Isolation and properties of a lectin from the roots of Pisum sativum (Garden pea)

Abstract The roots of pea (Pisum sativum L. ev. Feltham First) seedlings contained haemagglutinating activity and a protein which reacted with antibodies directed against pea seed lectin. This protein was shown to be present on the surface of root hairs and in the root cortical cells by immunofluorescence. Lectin (haemagglutinin) was purified from pea seedling roots by both immunoaffinity chromatography and affinity chromatography on Sephadex G-100. The pea root lectin was similar to the seed lectin when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was antigenically identical: however, the isoelectric focussing band patterns of the proteins differed. The sugar specificity of the root lectin differed from that of the seed lectin, and the haemagglutinating activity of the root lectin was less than the seed lectin. These results are discussed with reference to the hypothesis that lectins mediate in the symbiotic association of legume and Rhizobium through their carbohydrate-binding properties.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Determination of pea (Pisum sativum L.) root lectin using an enzyme-linked immunoassay

Root lectins are believed to participate in the recognition between Rhizobium and its leguminous host plant. Among other factors, testing this hypothesis is difficult because of the very low amounts in which root lectins are produced. A double-antibody-sandwich enzyme-linked immunoassay, was used to determine nanogram quantities of pea lectin in root slime and salt extracts of root cell-wall material when pea seedlings were 4 and 7 d old. In addition, a critical NO ₃ ^- concentration (20 mM) which inhibited nodulation was found, and the lectin present in root slime and salt extracts of root cell walls of 4- and 7-d-old peas supplied with 20 mM NO ₃ ^- was comparatively determined. With the enzyme-linked immunoassay, lectin quantities ranging between 20 and 100 nanograms could be determined. The assay is not affected by monomeric mannose and glucose (pealectin haptens). The slime of the 4-d-old roots contained more lectin than the slime of the 7-d-old roots. Salt-extractable, cell-wall-associated lectin accumulated in the older roots. Nitrate affected slime and cell-wall production, and the extractability of cell-wall material in both age groups. The presence of NO ₃ ^- increased lectin in the slime, most notably in the younger roots; the relative amount of lectin in the slime was almost doubled. The cell-wall-associated, salt-extractable lectin decreased two- to threefold compared with the control group.Abbreviations ELISA enzyme-linked immunoassay - PTN 0.01 M phosphate buffer (pH 7.4), containing 0.15 M NaCl, 0.05% Tween-20 and 0.02% NaN₃ Dedicated to Professor A. Quispel on the occasion of his retirement     

Detection of lectins in nodulated peanut and soybean plants

The direct double-antibody enzymelinked immunosorbent assay system was used in the detection and measurement of seed lectins from peanut (Arachis hypogaea L.) and soybean (Glycine max L.) plants (PSL and SBL, respectively) that had been inoculated with their respective rhizobia. Concentrations of PSL dropped to undetectable levels in peanut roots at 9 d and stems and leaves at 27 d after planting; SBL could no longer be detected in soybean roots at 9 d and in stems and leaves at 12 d. A lectin antigenically similar to PSL was first detected in root nodules of peanuts at 21 d reaching a maximum of 8 g/g at 29 d then decreasing to 2.5 g/g at 60 d. There was no evidence of a corresponding lectin in soybean nodules.Sugar haemagglutination inhibition tests with neuraminidase-treated human blood cells established that PSL and the peanut nodule lectin were both galactose/lactose-specific. Further tests with rabbit blood cells demonstrated a second mannosespecific lectin in peanut nodule extracts that was not detected in root extracts of four-week-old inoculated plants or six-week-old uninoculated plants, although six-week-old root extracts from inoculated plants showed weak lectin activity. The root extracts from both nodulated and uninoculated plants contained another peanut lectin that agglutinated rabbit but not human blood cells. Haemagglutination by this lectin was, however, not inhibited by simple sugars but a glycoprotein, asialothyroglobulin, was effective in this respect.Abbreviations DAS double antibody sandwich - ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - PSL peanut seed lectin - SBL soybean lectin     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis andex -planta nitrogenase activity of cowpea rhizobia

The root exudate ofArachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpeaRhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1.5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore,ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides. Part of this work was presented at the colloquium session of the 4th Hederation of Asian and Oceanian Biochemists Congress, held at Singapore, in November 1986.     

