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PubMed2006

Identification of a Sed5-like SNARE gene LjSYP32-1 that contributes to nodule tissue formation of Lotus japonicus.

We identified a Sed5-like clone LjSYP32-1 which contributes to nodule tissue formation and plant growth in Lotus japonicus. In the L. japonicus expressed sequence tag (EST) clone databases of Kazusa DNA Research Institute, another syntaxin-related clone (LjSYP32-2) was also detected, and the nucleotide and amino acid sequences of these two clone are very similar to each other. Real-time PCR and promoter analysis indicated that expression of LjSYP32-1 was dominant compared with LjSYP32-2 in the various plant organs. Promoter analysis and in situ hybridization revealed that LjSYP32-1 was expressed significantly in the inner cortex cell layer surrounding the infected zone of young nodules and in the meristem area of developing lateral root. To explore the function and physiological role of LjSYP32-1 in nodules and other plant organs, stable transformation lines of L. japonicus expressing either sense or antisense LjSYP32-1 were prepared. The antisense plants showed a significantly retarded plant growth phenotype, suggesting a role for LjSYP32-1 in supporting plant growth. In the same transgenic lines, the plants were capable of forming nodules, but the acetylene reduction activity was reduced by around 50% per plant. The nodules were much smaller and some nodules were fused to each other by sharing the inner cortex. The rate of occurrence of such irregular nodules was twice that observed in wild-type plants. The data suggest that LjSYP32-1 contributes to the support of plant growth and normal nodule tissue differentiation.

Amino Acid Sequence↗

PubMed2001

Yellow lupine cyclophilin transcripts are highly accumulated in the nodule meristem zone.

Cyclophilin (CyP) is one of the enzymes that act as peptidylprolyl cis-trans isomerases (EC 5.2.1.8). The cDNA and an intronless gene coding for cytosolic CyP have been isolated from yellow lupine. The deduced amino acid sequence of the characterized open reading frame shows approximately 80% homology with cytosolic CyP from other organisms. Southern blots of genomic DNA indicate that there is a small family of genes for CyP-related genes in the yellow lupine genome. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs. The amount of CyP transcripts is dramatically increased in root nodules. In situ hybridization experiments indicate that CyP transcripts are localized mainly in meristematic tissues, with the highest level observed in the nodule meristem zone. The promoter of the sequenced gene contains 5' AAAGAT 3' and AT-rich motifs that are characteristic for some nodulin promoters.

Base Sequence↗

PubMed2002

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants.

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.

Carbon Dioxide↗

PubMedDate not supplied

[Formation and function of the bean-rhizobium symbiosis in soy plants upon introduction of strains of Azotobacter and Bacillus species].

The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicum strains on formation and function of the legume-rhizobium symbiosis of soybean plants were studied. The data showed that the bacterial compositions B. japonicum 634b + B. subtilis 5, B. japonicum 634b + A. chroococcum 20, and B. japonicum 10k + A. vinelandii 56 with a cell ratio of 1:0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all the cases by 22-105% compared with the control variants. These binary microbial cultures may be used for development of combined bacterial preparations for soybean.

Azotobacter↗

PubMed1991

Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2).

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O(2) (volume/volume) in N(2). Plant dry matter yield was unaffected by partial pressure of oxygen (pO(2)) and N(2) fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O(2). Slight inhibition of nitrogenase activity occurred at 1% O(2) and as much as 50% inhibition occurred at 80% O(2). Low pO(2) (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO(2) used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO(2) was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO(2) and decreased permeability in those from plants cultured with their roots in pO(2) greater than ambient. A range of structural alterations in nodules resulting from low pO(2) were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO(2) comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O(2). Long-term adaptation to different external pO(2) involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.

Journal Article↗

PubMed2002

A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) druce.

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or RHIZOBIUM: The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

Alphaproteobacteria↗

PubMed2003

cg12 expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

Arabidopsis↗

PubMed1992

Nucleosomal structure and histone H1 subfractional composition of pea (Pisum sativum) root nodules, radicles and callus chromatin.

Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression. With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea. Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources. The calculated values of nucleosomal repeat changed from 185 +/- 5 bp in the nuclei of radicles to 168 +/- 5 bp and 195 +/- 6 bp in nodules and callus respectively. The observed changes are due to alterations in linker DNA lengths. The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations. The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development.

Cell Nucleus↗

PubMed1978

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome.

In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.

DNA↗

PubMed2004

Six nonnodulating plant mutants defective for Nod factor-induced transcriptional changes associated with the legume-rhizobia symbiosis.

