根粒菌の根粒形成に関する遺伝子工学的研究

I INTRODUCTION II PHAGE INDUCTION OF LYSOGENIC RHIZOBWM LEGUMINOSARUM BIOVAR TRIFOLII IN BOTH THE FREE-LIVING AND THE SYMBIOTIC FORM III A CHROMOSOMAL INTEGRATIVE VECTOR SYSTEM UTILIZING DNA FRAGMENTS OF A LYSOGENIC PHAGE OF RIIIZOBIUM LEGUMINOSARUM IV INTEGRATION OF A PLASMID CARRYING THE ATTP SITE INTO THE CHROMOSOME OF RHIZOBIUM INSENSITIVE TO THE LYSOGENIC PHAGE INFECTION V NODULE FORMATION BY CLOVER-RIIIZOBIUM CARRYING CHROMOSOMAL NOD GENES VI CONCLUDING REMARKS VII REFERENCES VIII ACKNOWLEDGMENTSMade available in DSpace on 2012-09-06T04:54:29Z (GMT). No. of bitstreams: 1 uchiumi.pdf: 15510521 bytes, checksum: 311dabb07c491af11958c7bbf20d3536 (MD5) Previous issue date: 1995-04-19主1-参

Hemoglobin LjGlb1-1 is involved in nodulation and regulated the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia–legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5′-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo. The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs

Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1

Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin

Elevated Nitrogen Priming Induced Oxinitro-Responses and Water Deficit Tolerance in Rice

Under water deficit conditions, the essential macronutrient nitrogen becomes limited as a result of reduced dissolved nitrogen and root nitrogen uptake. An elevated nitrogen level might be able to mitigate these effects, integrated with the idea of using nitric oxide as abiotic stress tolerant inducers. In this study, we evaluated the potential of using elevated nitrogen priming prior to water shortage to mitigate plant stress through nitric oxide accumulation. We grew rice plants in 300 mg L−1 nitrogen for 10 weeks, then we primed plants with four different nitrogen concentrations: 100, 300 (control), 500 and 1000 mg L−1 nitrogen prior to inducing water deficit conditions. Plants primed with 500 mg L−1 nitrogen possessed a higher photosynthetic rate, relative water content, electrolyte leakage and lipid peroxidation under water deficit conditions, compared to control plants. The induction of water deficit tolerance was supported with the activation of antioxidant defense system, induced by the accumulation of nitric oxide in leaves and roots of rice plants. We originally demonstrated the accumulation of nitric oxide in leaves of rice plants. The elevated nitrogen priming can be used to enhance water deficit tolerance in irrigated paddy fields, instead of nitric oxide donors

Ectopic or Over-Expression of Class 1 Phytoglobin Genes Confers Flooding Tolerance to the Root Nodules of Lotus japonicus by Scavenging Nitric Oxide

Flooding limits biomass production in agriculture. Leguminous plants, important agricultural crops, use atmospheric dinitrogen gas as nitrogen nutrition by symbiotic nitrogen fixation with rhizobia, but this root-nodule symbiosis is sometimes broken down by flooding of the root system. In this study, we analyzed the effect of flooding on the symbiotic system of transgenic Lotus japonicus lines which overexpressed class 1 phytoglobin (Glb1) of L. japonicus (LjGlb1-1) or ectopically expressed that of Alnus firma (AfGlb1). In the roots of wild-type plants, flooding increased nitric oxide (NO) level and expression of senescence-related genes and decreased nitrogenase activity; in the roots of transgenic lines, these effects were absent or less pronounced. The decrease of chlorophyll content in leaves and the increase of reactive oxygen species (ROS) in roots and leaves caused by flooding were also suppressed in these lines. These results suggest that increased levels of Glb1 help maintain nodule symbiosis under flooding by scavenging NO and controlling ROS