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At least 73 records · Page 4Linked to original sources

PubMed2004

N-hexanoyl-L-homoserine lactone, a mediator of bacterial quorum-sensing regulation, exhibits plant-dependent stability and may be inactivated by germinating Lotus corniculatus seedlings.

The half-life of N-hexanoyl-l-homoserine lactone (C6-HSL) was determined under various pH and temperature conditions, and in several plant environments. C6-HSL was sensitive to alkaline pH, a process that was also temperature-dependent. In addition, C6-HSL disappeared from plant environments, i.e. axenic monocot and dicot plants cultivated under gnotobiotic, hydroponic conditions, albeit with variable kinetics. The disappearance was rapid at the root system of legume plants such as clover or Lotus, and slow or non-existent at the root system of monocots such as wheat or corn. These variable kinetics were not dependent upon pH changes that may have affected the growth media of the plants. Furthermore, C6-HSL did not accumulate in the plant, and the plant did not produce inhibitors of the C6-HSL signal. HPLC analyses revealed that C6-HSL disappeared from the media, and hence, Lotus exhibited a natural C6-HSL inactivating ability. This ability was not specific for C6-HSL and allowed the degradation of other N-acyl-homoserine lactones such as 3-oxo-C6-HSL, 3-oxo-octanoyl-HSL and 3-oxo-decanoyl-HSL. Preliminary investigation revealed that the inactivating ability is temperature-dependant and possibly of enzymatic origin.

4-Butyrolactone↗

PubMed2005

A comparison of two strategies to modify the hydroxylation of condensed tannin polymers in Lotus corniculatus L.

A full-length sense Antirrhinum majus dihydroflavonol reductase (DFR) sequence was introduced into birdsfoot trefoil (Lotus corniculatus L.) in experiments aimed at modifying condensed tannin content and polymer hydroxylation in a predictable manner. Analysis of transgenic plants indicated lines that showed enhanced tannin content in leaf and stem tissues. In contrast to previous data from root cultures, levels of propelargonidin units were not markedly elevated in lines with enhanced tannin content. RT-PCR analysis of four selected lines indicated a correlation between enhanced tannin content and expression of the introduced DFR transgene. Using a contrasting approach we introduced a flavonoid 3'5' hydroxylase (F3'5'H) sequence derived from Eustoma grandiflorum into Lotus root cultures. Expression of the transgene was associated with increased levels of condensed tannins and in this case there was also no alteration in polymer hydroxylation. These results suggest that additional mechanisms may exist that control the hydroxylation state of condensed tannins in this model species.

Alcohol Oxidoreductases↗

PubMed2003

Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus.

Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear. Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins. We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium. The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux. A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.

Animals↗

PubMed2003

Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus.

Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a beta-amyrin synthase from M. truncatula (MtAMY1) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a beta-amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.

Amino Acid Sequence↗

PubMed2003

The Lotus japonicus ndx gene family is involved in nodule function and maintenance.

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndr genes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

Cell Nucleus↗

PubMed2006

Control of petal shape and floral zygomorphy in Lotus japonicus.

Zygomorphic flowers, with bilateral (dorsoventral) symmetry, are considered to have evolved several times independently in flowering plants. In Antirrhinum majus, floral dorsoventral symmetry depends on the activity of two TCP-box genes, CYCLOIDEA (CYC) and DICHOTOMA (DICH). To examine whether the same molecular mechanism of floral asymmetry operates in the distantly related Rosid clade of eudicots, in which asymmetric flowers are thought to have evolved independently, we investigated the function of a CYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus. We showed a role for LjCYC2 in establishing dorsal identity by altering its expression in transgenic plants and analyzing its mutant allele squared standard 1 (squ1). Furthermore, we identified a lateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays a key role in the control of lateral petal identity, and found LjCYC2 interacted with Kew1, resulting in a double mutant that bore all petals with ventralized identity to some extents. Thus, we demonstrate that CYC homologues have been independently recruited as determinants of petal identities along the dorsoventral axis in two distant lineages of flowering plants, suggesting a common molecular origin for the mechanisms controlling floral zygomorphy.

