Ultrastructure and functioning of the transport system of the leguminous root nodule

Summary The structure of the vascular tissues of nitrogen-fixing nodules of 27 genera of legumes and some non-legumes has been investigated by light microscopy. Pisum and Trifolium nodules have been examined by electron microscopy.Attention is directed to the presence of a pericycle in the vascular bundles of the nodules. In 7 of the legumes the pericycle cells possess a wall labyrinth consisting of branched filiform protuberances. The ultrastructure of the pericycle cell cytoplasm is described: its most striking feature is its abundant rough endoplasmic reticulum. These cells surround the xylem and phloem of the bundles, and are in turn surrounded by a layer of endodermal cells with Casparian strips. The pericycle cells develop their wall labyrinth in the levels of the nodule at which the bacterial tissue becomes pigmented; in nodule senescence their cytoplasm is disrupted level with the breakdown of the bacterial tissue.A pathway for symplastic lateral transfer of assimilates exists, from the sieve elements through the pericycle, endodermis and cortex to the bacterial tissue. The apoplast within the endodermis consists largely of the pericycle wall labyrinth and the xylem. The ultrastructure of the Casparian strip resembles that of roots.Intact, detached nodules can be induced to bleed a fluid from their severed vascular tissue. This fluid is exceptionally rich in organic nitrogen, particularly amides, but does not appear to contain sugars. Comparison between its amino acid composition and that of other parts of the nodule suggests that an active uptake or secretion of nitrogenous compounds precedes export from the nodule. Special functions are suggested for the nodule endodermis and the pericycle cells in this export process.     

Compensatory response of a legume root-nodule system to nodule herbivory by Sitona hispidulus

A laboratory study was conducted to examine the hypothesis that herbivory of nitrogen-fixing root nodules on legumes causes an exact compensatory response in nodule growth. Plants of Medicago sativa (L.) were grown hydroponically in clear plastic growth pouches so that the number and biomass of root nodules could be estimated nondestructively before, and 10 and 18 days after, partial denodulation. For treatments, plants were subjected to 23% denodulation by first-instar larvae of Sitona hispidulus (F.) (a common herbivore of Medicago and Trifolium) or 50% nodule pruning; additional plants were left untreated. Results indicated that nodule herbivory and nodule pruning caused an overcompensatory response in number of nodules. This was also true for number of nodule units (an indirect measure of nodule biomass) per plant at 10 days after denodulation but had changed to an exact compensatory response by day 18. An inverse relationship between change in number of nodule units and initial number of nodules indicated that compensatory nodulation was regulated by a feedback mechanism. Shoot and root biomasses were not affected by denodulation in this study.     

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.     

The occurrence of haemoglobin and hydrogenase in nodules of twelve Casuarina-Frankia symbiotic associations

Seedlings of Casuarina cunninghamiana Miq., C. equisetifolia L. and C. glauca Sieber growing in N-deficient potting mix were inoculated with Frankia sp. from each of 4 different sources. After ca 4 months, plants were harvested and nodules from the 12 Casuarina-Frankia combinations evaluated for (1) concentrations of haemoglobin (measured as CO-reactive haem) and (2) occurrence of hydrogenase. The aim was to determine if these factors were related to nitrogen accumulation and biomass production. There were marked host-Frankia source interactions with up to 10-fold differences in plant dry weight and 50-fold differences in the efficiency of nitrogen fixation (as estimated by N₂ accumulated mg^?1 nodule dry weight). Differences in plant growth and nitrogen accumulation were apparently related to nodule specific activity, because the 12 associations had similar nodulation characteristics, e.g. time for nodulation to occur. The concentration of haemoglobin in Casuarina nodules ranged from 0 to 27 nmol haem (g FW)^?1. There was a strong linear correlation between concentrations of haemoglobin and dry weights of the whole plants (r=0.77, 0.92 and 0.97, P≤0.05) for C. cunninghamiana, C. equisetifolia and C. glauca symbiotic associations, respectively. However, the linear correlation between concentration of haemoglobin and nitrogen content of whole plant was lower (r=0.60, 0.64 and 0.71, P≤0.05) for the three Casuarina symbioses, respectively, and there was only a poor correlation between haemoglobin concentration in nodules and the rate of nitrogen accumulation on nodule weight basis. This indicates that haemoglobin concentration is not the sole physiological determinant of nitrogen fixation in Casuarina. All the Casuarina-Frankia symbiotic associations studied also showed the presence of a hydrogen uptake enzyme. The activity of the enzyme ranged from 5.1 to 34.1 μmol H₂ (g FW)^?1 h^?1, and hydrogen uptake was not correlated with plant dry weight, nitrogen content or the rate of nitrogen fixation. Hydrogen evolution could not be detected in any of the associations.     