As the legume-rhizobia symbiosis is established, the plant recognizes bacterial-signaling molecules, Nod factors (NFs), and initiates transcriptional and developmental changes within the root to allow bacterial invasion and the construction of a novel organ, the nodule. Plant mutants defective in nodule initiation (Nod(-)) are thought to have defects in NF-signal transduction. However, it is unknown whether WT plants respond to NF-independent bacterial-derived signals or whether Nod(-) plant mutants show defects in global symbiosis-associated gene expression. To characterize plant gene expression in the establishment of the symbiosis, we used an Affymetrix oligonucleotide microarray representing 9,935 Medicago truncatula expressed sequences. We identified 46 sequences that are differentially expressed in plants exposed for 24 h to WT Sinorhizobium meliloti or to the invasion defective S. meliloti mutant, exoA. Eight of these genes encode nucleolar proteins, which are implicated in ribosome biogenesis. We also identified differentially expressed transcription factors, signaling components, defense response proteins, stress response proteins, and several previously uncharacterized genes. NF appears both necessary and sufficient to induce most changes. Six of seven Nod(-) M. truncatula mutants (nfp, dmi1, dmi2, dmi3, nsp1, and nsp2) showed no transcriptional response to S. meliloti, suggesting that the encoded proteins are required for initiating new transcription. The Nod(-) mutant hcl, however, exhibits a reduced transcriptional response to S. meliloti, indicating that the machinery responsible for initiating new transcription is at least partially functional in this mutant.

Lipopolysaccharides↗

PubMed2002

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).

Ethyl Methanesulfonate↗

PubMed1971

Formation of tumor-like structures on legume roots by Rhizobium.

Tumor-like structures appeared on the roots of Medicago sativa, Alysicarpus vaginalis, and Trifolium pratense inoculated with a non-nodulating strain of Rhizobium trifolii or with irradiated cultures of either of two nodulating Rhizobium strains. The structures were composed of disorganized plant tissues which, on the basis of microscopic examination, were devoid of bacterial cells. Rhizobia which could nodulate legumes of one cross-inoculation group and which were able to induce formation of such tumor-like structures on plants of a second cross-inoculation group were isolated from extracts of these root growths. The apparent tumorogenic activity of some of the rhizobia, but not their nodulating capacity, was lost when the bacteria were transferred in laboratory media.

Agglutination Tests↗

PubMed1986

Serine hydroxymethyltransferase from soybean root nodules : purification and kinetic properties.

Serine hydroxymethyltransferase has been purified 1,550-fold from the plant fraction of soybean (Glycine max [L]. Merr. cv Williams) nodules. The pH optimum for the enzyme was at 8.5. The native molecular weight was 230,000 with a subunit molecular weight of 55,000 which suggested a tetramer of identical subunits. The enzyme kinetics for the enzyme were Michaelis-Menten; there was no evidence for cooperativity in the binding of either substrates or product inhibitors. There were two K(m) values for serine at 1.5 and 40 millimolar. The K(m) for l-tetrahydrofolate was 0.25 millimolar. l-Methyl-, l-methenyl-, and l-methylene-tetrahydrofolates were all noncompetitive inhibitors with l-tetrahydrofolate with K(i) values of 1.8, 3.0, and 2.9 millimolar, respectively. Glycine was a competitive inhibitor with serine with a K(i) value of 3.0 millimolar. The intersecting nature of the double reciprocal plots together with the product inhibition data suggested an ordered mechanism with serine the first substrate to bind and glycine the last product released. The enzyme was insensitive to a wide range of metabolites which have previously been reported to affect its activity. These results are discussed with respect to the roles of serine hydroxymethyltransferase and the one-carbon metabolite pool in control of the carbon flow to the purine biosynthetic pathway in ureide biogenesis.

Journal Article↗

PubMed1991

Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains.

Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes. The common nodABC genes form an operon or are physically mapped together in all species studied thus far. Rhizobium leguminosarum bv. phaseoli strains are classified in two groups. The type I group has reiterated nifHDK genes and a narrow host range of nodulation. The type II group has a single copy of the nifHDK genes and a wide host range of nodulation. We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes. This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions. Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R. leguminosarum bv. phaseoli type I strains. Despite the separation, the coordination of the expression of these genes seems not to be altered.

Acyltransferases↗

PubMed1986

Physical association between the peribacteroid membrane and lipopolysaccharide from the bacteroid outer membrane in Rhizobium-infected pea root nodule cells.

