Regulators and Regulation of Legume Root Nodule Development

Nitrogen is the nutrient plants require in the highest amount, and in agriculture nitrogen availability has a major influence on both yield and product quality. In nature plants acquire nitrogen by assimilation of nitrate and ammonium or from dinitrogen through association with nitrogen-fixing bacteria. Symbiotic nitrogen fixation, where the plant supplies the carbon source for the energy-dependent reduction of dinitrogen and protects the oxygen-sensitive nitrogenase enzyme, is among the most effective fixation systems. To establish a symbiosis, the bacterial microsymbionts gain access to single plant cells and install themselves in compartments surrounded by a plant membrane. In Gunnerasp. the cyanobacterium Nostoc sp. invades pre-existing stem glands and forms nitrogen-fixing heterocysts in infected cells. In most other symbiotic interactions, a specialized plant organ, the root nodule, is developed to provide optimal conditions for the nitrogen-fixing bacteria. Among woody plant species belonging to eight different families, an interaction with the gram-positive genus Frankia leads to the development of actinorhizal root nodules. In legumes, gram-negative soil bacteria belonging to the family Rhizobiaceae (here collectively calledRhizobium) infect root tissue and induce the formation of the nitrogen fixing nodules. Why certain plants are able to develop root nodules is unclear, but recent phylogenetic studies based on DNA sequence analysis place all plants involved in rhizobial or actinorhizal symbiosis in the same lineage and suggest that the predisposition for nodulation evolved only once (Soltis et al., 1995;Doyle, 1998). The relationship between Rhizobium and legume plants is selective. Individual species of rhizobia have a distinct host range allowing nodulation of a particular set of legume plants. For example,Rhizobium leguminosarum bv viciae nodulates pea and vetch, whereas Bradyrhizobium japonicum nodulates soybean. At the other extreme, the exceptionally broad host-rangeRhizobium sp. NGR234 nodulates 353 legume species representing 122 genera (Pueppke and Broughton, 1999). Differences in both infection processes and organogenic programs are reflected in variations in root nodule morphology (Doyle, 1998), but overall there are pronounced developmental similarities as would be expected from a common ancestry. To cover most aspects of this unusual plant-prokaryote symbiosis, the study of nodulation is a multifaceted research area aiming to understand this plant-microbe interaction in a framework of physiological and developmental processes underlying infection and organogenesis. With this perspective, thisUpdate draws on observations from differentRhizobium-legume interactions. The following sections focus on plant control of root nodule organ formation and sketch the way plant genetics and functional genomics are changing our thinking. The early signal exchange as well as the biosynthesis and properties of the bacterial Nod factor signal molecules have been reviewed extensively (Dénarié et al., 1996; Spaink, 1996; Downie and Walker, 1999, and refs. therein) and will be presented only briefly. A, Whole mount of a developing Lotus japonicus root nodule invaded by two infection threads. Fluorescence microscopy shows the autofluorescent nodule cells and theMesorhizobium loti bacteria stained for β-galactosidase activity expressed from a lacZ reporter gene. The determinate root nodules of L. japonicus are initiated from cell division in the outer cortex, whereas indeterminate nodules (of pea, for example) are initiated from cell divisions in the inner cortex. Conditions or factors influencing nodule initiation or development are listed to the right and left of the root nodule. Where different responses to the factor or condition have been reported in individual legume species, this is indicated with the following: +, positive effect; −, negative effect; or 0, lack of response. EPS, Exopolysaccharides; LPS, lipopolysaccharides. B, Structure of a bacterial LCO Nod factor. The acetylated fucosyl in blue results from NodZ and NolL modification of the R. leguminosarumLCO. The early plant host signals secreted into the rhizosphere can be (iso)flavonoids, stachydrines, or aldonic acids. Best studied are the flavonoids that, in conjunction with the rhizobial NodDtranscriptional activator, induce expression of the nod gene regulon. In turn, nod gene products synthesize and transport the Nod factor (Fig. 1B), the major early signal molecule perceived by the host plant. A flexible interaction with the host is enabled by several mechanisms. Alternative NodD activators recognizing different plant flavonoids provide an extended host range in some bacterial strains. B. japonicum even has an alternative two component regulatory pathway for activating its nod regulon, and in Sinorhizobium meliloti nod gene expression is fine-tuned by positive- and negative-control circuits. Nod factors are low-M r β,1-4-linkedN-acetyl glucosamine compounds (Lerouge et al., 1990) typically carrying a fatty acid on the non-reducing sugar and sulfuryl, fucosyl, mannosyl, or arabinosyl groups at the reducing terminal sugar. Additional substitutions include carbamoyl, glycerol, and fucosyl derivatives (Dénarié et al., 1996; Spaink, 1996). When purified and applied in the absence of bacteria these lipochitooligosaccharides (LCOs) function