodule cells. Extant legumes have a wide range of nodule structures and at least 25% of them do not have infection threads. The latter have uniform infected tissue whereas those that have infection threads have infected cells interspersed with uninfected (interstitial) cells. Each type of nodule may develop indeterminately, with an apical meristem, or show determinate growth. These nodule structures are host determined and are largely congruent with taxonomic position. In addition to variation on the plant side, the last 10 years have seen the recognition of many new types of 'rhizobia', bacteria that can induce nodulation and fix nitrogen. It is not yet possible to fit these into the emerging pattern of nodule evolution.     

Signaling in symbiosis

In recent years, the major focus in nodulation research has been on the genetic dissection of Nod-factor signaling. Components of this pathway appear to be shared with signaling processes that are induced during the formation of mycorrhiza. With the cloning of orthologs of the NIN and DMI2 genes from several legumes, the molecular characteristics of components of the Nod-factor-signaling pathway are now starting to be revealed. Orthologs of HAR1, a key player in the systemic autoregulatory mechanism controlling nodule numbers, have also been cloned recently. The mechanism by which nodulation is autoregulated is related to that by which fixed nitrogen inhibits nodulation. Genes that are involved in Nod-factor signaling may be targets for mechanisms that suppress nodulation. If this is the case, it would bring two fascinating areas of symbiosis together.     

Search for nodulation-related CLE genes in the genome of Glycine max

CLE peptides are potentially involved in nodule organ development and in the autoregulation of nodulation (AON), a systemic process that restricts nodule number. A genome-wide survey of CLE peptide genes in the soybean glycine max genome resulted in the identification of 39 GmCLE genes, the majority of which have not yet been annotated. qRT-PCR analysis indicated two different nodulation-related CLE expression patterns, one linked with nodule primordium development and a new one linked with nodule maturation. Moreover, two GmCLE gene pairs, encoding group-III CLE peptides that were previously shown to be involved in AON, had a transient expression pattern during nodule development, were induced by the essential nodulation hormone cytokinin, and one pair was also slightly induced by the addition of nitrate. Hence, our data support the hypothesis that group-III CLE peptides produced in the nodules are involved in primordium homeostasis and intertwined in activating AON, but not in sustaining it.     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

Regulation of nodulation in Alnus incana-Frankia symbiosis

We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant⁻¹). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.     

Root-hair infection and nodulation of four grain legumes as affected by the form and the application time of nitrogen fertilizer

The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in bothVicia faba andPisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation ofV. faba, Phaseolus vulgaris andVigna sinensis. The inhibitory effect of urea on nodulation ofP. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events ofRhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.     

Long-distance transport of signals during symbiosis: Are nodule formation and mycorrhization autoregulated in a similar way?

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions.Key words: arbuscular mycorrhiza, autoregulation, CLE peptides, mutant, nodulation, split-root systemUnder natural conditions, growth of plants is often limited by the availability of nutrients such as nitrogen and phosphorous. Plants have therefore developed strategies to acquire nutrients with the help of soil microorganisms. Most land plants enter mutualistic root symbioses with arbuscular mycorrhizal (AM) fungi, whereas legumes form special root nodules containing nitrogen-fixing bacteria, so-called rhizobia.^1–4 Establishment and maintenance of symbiosis requires plant resources, such as photosynthetically assimilated carbon. To minimize these costs, host plants are able to control the degree of their symbiotic interactions. Above a critical threshold level further establishment of symbiosis is restricted—a feedback phenomenon termed autoregulation of symbiosis. Autoregulation can be experimentally demonstrated in split-root systems. When legume roots are already infected by rhizobia on one side of a split-root, further nodule development is “systemically” inhibited on the other side. Similarly, prior colonization of split-roots by AM fungi on one half suppresses later fungal root colonization on the other half. Hence, important elements of the symbiotic autoregulation circuit are not only localized in roots, but also in aerial parts of the plant, implicating transport of signals in vascular bundles (Fig. 1). Whereas autoregulation of nodulation in legumes has been studied for many decades,^5–9 the first publications clearly stating a shoot-controlled autoregulation of mycorrhization in split-root systems appeared in 2000 for the non-legume barley (Hordeum vulgare) and thereafter for alfalfa (Medicago sativa) and soybean (Glycine max).^10–13 The data from these split-root experiments are supported by the findings that supernodulating (or hypernodulating) loss-of-autoregulation mutants displayed either an increased degree of AM colonization and/or a higher abundance of arbuscules.^14–16Open in a separate windowFigure 1Proposed model of shoot-controlled autoregulation of symbiosis in a split-root system. Prior infection of root A by rhizobia or AM fungi systemically suppresses later establishment of symbiosis in root B. Expression of specific CLE peptides (and/or other peptide hormones) is induced in response to rhizobial nodulation signals (Nod factors) and perhaps also in response to colonization by AM fungi (stage 1). The CLE peptides (and/or other signals) are then presumed to be transported in the xylem to the shoot, where they are perceived by leucine-rich repeat (LRR) autoregulation receptor kinases (stage 2). As a result of autoregulation signaling in the shoot, an unknown shoot-derived inhibitor (SDI) is produced (stage 3) and transported as a phloem-mobile signal to the root. Perception and action of the SDI signal in roots would then inhibit nodulation and root colonization by AM fungi (stage 4).     

Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza.

Genetic and molecular mechanisms of development are compared for two major plant-microbe endosymbioses: N(2)-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N(2)-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripherical in legumes or central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia and located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps comprising four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. The origin of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g., inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). An analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N(2)-fixing symbioses.     

Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro

The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant NARK proteins showed autophosphorylation activity in vitro. Several regions of the NARK kinase domain were shown by mass spectrometry to possess phosphoresidues. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole plant autoregulation of nodulation. A wild-type NARK fusion protein transphosphorylated a kinase-inactive mutant NARK fusion protein, suggesting that it is capable of intermolecular autophosphorylation in vitro. In addition, Ser-861 and Thr-963 in the NARK kinase catalytic domain were identified as phosphorylation sites through site-directed mutagenesis. The genes coding for the kinase-associated protein phosphatases KAPP1 and KAPP2, two putative interacting components of NARK, were isolated. NARK kinase domain phosphorylated recombinant KAPP proteins in vitro. Autophosphorylated NARK kinase domain was, in turn, dephosphorylated by both KAPP1 and KAPP2. Our results suggest a model for signal transduction involving NARK in the control of nodule development.     

Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation

Legumes evolved about 60 million years ago (Ma), and nodulation 58 Ma. Nonnodulation remains common in Caesalpinioideae, with smaller numbers in Mimosoideae and Papilionoideae. The first type of infection by bacteria may have been at junctions where lateral roots emerged, followed by formation of infection threads to confine bacteria and convey them to some cells in the developing nodule, where they were generally released into symbiosomes. Infection threads were a prerequisite for root-hair infection, a process better controlled by the host, leading to a higher degree of specificity between symbionts. An alternative process, dating from the same time and persisting in about 25% of legumes, did not involve infection threads, bacteria entering a few host cells, surrounded by an undefined matrix. These cells divided repeatedly to give uniform infected tissue, with bacteria released into symbiosomes. Such legumes may have less stringent control of nodulation processes, and are found mainly in tropical and warm temperate areas. In each type of nodule, meristems may or may not be retained, leading to indeterminate or determinate forms. Nodule morphology and structure are host-determined, but the effectiveness of nitrogen fixation is largely controlled by the bacterial symbionts, which vary greatly in genotypic and phenotypic characters.     

Comparative genetics and evolutionary morphology of symbiosis formed by plants with nitrogen-fixing microbes and endomycorrhizal fungi

Results of comparative morphological and genetic analyses are described for two major plant-microbe endosymbioses: N2-fixing nodules (with rhizobia or actinomycetes Frankia) and arbuscular mycorrhiza (with Glomales fungi). Development from the primordia formed de novo in root tissues is common for all known types of N2-fixing nodules. However, their structure varies greatly with respect to: (i) tissue topology (location of vascular bundles is peripheral in legumes but central in non-legumes); (ii) position of nodule primordium (inner or outer cortex in legumes, whereas pericycle in non-legumes); (iii) stability of apical meristem (persistent in the indeterminate nodules, transient in the determinate ones). In addition, legumes vary in ability to form compartments harboring endosymbiotic rhizobia that can be located intercellularly (infection threads) and intracellularly (symbiosomes). Using pea (Pisum sativum) symbiotic mutants, the nodule developmental program is dissected into a range of spatially and temporarily differentiated steps composing four sub-programs (development of endosymbiotic compartments; nodule histogenesis; autoregulation of nodulation; bacteroid differentiation). The developmental mutations are suggested in some cases to reverse the endosymbiotic system into the morphologically simpler forms some of which may correspond to the ancestral stages of nodule evolution. Origination of legume-rhizobial and actinorhizal symbioses is suggested to be based on a set of preadaptations many of which had been evolved in angiosperms during coevolution with arbuscular mycorrhizal fungi (e.g. inter- and intracellular maintenance of symbionts, their control via defence-like reactions and recognition of chitin-like molecules). Analysis of parallel morphological variation in symbiotic mutants and wild-growing legume species enables us to reconstruct the major stages of evolution for N2-fixing symbioses. This evolution proceeded to a sufficient degree independently from the basic physiological function of nodules (symbiotic N2-fixation) and possibly a recruiting of plant genes that initially fulfilled various "non-symbiotic" functions into the genetic networks monitoring plant-microbe interactions.     

Dissecting Symbiosis: Developments in Nod Factor Signal Transduction

The interaction between legumes and rhizobial bacteria resultsin the formation of a unique organ, the nodule, on roots ofthe host plant. The nodule has evolved to harbour the bacterialsymbiont and provide conditions appropriate for the fixationof atmospheric nitrogen. Nod factor, generated by rhizobia,is sufficient to activate many of the responses involved inthe initiation of nodule development in the plant. Dissectingthe Nod factor signal transduction pathway has been greatlyaided by the adoption of genetically tractable model legumes.Recent studies have identified a number of genes involved inthis pathway and candidate proteins for the Nod factor receptor.Furthermore, a plethora of cellular responses have been linkedwith Nod factor perception. This Botanical Briefing covers recentadvances in the dissection of Nod factor signal transductionin the plant.Copyright 2001 Annals of Botany Company Review, Nod factor, rhizobia, nodulation, signal transduction     

Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.