Trehalose metabolism in root nodules of the model legume Lotus japonicus in response to salt stress

In wheat ( Triticum aestivum L), the leaves particularly flag leaves have been considered to be the key organs contributing to higher yields, whereas awns have been considered subsidiary organs. Compared with extensive investigations on the assimilation contribution of leaves, the photosynthetic characteristics of awns have not been well studied. In this study, we investigated the ultrastructure of chloroplasts, oxygen evolution, and phosphoenolpyruvate carboxylase [phosphoenolpyruvate carboxylase (PEPCase) EC 4.1.1.31)] activity in both flag leaves and awns during the ontogenesis of wheat. Transmission electron microscope observations showed initial increases in the sizes of grana and the degree of granum stacks from the florescence-emergence stage both in flag leaves and in awns, followed by the breakdown of membrane systems after the milk-development stage. The results of oxygen evolution assays revealed that in both organs, the rate of photosynthesis increased in the first few stages and then decreased, but the decrease occurred much earlier in flag leaves than in awns. A PEPCase activity assay demonstrated that the activity of PEPCase was much higher in awns than in flag leaves throughout ontogeny; the value was particularly high at the late stages of grain filling. Our results suggest that awns play a dominant role in contributing to large grains and a high grain yield in awned wheat cultivars, particularly during the grain-filling stages.     

Chromosomal map of the model legume Lotus japonicus

Lotus japonicus is a model plant for the legume family. To facilitate map-based cloning approaches and genome analysis, we performed an extensive characterization of the chromosome complement of the species. A detailed karyotype of L. japonicus Gifu was built and plasmid and BAC clones, corresponding to genetically mapped markers (see the accompanying article by Sandal et al. 2002, this issue), were used for FISH to correlate genetic and chromosomal maps. Hybridization of DNA clones from 32 different genomic regions enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and the partial characterization of different repetitive sequences, including telomeric and centromeric repeats. Additional analysis of L. filicaulis and its F(1) hybrid with L. japonicus demonstrated the occurrence of inversions between these closely related species, suggesting that these chromosome rearrangements are early events in speciation of this group.     

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     

Root, root hair, and symbiotic mutants of the model legume Lotus japonicus

To gain an overview of plant factors controlling nodule number and organogenesis, an extensive screening using model legume Lotus japonicus was carried out. This screening involved 40,000 M2 seeds, and 32 stable mutant lines were isolated. From these, 16 mutant lines maintaining the phenotypic variation were selected and genetically analyzed. With respect to nodule number, four loci were identified, Ljsym77, Ljsym78, slippery root (slp), and radial organization1 (rdo1). The former two mutants have an increased number of nodules, while the latter two have a decreased number. Ljsym78-1 and Ljsym78-2 are hypernodulating mutants with a branched root system and were found to be allelic to Ljsym16. The phenotype of the Ljsym77 mutant was highly pleiotropic, being deficient in light and gravity responses. The slp mutant was isolated as a low-nodulating mutant lacking root hairs. Concerning nodule organogenesis, nine symbiotic loci were identified, including the two loci alb1 and fen1. Mutants affecting the developmental process of nodule organogenesis were placed in three phenotypic categories: Nod- (Ljsym70 to Ljsym73), Hist- (alb1-1, alb1-2, and Ljsym79), and Fix- (fen1, Ljsym75, and Ljsym81).     

Lotus japonicus: a new model to study root-parasitic nematodes

Sedentary plant-parasitic nematodes engage in complex interactions, and induce specialized feeding structures by redirecting plant developmental pathways, and parallels have been observed with rhizobial nodule development on legumes. A model legume would greatly facilitate a better understanding of the differences between parasitic (nematode) and mutualistic (rhizobia and mycorrhizae) symbioses, and we have developed Lotus japonicus as such a model. Conditions for efficient parasitism by root-knot nematode (Meloidogyne spp.) of the widely used Lotus "Gifu" ecotype were established. Features of Lotus biology, such as thin and translucent roots, proved ideal for monitoring the progress of nematode infection both on live specimens and post-staining. We examined L. japonicus mutants with nodulation phenotypes. One, har1, which is a hypernodulated mutant defective in a CLAVATA1-like receptor kinase gene, was found to be hyperinfected by M. incognita. However, another hypernodulated Lotus mutant exhibited the same level of M. incognita infection as wild-type plants. We also established conditions for infection of Lotus by soybean cyst nematode (Heterodera glycines). In contrast to the response to root-knot nematode, the Gifu ecotype is resistant to H. glycines, and elicits a hypersensitive response. This pattern of resistance recapitulates that seen on nematode-resistant soybean plants. We conclude that L. japonicus is a powerful model legume for studying compatible and incompatible plant-nematode interactions.     