as mitogens or “morphogens” on some legume roots. For example, addition of nanomolar concentrations induces root hair deformation in most legumes. In more responsive plants, pre-infection threads (cytoplasmic bridges in G2 arrested cortical cells), cortical cell divisions, and empty nodule structures with an anatomy comparable to rhizobial-induced nodules also develop (Dénarié et al., 1996; Spaink, 1996, and references therein). These responses show that some legumes encode all functions necessary to develop the nodule once the process has been switched on. Spontaneous nodule development on certain alfalfa mutants grown axenically supports this idea. Schematic representation of the early communication between legume host and Rhizobium. A, Symbiotic development is initiated only when the correct plant and bacterial signal molecules are synthesized, presented, and perceived. B, Changes of the genetic repertoire of R. leguminosarum bvviciae leads to the synthesis of different LCOs and changes of host specificity. Stepwise addition of a flavonoid-independent NodD activator plus NodZ fucosyl transferase and NolL acetyl transferase allows R. leguminosarum bvviciae to nodulate L. japonicus. −, No nodulation; +, slow nodulation; +++, normal nodulation. Early physiological changes detected in root hairs following LCO treatment or rhizobial inoculation. The order of events roughly follows the timing of changes but does not indicate a causal relationship. Pharmacological experiments using agonists and antagonists [e.g. mastoparan, pertussis toxin, EGTA, 2,5-di(t-butyl)-1,4-benzohydroquinone, and La3+] of possible signal transduction components suggested that small trimeric GTPases together with phospholipase C and phosphoinositides are involved (Pingret et al., 1998). The causal relation between the various physiological changes now needs to be established and related to activation of downstream plant genes. For this work plant symbiotic mutants defining genetically separable steps and root hair expressed genes will be useful tools. It is interesting that a recently cloned LjCbp1 gene encoding a putative Ca2+-binding protein is expressed in an LCO-dependent fashion in root epidermal cells (Webb et al., 2000). The plant preference for particular rhizobial partners as well as the low operational concentrations and structural specificity of bacterial LCO signals suggest that signal perception is mediated by a plant receptor. At present a bona fide receptor for binding the LCO and amplifying the bacterial signal has not been identified. Two binding sites (NFBS1 and NFBS2) were described in microsomal fractions from alfalfa roots and tissue culture cells. NFBS1 has low substrate affinity, whereas NFBS2 binds LCO with higher affinity (Gressent et al., 1999). Both sites have specificity for LCO but will also bind derivatives without the sulfuryl substitution needed for in vivo activity. This discrepancy could result from a receptor that in vitro was depleted of a subunit adding specificity. A novel lectin type protein was recently purified from Dolichos biflorus and shown to bind LCO with high affinity (Etzler et al., 1999). This lectin also has apyrase (nucleotide phosphohydrolase) activity, suggesting a function at the start of a signal transduction (phosphorylation) pathway and thus adding a new twist to the study of lectins. Expression of pea or soybean genes encoding seed lectins with non-catalytic sugar-binding sites was previously shown to extend the host range of clover and Lotus corniculatus. This effect was attributed to the lectin's ability to attach sufficient bacteria at the root hair tip prior to infection or enhancement of the LCO induced mitogenic response rather than being mediated by an LCO-binding/receptor function (van Rhijn et al., 1998; Dı́az et al., 2000). The question concerning the nature and localization of the Nod factor receptor is still open, leaving room for alternative models (Hirsch, 1992; Ardourel et al., 1994; Schultze and Kondorosi, 1998). Intriguing results from studies with bacterial mutants, non-specific LCOs, and chitin-oligomers suggest a perception mechanism using two receptors of different stringency or a receptor with a complex substrate interaction. Simple LCO derivatives (O-acetylated chitin oligosaccharides) can, for example, induce cortical cell divisions after microtargeting into Vicia roots and in soybean, transient induction of the early nodulin Enod40 gene can be obtained with an unsubstituted chitin pentamer. This points toward a hierarchy of plant responses requiring different LCO specificity for execution and a mechanism where LCO transport, localization, and receptor affinity may be influenced by plant chitinases or glycosyl hydrolases can be envisaged. Uncoupling of cell division and infection thread formation seen for example in sym5 mutants of pea and sym4 of sweetclover could lend support for a two receptor model, but the abundance of generally unresponsive plants mutants indicate that common steps or interactive pathways are involved. Future map-based cloning of loci believed to be involved in LCO perception, for example,Sym2 from pea and Sym5 from L. japonicus are likely to clarify some of the questions and may also explain why infection threads are only observed in the presence of bacteria. Development of functional root nodules relies on synchronized activation of particular gene sets in both symbionts. The