Genome Structure of the Legume, Lotus japonicus

The legume Lotus japonicus has been widely used as a model system to investigate the genetic background of legume-specific phenomena such as symbiotic nitrogen fixation. Here, we report structural features of the L. japonicus genome. The 315.1-Mb sequences determined in this and previous studies correspond to 67% of the genome (472 Mb), and are likely to cover 91.3% of the gene space. Linkage mapping anchored 130-Mb sequences onto the six linkage groups. A total of 10 951 complete and 19 848 partial structures of protein-encoding genes were assigned to the genome. Comparative analysis of these genes revealed the expansion of several functional domains and gene families that are characteristic of L. japonicus. Synteny analysis detected traces of whole-genome duplication and the presence of synteny blocks with other plant genomes to various degrees. This study provides the first opportunity to look into the complex and unique genetic system of legumes.Key words: Lotus japonicus, genome structure, Fabaceae, comparative analysis     

Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

Preparation of a Monoclonal Antibody against Soybean Nodule Uricase (nod-35), and Immunoblot Analysis of the Expression of nod-35 in Tissues of Various Legumes

Immunoblot analysis showed that uricases in nonureide-transportingdeterminate nodules (Canavalia gladiata and Lotus japonicus)did not react with a monoclonal antibody against soybean noduleuricase, suggesting different immunological reactivities fromthose of uricases of ureide-transporting legumes. (Received June 1, 1996; Accepted September 17, 1996)     

High frequency transformation and regeneration of transgenic plants in the model legume Lotus japonicus

The molecular analysis of plant genes involved in nodulationhas been slowed by the inability to produce high numbers oftransgenic legume lines. The high efficiency gene transfer andplant regeneration systems of the model legume Lotus japonicusis described. A collection of wild-type A. rhizogenes strainswas tested for infectivity and the most virulent strains, 9402and AR10, were selected for further use. Growth conditions forplantlets, induction of hairy roots and nodulation of compositeplants were optimized for large-scale screening in Petri dishes.A cluster of 3–10 nodules was regularly formed on transgenichairy roots 7–12 d after inoculation with the effectiveRhizobium loti strain NZP2235. There were no apparent morphologicaldifferences between nodulation of hairy and wildtype roots.To test the applicability of the hairy root system for the trappingof symbiotic genes, transformation experiments with binary vectorspossessing a ß-glucuronidase (gus, uidA) or a luciferase(luc) reporter driven by a cauliflower mosaic virus (CaMV) 35Spromoter were performed. The frequency of cotransfer of a binaryT-DNA with a root-inducing (Ri) T-DNA was 70%. Positive expressionsuggests that gus and luc trap vectors can be used for genetagging in L. japonicus. To open the possibility of searchingfor mutant phenotypes, a regeneration system has been developedenabling the regeneration of large numbers of transgenic plantsfrom hairy root cultures in about 5–6 months. At the sametime, the A. tumefaciens hypocotyl transformation regenerationin L. japonicus has been improved. This new version providesfertile transgenic plants in about 4 months. Key words: Agrobacterium, luciferase, nodulation, Rhizobium, symbiosis     

Split-root study of autoregulation of nodulation in the model legume Lotus japonicus

We used a split-root system to determine the timing for induction of the autoregulation of nodulation (AUT) in Lotus japonicus (Regel) Larsen after inoculation with Mesorhizobium loti. The signal took at least five days for full induction of AUT and inhibition of infection thread formation. Strain ML108 (able to nodulate but unable to fix nitrogen) induced full AUT, but ML101 (unable to nodulate or to fix nitrogen) did not induce autoregulation. These results indicate that Nod factor-producing strains induce AUT, but that the nitrogen fixed by rhizobia and supplied to the plant as ammonia does not elicit the AUT in L. japonicus.     

Physiological characterization of four model Lotus diploid genotypes: L. japonicus (MG20 and Gifu), L. filicaulis, and L. burttii under salt stress

The genus Lotus comprises a heterogeneous group of annual and perennial species. Lotus japonicus (with MG20 and Gifu ecotypes) has been adopted as one of the model legumes in genetic and genomic studies. Other Lotus species, such us Lotus burttii and Lotus filicaulis, have also been used in genetic and genomic studies because of their capacity to produce fertile progenies in crosses with L. japonicus. In the present work, physiological responses to salt stress in four Lotus genotypes were evaluated on the basis of growth and associated parameters, such as photosynthesis, ions, relative water content, oxidative damage and antioxidant system responses, using two NaCl levels applied by acclimation for up to 28 and 60 d. Growth responses varied with plant developmental stage in the four Lotus genotypes. L. japonicus MG20 was found to be a salt-tolerant genotype, mainly when exposed to salt stress at the young plant stage. The capacity of Lj MG20 to sustain growth under salt stress was correlated with enhancement of Superoxide dismutase and Glutathione reductase activities, as well as with increases in total and reduced glutathione content and lower Na⁺ accumulation in leaves. These results suggest that enhancement of antioxidant responses in Lj MG20 contributed to improve salt stress tolerance at early stages. On the other hand, after long-term high NaCl stress treatment, L. filicaulis exhibited lower biomass reduction, lower oxidative damage and Na⁺ accumulation in leaves than the control treatment; hence, this genotype was considered salt-tolerant. These apparently ambiguous results remark that salt tolerance, as a development-related process, was differentially expressed among the Lotus genotypes and depended on stress duration and plant phenological stage.     