The transposable element landscape of the model legume Lotus japonicus

The largest component of plant and animal genomes characterized to date is transposable elements (TEs). The availability of a significant amount of Lotus japonicus genome sequence has permitted for the first time a comprehensive study of the TE landscape in a legume species. Here we report the results of a combined computer-assisted and experimental analysis of the TEs in the 32.4 Mb of finished TAC clones. While computer-assisted analysis facilitated a determination of TE abundance and diversity, the availability of complete TAC sequences permitted identification of full-length TEs, which facilitated the design of tools for genomewide experimental analysis. In addition to containing all TE types found in previously characterized plant genomes, the TE component of L. japonicus contained several surprises. First, it is the second species (after Oryza sativa) found to be rich in Pack-MULEs, with >1000 elements that have captured and amplified gene fragments. In addition, we have identified what appears to be a legume-specific MULE family that was previously identified only in fungal species. Finally, the L. japonicus genome contains many hundreds, perhaps thousands of Sireviruses: Ty1/copia-like elements with an extra ORF. Significantly, several of the L. japonicus Sireviruses have recently amplified and may still be actively transposing.     

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.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation     

The afterglow thermoluminescence band as an indicator of changes in the photorespiratory metabolism of the model legume Lotus japonicus

The afterglow (AG) luminescence is a delayed chlorophyll fluorescence emitted by the photosystem II that seems to reflect the level of assimilatory potential (NADPH+ATP) in chloroplast. In this work, the thermoluminescence (TL) emissions corresponding to the AG band were investigated in plants of the WT and the Ljgln2‐2 photorespiratory mutant from Lotus japonicus grown under either photorespiratory (air) or non‐photorespiratory (high concentration of CO₂) conditions. TL glow curves obtained after two flashes induced the strongest overall TL emissions, which could be decomposed in two components: B band (t_max = 27–29°C) and AG band (t_max = 44–45°C). Under photorespiratory conditions, WT plants showed a ratio of 1.17 between the intensity of the AG and B bands (I_AG/I_B). This ratio increased considerably under non‐photorespiratory conditions (2.12). In contrast, mutant Ljgln2‐2 plants grown under both conditions showed a high I_AG/I_B ratio, similar to that of WT plants grown under non‐photorespiratory conditions. In addition, high temperature thermoluminescence (HTL) emissions associated to lipid peroxidation were also recorded. WT and Ljgln2‐2 mutant plants grown under photorespiratory conditions showed both a significant HTL band, which increased significantly under non‐photorespiratory conditions. The results of this work indicate that changes in the amplitude of I_AG/I_B ratio could be used as an in vivo indicator of alteration in the level of photorespiratory metabolism in L. japonicus chloroplasts. Moreover, the HTL results suggest that photorespiration plays some role in the protection of the chloroplast against lipid peroxidation.     

Quantitative trait locus analysis of multiple agronomic traits in the model legume Lotus japonicus.

The first quantitative trait locus (QTL) analysis of multiple agronomic traits in the model legume Lotus japonicus was performed with a population of recombinant inbred lines derived from Miyakojima MG-20 x Gifu B-129. Thirteen agronomic traits were evaluated in 2004 and 2005: traits of vegetative parts (plant height, stem thickness, leaf length, leaf width, plant regrowth, plant shape, and stem color), flowering traits (flowering time and degree), and pod and seed traits (pod length, pod width, seeds per pod, and seed mass). A total of 40 QTLs were detected that explained 5%-69% of total variation. The QTL that explained the most variation was that for stem color, which was detected in the same region of chromosome 2 in both years. Some QTLs were colocated, especially those for pod and seed traits. Seed mass QTLs were located at 5 locations that mapped to the corresponding genomic positions of equivalent QTLs in soybean, pea, chickpea, and mung bean. This study provides fundamental information for breeding of agronomically important legume crops.     

Spontaneous root-nodule formation in the model legume Lotus japonicus: a novel class of mutants nodulates in the absence of rhizobia

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.