stage-characteristic arrest observed with rhizobial as well as plant mutants indicates that the processes are highly coordinated, but information on “late” signal exchange is somewhat sparse. However, continued expression of nod genes in bacteria contained within infection threads and localization of internalized immunoreactive LCO in cells of maturing root nodules, indicate a connection to the early LCO signaling. After endocytosis, the synthesis of LCOs is down-regulated in bacteroids. Endocytosis and bacterial differentiation appear therefore to mark a shift in plant-rhizobial communication. Bacterial surface polysaccharides are known to be involved in the infection process. In some symbiotic interactions, rhizobial mutants that are deficient in exo- or lipo-polysaccharides (EPS I, EPS II, and LPS) are defective in the infection process and may provoke increased host defense reactions suggesting that surface polysaccharides shield the bacteria. However, EPS mutants are partially rescued by exogenous application of picomolar concentrations of low-M r polysaccharide fractions of EPS I or EPS II, suggesting that these function as signal molecules (González et al., 1996). Secreted proteins also contribute to signaling. Strains of R. leguminosarum bv viciae excrete NodO, a protein shown to form Ca2+-transporting ion channels in vitro. In vivo a NodO-mediated enhancement of infection thread progression and nodulation was observed in partially compatible pea hosts. The NGR234 strain has a type-III protein secretion system known from pathogenic bacteria to export proteinaceous pathogenicity factors. Mutations preventing secretion of the NGR234 NolX and y4xl proteins (functions unknown) change the nodulation pattern on some but not all legume hosts, implying that aspects of protein signaling are shared between pathogens and symbionts (Viprey et al., 1998). It will be interesting to determine the targets of these “late” signals and address the possibility of positive roles in nodule development as well as defense avoidance. Most of the plant architecture is formed by post-embryonic development, and changes in relative hormone concentrations under the influence of biotic and abiotic factors strongly influence the developmental fate of cells and organs. Formation of root nodules is no exception, and several lines of evidence suggest a role for phytohormones in secondary signaling. Incubation with auxin transport inhibitors results in development of empty nodule-like structures on roots of some legumes and expression of genes such asEnod12, Enod40, and Enod2, which are normally expressed during early phases of root nodule organogenesis (Fang and Hirsch, 1998). Using an auxin sensitive reporter gene, it was shown that Rhizobium or external addition of LCO leads to a rapid transient and local inhibition of acropetal auxin transport in clover roots (Mathesius et al., 1998). This indicates that a changed hormone balance follows the primary LCO signal at the site of nodule initiation possibly sensitizing cells for division. Flavonoids could act as secondary effectors in this process, because the phenylpropanoid pathway is during and inhibition of auxin transport has been For partially the Nod factor local by expression of the biosynthesis gene in a Rhizobium in the development of nodule-like In and the LCO genes as well as the gene are all by and cell divisions are in these roots. and LCO may thus be of or influence the same signal transduction during nodulation. there are in legume exogenous application of generally nodulation and biosynthesis or perception and nodulation. A in and of nodules developing in shows that is involved in a local of infection and However, a comparable of soybean was in the of these without a of the involved et al., 1999). has indeterminate nodules, whereas soybean has determinate could that the from indeterminate to determinate nodules observed on is of a response in the two nodule In pea nodules develop of by in cells between in with a of the from the may provide the information for this nodulation under conditions where a of the events into functional nodules. under optimal most infection threads are arrested in root cell and the nodule are by the plant. mechanism cortical cell divisions in nodule to cell division and mutants developing nodules and lack the normal nitrate that nitrate its local effect the suggested that be involved in nitrate in but from pea not support a role for A transport of a to the and of an was on the of experiments control of the root and The of these compounds as but both nitrate and act of of the from that of nodule is in the root development et al., 2000). Formation of a new organ and activity of genes and gene products in the organogenic process. Using or proteins were at in root nodules, and genes were highly induced in particular cell or nodule The expressed genes such as and in root whereas genes encoding proteins needed in the and of the nodule to be strongly induced of nitrogen involved in of is the example of this of “late” The of gene during nodulation is also by the gene family where some are expressed in root hairs and cells et al., 2000). the studies of induced genes only gene has with a regulatory role in nodule The Enod40 was suggested to encode a small to acid and a to control or In with a regulatory Enod40 is in root cells within after or LCO application and prior to division of cortical cells. With the