Miyakojima MG-20 Research Articles

SEN1 gene from Lotus japonicus MG20 improves nitrogen fixation and plant growth

ABSTRACT In Lotus japonicus, quantitative trait loci (QTLs) for nitrogen fixation activity (acetylene reduction activity, ARA) and seed weight are located in the same region on chromosome 4 as the gene responsible for the Fix− mutant sen1. The SEN1 gene of L. japonicus Gifu B129 has a deletion of three nucleotides in comparison with that of L. japonicus Miyakojima MG20, which results in a deletion of one amino acid. Here, we examined whether SEN1 is responsible for these QTLs. Because the MG20 allele of the QTL for ARA and seed weight has a positive effect, we generated a near-isogenic line (NIL) in which the SEN1 gene region of B129 was replaced with that of MG20. Growth parameters of this NIL (MG20-type) 21 days after inoculation with Mesorhizobium loti were significantly higher than those of B129-type, and ARA was also significantly higher. Nodules formed on hairy roots in which the sen1 mutant was complemented with the MG20 SEN1 gene had higher ARA than those of B129. The seeds of the MG20-type NIL were significantly heavier than those of B129-type. These results indicate that the SEN1 gene is a very likely candidate for the QTL for ARA and seed weight on chromosome 4.

Chlorophyll a fluorescence analysis reveals divergent photosystem II responses to saline, alkaline and saline–alkaline stresses in the two Lotus japonicus model ecotypes MG20 and Gifu-129

Saline and alkaline stresses affect more than 10% of the World’s arable land, limiting agricultural production. Salt-induced stress may affect the photosystem II (PSII) function, altering fluorescence emission. Therefore, changes in fluorescence are used to quantify and analyze abiotic stress responses in plants. So far, no study has focused on the response of PSII to saline, alkaline and saline–alkaline stresses in the model legume Lotus japonicus. For the saline, alkaline and saline–alkaline treatments, plants of the L. japonicus ecotypes MG20 and Gifu-129 were cultivated in sand with nutrient solution, added with NaCl and NaHCO3 in different proportions. Growth, gas exchange, and chlorophyll a fluorescence transient kinetic and OJIP parameters were measured, and chlorophyll a and b were determined. The analysis of the kinetic of chlorophyll a fluorescence showed that NaCl-derived stress sources affect the photochemical events in PSII in both ecotypes, being this effect more evident under higher pH condition, whereas alkalinity per se has a mild or no effect on these events. The saline–alkaline stress induced a more severe effect on Gifu B-129, compared with Miyakojima MG20, whereas NaCl improved primary photochemistry in MG20. Our results allow us to accept the hypothesis that both ecotypes deploy differential responses under the three stressful treatments and that the saline–alkaline stress causes higher damage levels than saline and alkaline stresses alone in relation with structures and sub-processes of the PSII.

Characterization of the primary metabolome during the long-term response to NaHCO3-derived alkalinity in Lotus japonicus ecotypes Gifu B-129 and Miyakojima MG-20

This study compares the response of two ecotypes of the model species Lotus japonicas, MG-20 and Gifu-B-129, to soil alkalinity, in terms of plant survival and changes in global primary metabolome profiles. After 54 days of treatment with 30 mM NaHCO3, a higher survival was registered in MG-20, with respect to Gifu-B-129 plants. Gas chromatography–mass spectrometry (GC–MS) analysis of shoot extracts from both ecotypes yielded 123 different analytes, 62 of which were identified, including organic acids (OA), amino acids (AA), sugars and polyols. Glycolysis, TCA cycle and amino acids metabolism pathways were differently affected by alkalinity according to the ecotype. The lower tolerance of Gifu B-129 plants to 10 mM NaHCO3, compared with MG-20 ones could be related, at least partially, to the differential accumulation of phosphoric, lactic, threonic, succinic and p-coumaric acids, as well as β-alanine and valine.