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N₂-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

ESI–MS and matrix-assisted laser desorption ionization–mass spectrometry imaging reveal alterations in Medicago truncatula nodules membrane lipid composition and spatial distribution in phosphorus deficiency.     

RNAi Phenotypes and the Localization of a Protein::GUS Fusion Imply a Role for Medicago truncatula PIN Genes in Nodulation

The symbiosis between legumes and rhizobia results in the development of a new plant organ, the nodule. A role for polar auxin transport in nodule development in Medicago truncatula has been demonstrated using molecular genetic tools. The expression of a DR5::GUS auxin-responsive promoter in uninoculated M. truncatula roots mirrored that reported in Arabidopsis, and expression of the construct in nodulating roots confirmed results reported in white clover. The localization of a root-specific PIN protein (MtPIN2) in normal roots, developing lateral roots and nodules provided the first evidence that a PIN protein is expressed in nodules. Reduced levels of MtPIN2, MtPIN3, and MtPIN4 mRNAs via RNA interference demonstrated that plants with reduced expression of various MtPINs display a reduced number of nodules. The reported results show that in M. truncatula, PIN proteins play an important role in nodule development, and that nodules and lateral roots share some early auxin responses in common, but they rapidly differentiate with respect to auxin and MtPIN2 protein distribution.     

Aerial root nodules in the tropical legume,Pentaclethra macroloba

Summary Symbiotic nitrogen fixation in angiosperms normally occurs in buried root nodules and is severely inhibited in flooded soils. A few plant species, however, respond to flooding by forming nodules on stems, or, in one case, submerged roots with aerenchyma. We report here the novel occurrence of aerial rhizobial nodules attached to adventitious roots of the legume,Pentaclethra macroloba, in a lowland tropical rainforest swamp in Costa Rica. Swamp sapdings (1–10 cm diameter) support an average 12 g nodules dry weight per plant on roots 2–300 cm above water, and nodules remain in aerial positions at least 6 months. Collections from four swamp plants maintained linear activity rates (3–14 moles C₂H₄/g nodule dry weight/hr) throughout incubations for 6 and 13 hrs; excised nodule activity in most legumes declines after 1–2 hrs. Preliminary study of the anatomy and physiology suggest aerial nodules possess unusual features associated with tolerance to swamp conditions. High host tree abundance and nodulation in the swamp compared to upland sites indicate the aerial root symbiosis may contribute more fixed nitrogen to the local ecosystem than the more typical buried root symbiosis.     

Structure of root nodules and nitrogen fixation in Egyptian wild herb legumes

Five wild herb legumes (Trifolium resupinatum, Melilotus indica, Medicago intertexta, Trigonella hamosa, and Alhagi murarum) were collected from cultivated lands of the Nile Valley, and compared with clover (Trifolium alexandrinum), a cultivated forage legume. The wild herb legumes exhibited great variation in nodulation percentage, nodule number, nodule mass and acetylene reduction activity with regard to locality. Nodulation of T. resupinatum and M. indica ranged between 50 - 100% and 33 - 100%, respectively, compared to 50 - 100% for T. alexandrinum. The number of nodules formed on T. resupinatum was 9 - 128 and that of M. indica 6 - 39, compared to 13 - 122 nodule per plant for T. alexandrinum. Nodule mass was correlated with nodule number. In M. indica, a small number of nodules was compensated with high specific nitrogenase activity. The herb legumes formed nodules of small size, varying shape (globose, cylindrical, branched, etc.), and of different types (crotalaroid and astragaloid). Microscopic examination of root-nodules from T. resupinatum, M. indica and M. intertexta, showed that these legumes formed indeterminate and effective nodules, containing apical meristems, central symbiotic tissue with characteristic zonation and peripheral vascular bundles. The nodules harboured bacteroids with pleiomorphic morphology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