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

Summary The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.Abbreviations EH smallest emergent root hair - EM electron microscope - enod2 early nodulin2 gene - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract-mannitol-gluconate     

Characterization of LPS mutants of peanut specific Bradyrhizobium (GN17)

Two mutants of Bradyrhizobium sp. (Arachis) strain GN17 having altered lipopolysaccharide (LPS) composition were isolated upon random Tn5 mutagenesis to study their binding with peanut root lectin (PRA II). These mutant strains designated as GN17M1 and GN17M2 produced rough colonies and showed autoagglutination. Flow cytometric analyses indicated that strain GN17M1 bind to PRA II with highest efficiency. Both the mutants synthesized only high molecular weight lipopolysaccharides as observed by silver staining of polyacrylamide gel. The LPSs from both the mutants cross-reacted with anti-GN17 LPS, however, GN17M1 LPS showed 3 times higher cross-reactivity as detected by ELISA. Carbohydrate analysis by high performance anion exchange chromatography (HPAEC) showed that glucose was the major constituent of the purified LPS from the parent strain whereas mannose appeared as major component in the GN17M2 LPS. Equivalent amount of glucose and galactosamine with significant amount of mannose and galactose was the characteristics of the GN17M1 LPS. Purified LPS from GN17M1 and GN17M2 were respectively 17 and 10 times more potent inhibitors of PRA II activity than that of parent strain GN17. Similar binding efficiencies of the mutant LPS towards PRA II was also observed by ELISA. The results of this study indicate that the composition and the arrangement of the LPS are crucial for lectin binding.     

Distribution of glucose/mannose-specific isolectins in pea (Pisum sativum L.) seedlings

We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF isoelectric focusing - MW molecular weight - pI isoelectric point - Psl1, Psl2 and Psl3 pea isolectins - SDSPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions.     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ∼54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ∼26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% β-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

Aerenchyma formation and porosity in root of a mangrove plant,<Emphasis Type="Italic"> Sonneratia alba</Emphasis> (Lythraceae)

Aerenchyma gas spaces are important for plants that grow in flooded and anaerobic sites or habitats, because these gas spaces provide an internal pathway for oxygen transport. The objective of this study is to characterize the development of aerenchyma gas spaces and observe the porosity in roots of Sonneratia alba. Tissue at different developmental stages was collected from four root types, i.e. cable root, pneumatophore, feeding root and anchor root, of S. alba. In S. alba, gas space is schizogenously produced in all root types, and increases in volume from the root meristem to mature root tissues. The aerenchyma formation takes place immediately, or 3–5 mm behind the root apex. At first, cortical cells are relatively round in cross sections (near the root apex); they then become two kinds of cells, rounded and armed, which combine together, forming intercellular spaces behind the root apex. The average dimensions of cortical cells increased more than 1.3 times in the vertical direction and over 3.3 times in the horizontal direction. At maturity, aerenchyma gas spaces are long tuberous structures without diaphragms and with numerous small pores on the lateral walls. Within the aerenchyma, many sclereids grow intrusively. Root porosity in all root types ranged from 0–60%. Pneumatophores and cable roots had the highest aerenchyma area (50–60%).     

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Summary Five specific transposon-induced nodulation defective (Nod⁻) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod⁻ mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod⁻ on all their known plant hosts, (b), they could not induce root hair curling (Hac⁻) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.     