Enhanced hyphal growth of arbuscular mycorrhizae by root exudates derived from high R/FR treated Lotus japonicus

ABSTRACTRed/Far Red (R/FR) sensing positively influences the arbuscular mycorrhizal (AM) symbiosis of both legume and nonlegume plants through jasmonic acid (JA) and strigolactone signaling. We previously reported that root exudates obtained from high R/FR-grown plants contained more strigolactone than low R/FR-grown plants. To determine whether JA and JA derivatives were secreted from roots, we investigated the expression levels of JA-responsive genes in L. japonicus Miyakojima MG20 plants treated with root exudates prepared from either high or low R/FR light-treated plants. The root exudates from high R/FR light-treated plants were found to enhance the expression levels of JA-responsive genes significantly. Moreover, exogenous JA increased AM fungal hyphal elongation as did the root exudates derived from high R/FR-grown L. japonicus plants. We conclude that increased JA accumulation and secretion into root exudates from high R/FR light-grown plants is the best explanation for increased colonization and enhanced mycorrhization under these conditions.

A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus.

Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops.

Response to long-term NaHCO3-derived alkalinity in model Lotus japonicus Ecotypes Gifu B-129 and Miyakojima MG-20: transcriptomic profiling and physiological characterization.

The current knowledge regarding transcriptomic changes induced by alkalinity on plants is scarce and limited to studies where plants were subjected to the alkaline salt for periods not longer than 48 h, so there is no information available regarding the regulation of genes involved in the generation of a new homeostatic cellular condition after long-term alkaline stress. Lotus japonicus is a model legume broadly used to study many important physiological processes including biotic interactions and biotic and abiotic stresses. In the present study, we characterized phenotipically the response to alkaline stress of the most widely used L. japonicus ecotypes, Gifu B-129 and MG-20, and analyzed global transcriptome of plants subjected to 10 mM NaHCO3 during 21 days, by using the Affymetrix Lotus japonicus GeneChip®. Plant growth assessment, gas exchange parameters, chlorophyll a fluorescence transient (OJIP) analysis and metal accumulation supported the notion that MG-20 plants displayed a higher tolerance level to alkaline stress than Gifu B-129. Overall, 407 and 459 probe sets were regulated in MG-20 and Gifu B-129, respectively. The number of probe sets differentially expressed in roots was higher than that of shoots, regardless the ecotype. Gifu B-129 and MG-20 also differed in their regulation of genes that could play important roles in the generation of a new Fe/Zn homeostatic cellular condition, synthesis of plant compounds involved in stress response, protein-degradation, damage repair and root senescence, as well as in glycolysis, gluconeogenesis and TCA. In addition, there were differences between both ecotypes in the expression patterns of putative transcription factors that could determine distinct arrangements of flavonoid and isoflavonoid compounds. Our results provided a set of selected, differentially expressed genes deserving further investigation and suggested that the L. japonicus ecotypes could constitute a useful model to search for common and distinct tolerance mechanisms to long-term alkaline stress response in plants.

Phenomic networks reveal largely independent root and shoot adjustment in waterlogged plants of Lotus japonicus

Waterlogging imposes severe stress to the plant, and the interplay between root and aerial organs in the adjustment to this stress is poorly understood. A set of recombinant inbred lines (RILs) of Lotus japonicus (Gifu B-129 × Miyakojima MG-20) was subjected to control and waterlogging conditions for 21 d, and 12 traits related to leaf physiological functioning, root aerenchyma formation, shoot and root morphology, and dry mass accumulation were assessed to generate phenomic networks. The phenomic network became more complex under waterlogging as a result of the incorporation of root aerenchyma and dark-adapted Fv/Fm. Significant waterlogging-induced variation was found for stomatal conductance, dark-adapted Fv/Fm and aerenchyma. The RILs with stronger induction of aerenchyma in response to waterlogging tended to show reduced negative impact of this stress on root growth but suffered average impact on shoot growth. The RILs that retained higher stomatal conductance under waterlogging tended to retain higher dark-adapted Fv/Fm and shoot growth under waterlogging conditions but showed average impact on root traits. We propose a model where, although the stress experienced by the roots during waterlogging is transmitted to the shoot, shoots and roots deal with waterlogging in a less interdependent manner than often assumed.

Micro-CT observations of the 3D distribution of calcium oxalate crystals in cotyledons during maturation and germination inLotus miyakojimaeseeds

The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels.

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume Lotus japonicus

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20×Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac-), shoot dry weight without inoculation (SWbac-), root length without inoculation (RLbac-), and root dry weight (RWbac-), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.

Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling.

