The origin, initiation and development of axillary shoot meristems in Lotus japonicus

BACKGROUND AND AIMS: Lotus japonicus 'Gifu' develops multiple axillary shoots in the cotyledonary node region throughout the growth of the plant. The origin, initiation and development of these axillary meristems were investigated. METHODS: Morphological, histological and mRNA in situ analyses were done to characterize the ontogeny of cotyledonary axillary shoot meristems in Lotus. Morphological characterization of a putative Lotus shoot branching mutant (super-accessory branches) sac, is presented. KEY RESULTS: By using expression of an L. japonicus STM-like gene as a marker for meristematic tissues, it was demonstrated that groups of cells maintained in the meristematic state at the cotyledonary axil region coincide with the sites where additional axillary meristems (accessory meristems) form. A Lotus shoot branching mutant, sac, is a putative Lotus branching mutant characterized by increased proliferation of accessory shoots in all leaf axils including the cotyledons. CONCLUSION: In Lotus, axillary shoot meristems continually develop at the cotyledonary node region throughout the growth of the plant. These cotyledonary primary and accessory axillaries arise from the position of a meristematic zone of tissue at the cotyledonary node axil region.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

Structural and expression analysis of uricase mRNA from Lotus japonicus

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.     

The small GTPase ROP6 interacts with NFR5 and is involved in nodule formation in Lotus japonicus

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.     

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.     

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.     

Novel SINEs families in Medicago truncatula and Lotus japonicus: bioinformatic analysis

Although short interspersed elements (SINEs) were discovered nearly 30 years ago, the studies of these genomic repeats were mostly limited to animal genomes. Very little is known about SINEs in legumes--one of the most important plant families. Here we report identification, genomic distribution and molecular features of six novel SINE elements in Lotus japonicus (named LJ_SINE-1, -2, -3) and Medicago truncatula (MT_SINE-1, -2, -3), model species of legume. They possess all the structural features commonly found in short interspersed elements including RNA polymerase III promoter, polyA tail and flanking repeats. SINEs described here are present in low to moderate copy numbers from 150 to 3000. Bioinformatic analyses were used to searched public databases, we have shown that three of new SINE elements from M. truncatula seem to be characteristic of Medicago and Trifolium genera. Two SINE families have been found in L. japonicus and one is present in both M. truncatula and L. japonicus. In addition, we are discussing potential activities of the described elements.     

Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

Lotus japonicus nodules are reviewed, and current research data on Nod-35 (uricase) gene, including that of Lotus japonicus as an example of a late nodulin gene are presented. Received 5 September 2000/ Accepted in revised form 2 October 2000     

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express beta-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.     

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.     

Nodule Organogenesis in Lotus japonicus

Lotus japonicus. Received 25 September 2000/ Accepted in revised form 9 October 2000     

百脉根嫁接方法的优化及其在结瘤自调控研究中的应用

嫁接试验是研究结瘤自调控的有效方法。本文优化了百脉根的嫁接方法,并应用该方法初步研究了百脉根突变体rel3根瘤数目减少的机理。结果表明,rel3突变体根瘤数目减少的表型是由来自于茎的信号决定。