Cloning and expression pattern of a novel microspore-specific gene encoding hypersensitive-induced response protein (LjHIR1) from the model legume, Lotus japonicus

In order to understand the microspore and pollen development, recently, we have isolated a number of anther-specific genes in the model legume, Lotus japonicus. From these anther-specific genes, we identified one novel microspore-specific gene, LjImfb-c82. In order to determine the molecular characterization of LjImfb-c82, full-length cDNA clone was first isolated and sequenced. It encoded a protein of 286 amino acids (LjHIR1), which had sequence similarity to Hypersensitive-Induced Response like protein. LjHIR1 was specifically expressed in microspore on the in situ hybridization experiment. From the sequence similarity to prohibitin-domain protein, the LjHIR1 might be related to ion channel regulation in microspore development.     

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).     

Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.     

Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Karyotypes and sex-determination in spider mites (Tetranychidae)

The chromosomes of 13 species of spider mites (Tetranychidae) are determined using the aceto-orcein squash technique in order to establish the haplo-diploid sex-determination. 12 species showed the existence of haploid and diploid eggs:Neotetranychus rubi (Trägårdh) with 7 and 14 chromosomes;Eurytetranychus buxi (Garman) with 5 and 10;Bryobia sarothamni (Geijskes),Eotetranychus tiliarium (Joh. Hermann) andE. carpini (Oudemans) with 4 and 8;Panonychus ulmi (Koch),Schizotetranychus schizopus (Zacher),Oligonychus ununguis (Jacobi).Tetranychus hydrangeae Pritch. & Baker, T.pacificus McGregor,T. urticae Koch andT. cinnabarinus (Boisduval) with 3 and 6 chromosomes.The progeny of virgin females in 7 different species consisted of eggs with the haploid complement. One species was shown to be thelytokous, viz.Tetranycopsis horridus (Canestrini & Franzago), having a diploid number of 4 chromosomes. Some eggs of an inbred line ofT. urticae showed a patchwork quilt of odd polyploidy in embryonic tissue.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

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.     

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.     

Ecotypes of the model legume Lotus japonicus vary in their interaction phenotypes with the root-knot nematode Meloidogyne incognita

BACKGROUND AND AIMS: Knowledge of host factors affecting plant-nematode interactions is scarce. Here, relevant interaction phenotypes between a nodulating model host, Lotus japonicus, and the endoparasitic root-knot nematode Meloidogyne incognita are assessed via a genetic screen. METHODS: Within an alpha experimental design, 4-week-old replicate plants from 60 L. japonicus ecotypes were inoculated with 1000 nematodes from a single egg mass population, and evaluated for galling and nematode egg masses 6 weeks after inoculation. KEY RESULTS: Statistical analysis of data for 57 ecotypes showed that ecotype susceptibilities ranged from 3.5 to 406 galls per root, and correlated strongly (r = 0.8, P < 0.001, log scale) with nematode reproduction (ranging from 0.6 to 34.5 egg masses per root). Some ecotypes, however, showed a significant discrepancy between disease severity and nematode reproduction. Necrosis and developmental malformations were observed in other infected ecotypes. CONCLUSIONS: The first evidence is provided of significant variability in the interactions between L. japonicus and root-knot nematodes that may have further implications for the genetic dissection and characterization of host pathways involved in nematode parasitism and, possibly, in microbial symbiosis.     

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.     

The advantages of cDNA microarray as an effective tool for identification of reproductive organ-specific genes in a model legume, Lotus japonicus

To understand the molecular mechanisms intrinsic to reproductive organ development a cDNA microarray, fabricated from flower bud cDNA clones, was used to isolate genes, which are specifically expressed during the development of the anther and pistil in Lotus japonicus. Cluster analysis of the microarray data revealed 21 and 111 independent cDNA groups, which were specifically expressed in immature and mature anthers, respectively. RT-PCR was performed to provide a direct assessment of the accuracy and reproducibility of our approach. Confirmation of our results suggests that cDNA microarray technology is an effective tool for identification of novel reproductive organ-specific genes.     

Induced production of extrafloral nectar in intact lima bean plants in response to volatiles from spider mite-infested conspecific plants as a possible indirect defense against spider mites

We found that intact lima bean plants increased the secretion of extrafloral nectar (EFN) after exposure to Tetranychus urticae-induced plant volatiles. Predatory mites, Phytoseiulus persimilis, dispersed more slowly from an exposed intact plant than from a control plant (plant exposed to volatiles from intact conspecific). The predators also dispersed more slowly from those plants that were provided with extra EFN than from untreated plants. We further show that EFN was a potential alternative food source for P. persimilis. From these results, we concluded that increased EFN was involved in the slow dispersal of P. persimilis from the plants exposed to herbivore-induced plant volatiles. Our data suggest that the increase of EFN in an HIPV-exposed intact plant could be an induced indirect defense against spider mites.