Growth of an invasive legume is symbiont limited in newly occupied habitats

Mutualisms may play an important role in the establishment and invasion success of introduced species, but their influence is little studied. To test whether a lack of root nodule symbionts may limit the performance of invasive legumes, seedlings of Cytisus scoparius were introduced to an old-field habitat and then either inoculated with Bradyrhizobium strains from existing C. scoparius populations, or left uninoculated. In two separate years, inoculation more than doubled average plant biomass. For uninoculated transplants, nodule formation was positively correlated with proximity to plants of the native legume Desmodium canadense , but not related to distance from a second legume species, Apios americana. Polymerase chain reaction assays and DNA sequencing confirmed that bacteria isolated from uninoculated C. scoparius plants were indistinguishable from Bradyrhizobium strains in root nodules of D. canadense . By contrast, bacterial strains associated with A. americana were never found in C. scoparius nodules. Transplants in seven other habitats across a 160 km region also showed a highly significant, fivefold biomass increase in response to inoculation. Thus, colonizing legumes can suffer from a scarcity of nodule symbionts. However, certain indigenous legumes may create favourable microhabitats for invasion, by increasing symbiont availability in their vicinity.     

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.     

Vascular transport and soybean nodule function: II. A role for phloem supply in product export*

Abstract The ureide content of soybean (Glycine max (L.) Merr.) nodules was unaffected by variations in the transpirational rate, while whole plant manipulations designed to decrease phloem supply to nodules resulted in lower rates of nitrogenase activity and an increase in the ureide content of the nodules. The rate of ureide export from the nodule was estimated from the exponential rate of decrease in the pool size of ureides in nodules, following exposure to an N₂-free atmosphere (Ar:O₂). Export was greatly reduced under treatments which reduced phloem supply to the nodule. A water budget for nodules suggested that the delivery of water to the nodule via mass flow in the phloem was comparable to that required for export of ureides from the nodule in the xylem from the nodule. Therefore, we suggest that xylem export from nodules is related to the phloem supply to the nodule rather than to the transpirational flux in the parent root. This suggestion is related to the reported decreases in nodule permeability to gases under conditions of phloem deprivation.     

Belowground mutualists and the invasive ability of Acacia longifolia in coastal dunes of Portugal

The ability to form symbiotic associations with soil microorganisms and the consequences for plant growth were studied for three woody legumes grown in five different soils of a Portuguese coastal dune system. Seedlings of the invasive Acacia longifolia and the natives Ulex europaeus and Cytisus grandiflorus were planted in the five soil types in which at least one of these species appear in the studied coastal dune system. We found significant differences between the three woody legumes in the number of nodules produced, final plant biomass and shoot ¹⁵N content. The number of nodules produced by A. longifolia was more than five times higher than the number of nodules produced by the native legumes. The obtained ¹⁵N values suggest that both A. longifolia and U. europaeus incorporated more biologically-fixed nitrogen than C. grandiflorus which is also the species with the smallest distribution. Finally, differences were also found between the three species in the allocation of biomass in the different studied soils. Acacia longifolia displayed a lower phenotypic plasticity than the two native legumes which resulted in a greater allocation to aboveground biomass in the soils with lower nutrient content. We conclude that the invasive success of A. longifolia in the studied coastal sand dune system is correlated to its capacity to nodulate profusely and to use the biologically-fixed nitrogen to enhance aboveground growth in soils with low N content.