Alleles↗

PubMed2002

A Lotus basic leucine zipper protein with a RING-finger motif negatively regulates the developmental program of nodulation.

The developmental program of nodulation is regulated systemically in leguminous host species. A mutant astray (Ljsym77) in Lotus japonicus has lost some sort of its ability to regulate this symtem, and shows enhanced and early nodulation. In the absence of rhizobia, this mutant exhibits characteristics associated with defects in light and gravity responses. These nonsymbiotic phenotypes of astray are very similar to those observed in photomorphogenic Arabidopsis mutant hy5. Based on this evidence, we predicted that astray might contain a mutation in the HY5 homologue of L. japonicus. The homologue, named LjBzf, encodes a basic leucine zipper protein in the C-terminal half that shows the highest level of identity with HY5 of all Arabidopsis proteins. It also encodes legume-characteristic combination of motifs, including a RING-finger motif and an acidic region in the N-terminal half. The astray phenotypes were cosegregated with LjBzf, and the failure to splice the intron was detected. Nonsymbiotic and symbiotic phenotypes of astray were complemented by introduction of CaMV35SLjBzf. It is noteworthy that although Arabidopsis hy5 showed an enhancement of lateral root initiation, Lotus astray showed an enhancement of nodule initiation but not of lateral root initiation. Legume-characteristic combination of motifs of ASTRAY may play specific roles in the regulation of nodule development.

Amino Acid Sequence↗

PubMed2004

The galactolipid digalactosyldiacylglycerol accumulates in the peribacteroid membrane of nitrogen-fixing nodules of soybean and Lotus.

The peribacteroid membrane (PBM) surrounding nitrogen fixing rhizobia in the nodules of legumes is crucial for the exchange of ammonium and nutrients between the bacteria and the host cell. Digalactosyldiacylglycerol (DGDG), a galactolipid abundant in chloroplasts, was detected in the PBM of soybean (Glycine max) and Lotus japonicus. Analyses of membrane marker proteins and of fatty acid composition confirmed that DGDG represents an authentic PBM lipid of plant origin and is not derived from the bacteria or from plastid contamination. In Arabidopsis, DGDG is known to accumulate in extraplastidic membranes during phosphate deprivation. However, the presence of DGDG in soybean PBM was not restricted to phosphate limiting conditions. Complementary DNA sequences corresponding to the two DGDG synthases, DGD1 and DGD2 from Arabidopsis, were isolated from soybean and Lotus. The two genes were expressed during later stages of nodule development in infected cells and in cortical tissue. Because nodule development depends on the presence of high amounts of phosphate in the growth medium, the accumulation of the non-phosphorus galactolipid DGDG in the PBM might be important to save phosphate for other essential processes, i.e. nucleic acid synthesis in bacteroids and host cells.

Arabidopsis Proteins↗

PubMed2003

Sn, a maize bHLH gene, modulates anthocyanin and condensed tannin pathways in Lotus corniculatus.

Anthocyanins and condensed tannins are major flavonoid end-products in higher plants. While the transactivation of anthocyanins by basic helix-loop-helix (bHLH) transcription factors is well documented, very little is known about the transregulation of the pathway to condensed tannins. The present study analyses the effect of over-expressing an Sn transgene in Lotus corniculatus, a model legume, with the aim of studying the regulation of anthocyanin and tannin end-products. Contrary to expectation, effects on anthocyanin accumulation were subtle and restricted to the leaf midrib, leaf base and petiole tissues. However, the accumulation of condensed tannin polymers was dramatically enhanced in the leaf blade and this increase was accompanied by a 50-fold increase in the number of tannin-containing cells in this tissue. A detailed analysis of selected lines indicated that this transactivational phenotype correlated with high steady-state transcript levels of the introduced transgene and the introduction of a single copy of the CaMV35S-Sn construct into these clonal genotypes. While the levels of condensed tannins in leaves were increased by up to 1% of the dry weight, other major secondary end-products (flavonols, lignins and inducible phytoalexins) were unaltered in transactivated lines. These results give an initial insight into the developmental and higher-order regulation of polyphenolic metabolism in Lotus and other higher plant species.