Acceleration of Soybean Genomics Using Large Collections of DNA Markers for Gene Discovery

Legumes are known to provide nutritious proteins and vegetableoils while at the same time providing industrial products suchas biodiesel. It is estimated that approximately 25% of worldcrop production is derived from legumes. Recently, knowledgeof the molecular biology and genomics of legumes have been extendedsignificantly using two model species, Lotus japonicus (http://www.kazusa.or.jp/lotus/)and Medicago     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

Identification of the centromere-specific histone H3 variant in Lotus japonicus

The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species.     

Seed Set in Lotus berthelotii Masferrer

Self-pollination in L. berthelotii results in a very low seedset and so the species is considered to be self-incompatible.Pollen tubes have been observed in the ovary. The situationis complicated because ovules in individual ovaries possessembryo sacs at various stages of development from two-celledto eight-celled and this must affect the numbers of seeds whichset. Successful pollination requires rupture of the stigmaticcuticle. The conservation of L. berthelotii by seed propagation is possiblebut the genetic base of the species remains restricted. Lotus berthelotii stigma, style, self-incompatibility, seed set     

Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus

Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a -amyrin synthase from M. truncatula (MtAMYI) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a -amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.     

Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation

Host legumes control root nodule numbers by sensing externaland internal cues. A major external cue is soil nitrate, whereasa feedback regulatory system in which earlier formed nodulessuppress further nodulation through shoot–root communicationis an important internal cue. The latter is known as autoregulationof nodulation (AUT), and is believed to consist of two long-distancesignals: a root-derived signal that is generated in infectedroots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In Lotus japonicus, theleucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANTROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibitionof nodulation, and is hypothesized to recognize the root-derivedsignal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1)and LjCLE-RS2 as strong candidates for the root-derived signal.A hairy root transformation study shows that overexpressingLjCLE-RS1 and -RS2 inhibits nodulation systemically and, furthermore,that the systemic suppression depends on HAR1. Moreover, LjCLE-RS2expression is strongly up-regulated in roots by nitrate addition.Based on these findings, we propose a simple model for AUT andnitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2peptides and the HAR1 receptor-like kinase.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.     

Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.     

Somatic Hybrids between the Forage Legumes Lotus corniculatus L. and L. tenuis Waldst et Kit

A cell suspension of Lotus tenuis was established, as a sourceof protoplasts, from kanamycin resistant callus derived fromroots transformed by Agrobacterium rhizogenes LBA9402 (pRil855-pBinl9).Such protoplasts were treated with a sublethal dose of sodiumiodoacetate prior to their electrofusion with green cotyledonprotoplasts of L. corniculatus. Putative somatic hybrid colonieswere selected on medium containing kanamycin sulphate. The hybridityof plants regenerated from these selected colonies was confirmedby their morphology, esterase banding patterns, the presenceof condensed tannins in leaves and stems, and chromosome complements.The latter approximated to the expected allohexaploid numberof 2n = 6x = 36. Key words: Forage legumes, Lotus corniculatus, L. tenuis, protoplasts, electrofusion, kanamycin resistance, sodium iodoacetate, somatic hybridization     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Mesorhizobium loti is a Gram negative bacterium that induces N₂-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.     

Seasonality of Rhizome and Shoot Production and Nitrogen Fixation in Lotus uliginosus under Upland Conditions in South-west Scotland

Development, growth and nitrogen fixation were monitored over3 years in marsh birdsfoot trefoil (Lotus uliginosus Schkuhr),at an upland site in south-west Scotland Plants exhibited markedseasonality of growth, with production of both aerial shootsand rhizome reaching a peak in September and October Renewedaerial shoot growth in spring was initiated mainly from nodeson rhizomes formed in the previous autumn Nitrogen fixationwas measured in soil cores using the acetylene reduction techniqueFixation commenced in early June, coinciding with increasingsoil temperature, and thereafter rose to a peak in mid-summerThe subsequent decline in fixation was associated with increasingrainfall, decreasing soil temperature, flowering and the onsetof rhizome production Lotus uliginosus Schkuhr, marsh birdsfoot trefoil, seasonality, rhizome production, nitrogen fixation, acetylene reduction     

Two Ineffective-Nodulating Mutants of Lotus japonicus--Different Phenotypes Caused by the Blockage of Endocytotic Bacterial Release and Nodule Maturation

Mutants defective in nodule development and nitrogen fixationof Lotus japonicus B-129 ‘Gifu’ were obtained byinduced mutagenesis with EMS (ethylmethane sulfonate) treatment.Using a symbiont of L. japonicus, Rhizobium loti JRLS01, 17,000M2 seeds were screened for plants affected in their symbioticphenotype, resulting in the successful isolation of eleven stablemutants. In this paper, we report two ineffective nodulatingmutants among them. Reciprocal crossing between wild type 'Gifu'and these mutants indicated that their phenotypes are undermono-genic and recessive control. Furthermore, tests for alle-lismwith these mutants showed that the mutated genes are non-allelic.Ultrastructural analysis revealed that these mutants were inhibitedat different stages of nodule development and maturation. Basedon histological characteristics of the nodules, two ineffectivenodulating mutants were named albl (aberrant localization ofbacteria inside nodule) and fenl (fail in enlargement of infectedcells), respectively. In the nodules of albl, most of the bacteriafailed to invade the cytoplasm of cortical cells and were tightlyconfined inside infection threads or localized in intercellularspaces of nodules. Following prolonged inoculation, albl mutantalso formed pale-pink colored nodules with a low frequency,in which bacteria differentiated into bacteroid and fixed nitrogennormally. Although the infected cells in the nodules of fenlmutant contained numerous differentiated bacteroids, they failedto enlarge by cell expansion and showed a low activity of nitrogenfixation. (Received March 18, 1997; Accepted May 8, 1997)