Monoclonal antibodies were used as cytochemical markers to study surface interactions between endosymbiotic Rhizobium bacteroids from pea root nodules and the encircling peribacteroid membranes, which are of plant origin. Monoclonal antibodies that react with Rhizobium lipopolysaccharide (LPS) or with a plant membrane glycoprotein were used as markers for material from the bacteroid outer membrane or the peribacteroid membrane, respectively. Membrane-enclosed bacteroids were isolated from nodule homogenates by sucrose gradient centrifugation, and the encircling peribacteroid membrane was released by mild osmotic shock treatment. Using an immunochemical technique (sandwich ELISA), it was shown that 1-5% of the LPS antigen released into the peribacteroid fraction by mild osmotic shock treatment was physically associated with peribacteroid membrane through a detergent-sensitive linkage. This association could be visualized when freshly prepared peribacteroid material was immobilized on gold grids and examined by electron microscopy after dual antibody immunogold treatment and subsequent negative staining. The distribution of LPS antigen within infected nodule cells was also investigated by immunogold staining for thin sections of nodule tissue fixed in glutaraldehyde, and a close association between LPS antigen and peribacteroid membrane was often seen.

Cell Membrane↗

PubMed2003

Recent advances in Rhizobium-legume symbiosis.

The research findings in the field of Rhizobium-legume symbiosis reported worldwide during the years 2002 and 2003 (up to September) have been summarized. The information is presented under the various topics, viz., isolation and characterization of rhizobial strains, physiological aspects of nitrogen fixation, rhizosphere interactions and root surface signals, genomics and proteomics, plant genes involved in nodule formation, bioremediation and biocontrol, and review articles and conference reports. The postal and e-mail addresses of the concerned scientists have also been included.

Fabaceae↗

PubMed2005

[Effects of selenium on Chamaecrista rotundifolia growth, nutrient absorption and nitrogen-fixing ability].

In this paper, a field experiment was conducted on a red soil of South China to study the effects of applying selenium fertilizer on the growth, forage quality and nitrogen-fixing ability of Chamaecrista rotundifolia. The results showed that applying 75, 150, 225 and 300 g Se x hm(-2) could increase the plant height, branch number, dry root weight and dry forage yield by 0.3% - 6.2%, 65.1% - 79.5%, 155% - 252% and 30.6% - 54.1%, respectively. The total nitrogen, total phosphorus and total potassium contents of plant were increased by 21.79% - 41.46%, 20.74% - 34.67% and 34.3% - 62.4%, respectively, and the raw protein, raw fat, raw fiber and amino acid contents were increased by 21.79% - 41.46%, 1.4% - 89.6%, 34.1% - 56.6% and 6.33% 63.24%, respectively. Among the 4 doses, 150 g Se x hm(-2) was the most effective and suitable application amount. The selenium content in plant after applying 300 g Se x hm(-2)(S4) was 0.695 mg x kg(-'), with an increase of 0.658 mg x kg(-1) compared to no selenium fertilization. There was a significantly positive correlation between plant selenium content and applied selenium (R2 = 0.9666**). The root nodule weight, number and nitrogenase activity in treatment 150 g Se x hm(-2)(S2) were respectively 131.7%, 114.3% and 1417.9% higher than those of no selenium fertilization. The correlation between applied selenium and nitrogenase activity was also significant (R2 = 0.9606*).

Biomass↗

PubMed2003

A Medicago sativa haem oxygenase gene is preferentially expressed in root nodules.

Haem oxygenases (HO) are ubiquitous enzymes catalysing the oxidative degradation of haem into biliverdin, iron and carbon monoxide. Whereas animal HOs participate in multiple cellular functions including haemoglobin catabolism, antioxidant defence and iron homeostasis, to date, plant HOs have so far only been involved in phytochrome metabolism. The expression of the HO1 gene was studied in Medicago sativa, especially during the interaction with its symbiotic partner, Sinorhizobium meliloti. Transcript accumulation was higher in mature root nodules than in roots and leaves and was correlated to HO1 protein immunodetection. The analysis of HO1 expression following alfalfa root inoculation with S. meliloti indicates that transcripts do not accumulate during the early steps of symbiosis, but rather in the mature nodules. These results correlate with the expression of the leghaemoglobin gene, which encodes the major haem-containing protein present in the nodule. Contrary to its animal counterpart, alfalfa HO1 was not induced by pro- oxidant compounds including H(2)O(2), paraquat and sodium nitroprusside, suggesting that it is not involved in the antioxidant defence. The results suggest that HO1 could play a role in the alfalfa mature nodule and its involvement in leghaemoglobin metabolism is hypothesized.

Gene Expression Regulation, Enzymologic↗