Anthocyanins↗

PubMed2005

Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.

The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.

Amino Acid Sequence↗

PubMed2005

Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions.

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

Gas Chromatography-Mass Spectrometry↗

PubMed2005

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Amino Acid Sequence↗

PubMed2002

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus.

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.

Expressed Sequence Tags↗

PubMed2004

Cloning of LjCYC1 gene and nuclear localization of LjCYC1 protein in Lotus japonicus.

The LjCYC1 (Lotus japonicus Cycloidea-like 1) gene, a homolog of CYC (Cycloidea) belonging to the TCP [TB1(teosinte branched 1), CYC, PCFs (PCF1 and PCF2)] gene family and encoding a predicted transcription factor and being proposed controlling different aspects of plant development, was isolated from the papilionaceous plant Lotus japonicus by screening the genomic DNA library, in order to test the functional conservation and divarication of CYC-like genes in legume. Sequence analyses indicate that LjCYC1 gene contains two exons and one intron and encodes a 370-AA peptide LjCYC1. The putative protein, LjCYC1, contains a TCP domain and an R domain, being a member of the CYC/TB1 subfamily of TCP family, and has 39.0% identity with and 42.6% similarity to CYC. LjCYC1-cDNA was cloned through RT-PCR. Different regions of the LjCYC1-cDNA were fused with the report gene GUS and then the fused constructs were transiently expressed in the onion epidermal cells through particle bombardment. Results of GUS and DAPI staining showed that the chimeric proteins with TCP domain were localized within the nucleus, confirming that LjCYC1 may act as a transcription factor. But the TCP domain itself could not confer the nuclear localization because the chimeric proteins with TCP domain alone were dispersed all over the transformed cells.

Amino Acid Sequence↗

PubMed1995

Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

Amino Acid Sequence↗

PubMed1995

The maize transposable element Ac is mobile in the legume Lotus japonicus.

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of beta-glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T2 generation and germinal excision events were scored in segregating T3 families as complete beta-glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T3 offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.

Base Sequence↗

PubMed1997

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development.

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses. These plants had a 50-80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH4+) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions. Labelling experiments using 15NH4+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium.

Cytosol↗

PubMed2000

Water-selective and multifunctional aquaporins from Lotus japonicus nodules.

By using reverse transcriptase-polymerase chain reaction, two cDNAs were isolated that encode major intrinsic membrane proteins (MIPs) that are expressed in nitrogen-fixing root nodules of Lotus japonicus. Lotus intrinsic membrane protein 1 (LIMP 1) is expressed at high levels in both nodule and root tissues and shows highest sequence similarity to members of the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Functional analysis of LIMP 1 by expression in Xenopus laevis oocytes show that it is a water-specific aquaporin. In contrast, LIMP 2 shows the highest sequence similarity to soybean nodulin 26 (67.8% amino acid sequence identity). LIMP 2 is also a nodulin, showing expression only in mature nitrogen fixing nodules of L. japonicus. LIMP 2 is a multifunctional aquaglyceroporin, and displays the ability to flux both water as well as glycerol upon expression in Xenopus oocytes. Additionally, the carboxyl terminal region of LIMP 2 has a conserved phosphorylation motif that is phosphorylated by a calmodulin-like domain protein kinase. Overall, the data show that L. japonicus nodules contain two structurally and functionally distinct MIP proteins: one (LIMP 2) which appears to be the nodulin 26 ortholog of L. japonicus and another (LIMP 1) which appears to be a member of the TIP subfamily.

Amino Acid Sequence↗