expressed sequence now for various legumes, a of the genes in root nodules will appear and change the nodulin a of the genes is only possible with and are therefore to and for access and of this information The is to function to genes of the and to provide a analysis of genes nodule formation and to known genes will the of this is from the present studied using genetics for gene have to establish in plants and functional studies in legumes on studies in the absence of a For example, some plants whereas other lines of the developed or nodules et al., 1999). with the gene were In a it was that absence not influence nodule development or the gene was or for the The recent that can gene activity in and may in the more effective in studies of gene function in legumes. of genes and proteins in to determine a role in root nodule development or function of genes and proteins in to determine a role in root nodule development or function The regulatory nodule development has been with studies of the early signaling or of nodulin gene these studies start at of the developmental process, aiming to in To the a genetic a more of and of pathways from studies is now The from plant genetics in gene and functional analysis was recently by the of the L. japonicus nodule gene from a symbiotic with the et al., 1999). Symbiotic mutants have been known in soybean and pea for but the genetically symbiotic at the and an example of the by legumes. mutants are cell divisions, the to nodule appear not to be the mutants root hair deformation after that LCO signal perception is These observations to a function at the of signal transduction and gene The presence of putative factor in the protein as well as to the protein in support this idea. Structure of the protein and a for the role in early phases of root nodule A, The LCO-dependent root hair deformation observed on the together with the lack of cell division in the outer cortex, place the gene in signal transduction or gene activation downstream of LCO in have to factor suggesting a function in activation of genes for nodule To explain the lack of infection thread formation in mutants, a secondary positive signal is to be necessary for this process. This signal may at the same root hair deformation the root hair response of the mutants et al., 1999). B, The present of events during nodule that Enod40, and involved at the same developmental activation putative Schematic representation of the and proteins of the and the pea positive of an suggested to with whereas the has two binding sites for the for function are in the and negative The protein nodule and factor bind at as sites of the In the a positive binding the protein is sufficient for LCO tissue proteins binding at or in the of these DNA were by studies using or in Two proteins were in nodules and A nodule protein binds at the soybean and a in nodules as well as the in is a soybean binding to a two present in proteins et al., 2000). proteins expression of developmental and there is some support for negative of expression by et al., 2000). Additional at the functional are proteins and activator proteins (Fig. The pea early nodulin from was by a A was to for both nodule activation in and interaction with an early protein When binding site were for binding in vitro and in vivo a between binding and activity was seen et al., but was not expression in plants. The protein shown to be for binding and a Both the and gene to LCO and but regulatory DNA in gene expression were not reported et al., 1996; and Hirsch, 1998). Additional analysis may a between and gene expression during nodule organogenesis. The of the LCO Nod factor molecules and on legume roots the possibility of a more role as a of plant it is still an question nodule induction is a specialized effect or a LCOs to the compounds by in plants and the evidence of a recent example, a study of pea activity in that the LCO perception mechanism is functional in a plant. et al., 1998; for Spaink, 1996). which has new evidence for the role of in plant development, has in relation to with to bacterial nod genes were not reported from the even a gene with to was in 1996; Schultze and Kondorosi, 1998). may still contribute but may also have to the of the legume LCO and signal transduction the question can be in The interaction is for symbiotic functions and genes with a more plant mutants are also in the together with expression of and in roots by points at in the programs In this analysis of symbiotic genes is to to the of plant as an example, several genes to the gene can be from the sequence and the functional analysis of these genes in is now an This analysis can be to in the function and its in legume development and nodulation. With the new for functional genomics in legumes, analysis of the plant to symbiosis will gain and together with map-based cloning of symbiotic loci are to novel genes in the root nodule regulatory The of sequence information by and expression analysis using or DNA will provide information of gene under various and pathway to be from will this analysis and to the In the will be to of the and studies of the and genomics will be major in of gene and gene function in With a more of legume symbiosis, the question why among the forms nodules with and the question of the genetic between legumes and other plants could be I to the or for this I to the I was to include or I for the in and and at the of Expression for on the