Quantitative trait locus analysis of root ferric reductase activity and leaf chlorosis in the model legume, Lotus japonicus

Background and aimsFerric reductase activity is a rate-limiting step in the accumulation of iron by Strategy I plants. Preliminary work with Lotus japonicus accessions Miyakojima MG-20 and Gifu B-129 identified differences in shoot chlorosis and ferric reductase activity. This study assessed the genetic basis for these differences.MethodsLines of a recombinant inbred population, derived from Miyakojima and Gifu, were tested for whole-root ferric reductase activity and shoot chlorosis following iron-limited growth. A ferric reductase gene (LjFRO1) was cloned from both parents. Protein sequence analysis, transcript abundance, and yeast complementation studies were conducted with the two parental alleles.ResultsA single quantitative trait locus (QTL) was identified for both ferric reductase activity and shoot chlorosis, with each QTL explaining ~30% of the variation and both overlapping across the same region of chromosome 3. LjFRO1 mapped to chromosome 3, but to a region adjacent to the reductase and chlorosis loci. Nucleotide variation in LjFRO1 parental alleles was identified, as were minor functional differences between the two proteins.ConclusionsThe results indicate that both allelic variation (providing potential functional differences) and unidentified molecular components (derived from non-LjFRO1 genetic loci) can contribute to the regulation of ferric reductase activity and chlorosis susceptibility.

Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.

Real-time imaging of ion uptake from root to above-ground part of the plant using conventional beta-ray emitters

We present real-time imaging of 32P-phosphate within a living plant as well as that in culture solution toward roots. The above-ground part of the plant was irradiated with light from LED and roots were kept in dark. The plant was Lotus japonicus, cv. Miyakojima MG20 and real-time 32P-phosphate uptake manners in young, flowering and adult phase were visualized. In the case of young plant, there was a preferential translocation of 32P in younger leaves. When the sample was treated with phosphate deficient condition, 32P uptake amount was much higher. In an adult phase, 32P uptake was high at pods.

Identification of nutrient and physical seed trait QTL in the model legumeLotus japonicus

Legume seeds have the potential to provide a significant portion of essential micronutrients to the human diet. To identify the genetic basis for seed nutrient density, quantitative trait locus (QTL) analysis was conducted with the Miyakojima MG-20 x Gifu B-129 recombinant inbred population from the model legume Lotus japonicus. This population was grown to seed under greenhouse conditions in 2006 and 2007. Phenotypic data were collected for seed calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and zinc (Zn) concentrations and content. Data for physical seed traits (average seed mass and seed-pod allocation values) were also collected. Based on these phenotypic data, QTL analyses identified 103 QTL linked to 55 different molecular markers. Transgressive segregation, identified within this recombinant inbred population for both seed nutrient and physical traits, suggests new allelic combinations are available for agronomic trait improvement. QTL co-localization was also seen, suggesting that common transport processes might contribute to seed nutrient loading. Identification of loci involved in seed mineral density can be an important first step in identifying the genetic factors and, consequently, the physiological processes involved in mineral distribution to developing seeds. Longer term research efforts will focus on facilitating agronomic breeding efforts through ortholog identification in related crop legumes.

A High-density Linkage Map of Lotus japonicus Based on AFLP and SSR Markers

A collection of 94 F6 individuals derived from crosses between Lotus japonicus, Gifu B-129 (G) and Miyakojima MG-20 (M) were used for mapping. By using the HEGS running system, 427 EcoRI/MseI primer pairs were selected to generate a total of 2053 markers, consisting of 739 G-associated dominant markers, 674 M-associated dominant markers, 640 co-dominant markers, 95 SSR markers and 2 dCAPS markers. Excluding heavily distorted markers, 1588 were mapped to six chromosomes of the L. japonicus genome based on the 97 reference markers. This linkage map consisted of 1023 unique markers (excluding duplicated markers) and covered a total of 508.5 cM of the genome with an average chromosome length of 84.7 cM and interval distance of 0.50 cM. Fifteen quantitative traits loci for eight morphological traits were also mapped. This linkage map will provide a useful framework for physical map construction in L. japonicus in the near future.