Programmed cell death in the root cortex of soybean root necrosis mutants

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis-related proteins and an increased tolerance to root-borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root-autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot-derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (T erminal deoxynucleotidyl transferase-mediated dU TP-digoxigenin n ick e nd l abeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL-positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

In-situ localization of chalcone synthase mRNA in pea root nodule development

In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it is hypothesized that the Rhizobium Nod factor induces cell division in the root cortex by stimulating the production of flavonoids that function as auxin transport inhibitors. In nodules CHS mRNA is predominantly present in a region at the apex of the nodule consisting of meristematic and cortical cells. These cells are not infected by Rhizobium. Therefore it is postulated that CHS plays a role in nodule development rather than in a defence response. In roots CHS mRNA is located at a similar position as in nodules, suggesting that CHS has the same function in both root and nodule development. When nodules are formed by mutants of Rhizobium leguminosarum bv. viciae that are unable to secrete β(1-2) glucan and to synthesize the O-antigen containing LPS I, CHS genes are also expressed in regions of the nodule that are infected by Rhizobium. It is postulated that the impaired development of nodules formed by these mutants is due to an induction of a plant defence response.     

Carbohydrate Analysis of Bradyrhizobial (NC 92) Lipopolysaccharides by High Performance-Anion Exchange Chromatography with Pulsed Amperometric Detection

Composition analysis of monosaccharides of Sepharose 4B purified NC 92 LPS and the polysaccharides fractions from Sephadex G-50 chromatography was performed by high performance anion exchange chromatography using pulsed amperometric detection. Rhamnose, mannose, galactose and glucose are present in a substantial amount in the purified LPS (Pk I). High molecular weight purified polysaccharides (PS I) obtained after sephadex gel filtration of the purified LPS (Pk I) acid hydrolysate showed an increase in glucose:galactose ratio. This indicates the presence of the peanut root lectin (PRA II) specific sugar in higher proportion on the O-antigen part of the LPS molecule, which may aid in the critical recognition reaction.     

The unique root-nodule symbiosis between Rhizobium and the aquatic legume,Neptunia natans (L. f.) Druce

We examined the development of the aquatic N₂-fixing symbiosis between Rhizobium sp. (itNeptunia) and roots of Neptunia natans L. f. (Druce) (previously N. oleracea Lour.) under natural and laboratory conditions. When grown in its native marsh habitat, this unusual aquatic legume does not develop root hairs, the primary sites of rhizobial infection for most temperate legumes. Under natural conditions, the aquatic plant floats and develops nitrogen-fixing nodules at emergence of lateral roots on the primary root and on adventitious roots at stem nodes, but not from the stem itself. Cytological studies using various microscopies revealed that the mode of root infection involved an intercellular route of entry followed by an intracellular route of dissemination within nodule cells. After colonizing the root surface, the bacteria entered the primary root cortex through natural wounds caused by splitting of the epidermis and emergence of young lateral roots, and then stimulated early development of nodules at the base of such roots. The bacteria entered the nodule through pockets between separated host cells, then spread deeper in the nodule through a narrower intercellular route, and eventually evoked the formation of infection threads that penetrated host cells and spread throughout the nodule tissue. Bacteria were released from infection droplets at unwalled ends of infection threads, became enveloped by peribacteroid membranes, and transformed into enlarged bacteroids within symbiosomes. In older nodules, the bacteria within symbiosomes were embedded in an unusual, extensive fibrillar matrix. Cross-inoculation tests of 18 isolates of rhizobia from nodules of N. natans revealed a host specificity enabling effective nodulation of this aquatic legume, with lesser affinity for Medicago sativa and Ornithopus sp., and an inability to nodulate several other crop legume species. Acetylene reduction (N₂ fixation) activity was detected in nodules of N. natans growing in aquatic habitats under natural conditions in Southern India. These studies indicate that a specific group of Rhizobium sp. (Neptunia) occupies a unique ecological niche in aquatic environments by entering into a N₂-fixing root-nodule symbiosis with Neptunia natans.We thank J. Whallon for technical assistance, G. Truchet, J. Vasse, S. Wagener, J. Beaman, F. DeBruijn, F. Ewers, and A. Squartini for helpful comments, and N.N. Prasad and G. Birla for assistance in conducting field observations. This work was supported by the Michigan Agricultural Experiment Station and National Science Foundation grants DIR-8809640 and BIR-9120006 awarded to the MSU Center for Microbial Ecology. This study is dedicated to the memory of Dr. Joseph C. Burton, a friend and colleague who made many contributions to the study of the Rhizobiumlegume symbiosis.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.