Isolation of a novel root-determined hypernodulation mutant rdh1 of Lotus japonicus

A mutant line that develops an excess number of small nodules was found in Lotus japonicus Miyakojima MG20 during a screening for mutants defective in nodule development and nitrogen fixation. Genetic analysis revealed that the phenotype is inherited in a monogenic, recessive manner. The gene's locus was mapped on chromosome 1 between 53.7 and 61.4 cM. This mutant formed 5–10-fold more nodules than the wild-type plant, and a grafting experiment revealed that the root regulated the hypernodulation. Except for the nodulation's phenotype no other differences were found between the mutant and the wild-type plant with respect to growth and morphological characteristics. In the mapped locus for the mutant no nodulation genes were reported, and this fact strongly suggests that the gene's locus is a new one. The gene was named root-determined hypernodulation (rdh) 1.

Metabolic profiling of flavonoids in Lotus japonicus using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

Flavonoids detected from a model legume plant, Lotus japonicus accessions Miyakojima MG-20 and Gifu B-129, were profiled using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR/MS). Five flavonols and two anthocyanidins were detected as aglycones. LC-FTICR/MS facilitated simultaneous detection of 61 flavonoids including compounds that have not been reported previously. Chemical information of the peaks such as retention time, λ max, m/ z value of the quasi-molecular ion, m/ z value of MS/MS fragment ions, and relative intensity of MS/MS fragments was obtained, along with the molecular formulas and conjugate structures. Fourteen were completely identified by comparison with authentic compounds. The high accuracy of m/ z values, being 0.081 ppm between observed and theoretical values, allowed prediction of molecular formulas of unknown compounds with the help of isotope peak information for determination of chemical composition. Based on a predicted elemental composition, the presence of a novel nitrogen-containing flavonoid was proposed. A comparison of flavonoid profiles in flowers, stems, and leaves demonstrated that the flowers yielded the most complex profile, containing 30 flower-specific flavonoids including gossypetin glycosides and isorhamnetin glycosides. A comparison of flavonoid profiles between MG-20 and B-129 grown under the same conditions revealed that the accumulation of anthocyanins was higher in B-129 than MG-20, particularly in the stem. Developmental changes in the flavonoid profiles demonstrated that kaempferol glycosides increased promptly after germination. In contrast, quercetin glycosides, predominant flavonoids in the seeds, were not detectable in growing leaves.

Quantitative trait locus analysis of multiple agronomic traits in the model legumeLotus 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.

Klavier (klv), A novel hypernodulation mutant of Lotus japonicus affected in vascular tissue organization and floral induction

A novel hypernodulation mutant line was isolated from Lotus japonicus Miyakojima MG-20 by irradiation with a helium ion beam. This mutant, named klavier (klv), had roots that were densely covered with small nodules. The nodulation zone of klv was significantly wider than that of the wild type. Grafting experiments showed that klv is impaired in the long-distance shoot-to-root autoregulatory mechanism. Thus the shoot genotype was found to be responsible for the negative regulation of nodule development by KLV. Nodulation of klv showed a higher tolerance to nitrogen (KNO3) than the wild type, which is a common feature of hypernodulating mutants. In addition to an increased number of nodules, the klv mutant showed convex leaf veins on the adaxial leaf surface, markedly delayed flowering and dwarf phenotypes. Microscopic examination of the leaf veins revealed that they were discontinuous. Other phenotypes such as fasciated stems, increased number of flowers and bifurcated pistils were also frequently observed in the klv mutant. Among these phenotypes, hypernodulation, aberrant leaf vein formation and significantly delayed flowering were all linked in a monogenic and recessive manner, indicating that these phenotypes are caused by either a single mutation, or tightly linked mutations. KLV was mapped within 0.29 cM on the long arm of chromosome 1.

Expression of a major house dust mite allergen gene from Dermatophagoides farinae in Lotus japonicus accession miyakojima MG-20

Transformation of a model legume Lotus japonicus accession Miyakojima MG-20 was examined using Agrobacterium tumefaciens with a binary expression vector. Using the improved transformation method, we introduced a major allergen gene from a house dust mite, Der f 1, into MG-20. Analyses by Southern hybridization, reverse transcription (RT)-PCR, and Western blotting showed that the Der f 1 gene was integrated into the genome of L. japonicus, expressing the gene product in the T1 lines. Our results imply future application of oral allergen-specific immunotherapy using legume plants.