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PubMed1988

Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234.

Mutant strain ANU2861, a transposon Tn5 mutant of the fast-growing, broad-host-range Rhizobium strain ANU280 (NGR234 Smr Rfr) overproduces polysaccharide, is an ade auxotroph, and induces poorly developed nodules on Leucaena leucocephala and Lablab purpureus (H.C. Chen, M. Batley, J.W. Redmond, and B.G. Rolfe, J. Plant Physiol. 120:331-349, 1985). Strain ANU2861 cannot form nodules on Macroptilium atropurpureum Urb. (siratro) or on Desmodium intortum and D. uncinatum and the nonlegume Parasponia. The parent strain, ANU280, effectively nodulates all these legume species except Parasponia, on which it forms ineffective nodules. Ultrastructural examination of infection sites on the legume siratro showed that mutant strain ANU2861 caused root hair curling (Hac+ phenotype), some cortical cell division (Noi+), but no infection threads (Inf-). Localized cellular responses, known to occur in phytopathological interactions, were observed in electron micrographs of the epidermal tissue at or near the infection zone after inoculation with strain ANU2861 but not the wild-type parental strain. These include (i) the rapid (within 20 h) accumulation of osmiophilic droplets attached to membranes at potential sites of strain ANU2861 penetration and (after 48 h) in the epidermal cells in the immediate region of the curled root hairs, and (ii) localized cell death of the epidermal cells. In addition, strain ANU2861 can initiate a systemic response in split-root siratro plants which prevents the successful nodulation of strain ANU280. A 6.3-kilobase fragment of wild-type genomic DNA, which includes the site of Tn5 insertion in strain ANU2861, was cloned and introduced to strain ANU2861. All the phenotypic defects of the mutant strain were corrected by the introduction of this DNA fragment. This indicates that the original Tn5 insertion is responsible for the phenotype.

Cloning, Molecular↗

PubMed1993

Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis.

The peribacteroid membrane (PBM) in legume root nodules is derived from plasma membrane following endocytosis of Rhizobium by fusion of newly synthesized vesicles. We studied the roles of plant Rab1p and Rab7p homologs, the small GTP-binding proteins involved in vesicular transport, in the biogenesis of the PBM. Three cDNAs encoding legume homologs of mammalian Rab1p and Rab7p were isolated from soybean (sRab1p, sRab7p) and Vigna aconitifolia (vRab7p). sRab1p was confirmed to be a functional counterpart of yeast Ypt1p (Rab1p) by complementation of a yeast ypt1-1 mutant. Both srab1 and vrab7 genes are induced during nodulation with the level of vrab7 mRNA being 12 times higher than that in root meristem and leaves. This induction directly correlates with membrane proliferation in nodules. Antisense constructs of srab1 and vrab7, under a nodule-specific promoter (leghemoglobin, Lbc3), were made in a binary vector and transgenic nodules were developed on soybean hairy roots obtained through Agrobacterium rhizogenes-mediated transformation. Both antisense srab1 and vrab7 nodules were smaller in size and showed lower nitrogenase activity than controls. The antisense srab1 nodules showed lack of expansion of infected cells, fewer bacteroids per cell and their frequent release into vacuoles. In contrast, antisense vrab7 expressing nodules showed accumulation of late endosomal structure and multivesicular bodies in the perinuclear region. These data suggest that both Rab1p and Rab7p are essential for the development of the PBM compartment in effective symbiosis.

Amino Acid Sequence↗

PubMed2000

Localized expression of cathepsin B-like sequences from root nodules of pea (Pisum sativum).

Cathepsin B is an ancient family of eukaryotic cysteine proteases. We describe PsCat1, a plant cathepsin B-like transcript, identified as an expressed sequence in Rhizobium-induced, nitrogen-fixing root nodules of pea. In situ hybridization studies in root nodules showed strong, extremely localized expression of PsCat1 in individual cells associated with the central infected tissue. Restriction fragment polymorphism mapping of the PsCat1 locus in pea shows no correlation with existing mutant lines defective in symbiosis.

Cathepsin B↗

PubMedDate not supplied

[Effect of plant growth stimulators on Rhizobium leguminosarum BV. VICIAE 263b and efficiency of symbiotic nitrogen-fixation in peas].

Effect of two plant growth stimulators: bactozol (drug of bacterial origin) and D1 (synthetic analog of phytohormones) on metabolism of pea rhizobia (Rhizobium leguminosarum bv. viciae 2636) and efficiency of their symbiosis with pea plants have been studied. The D1 drug in concentration 0.1% suppressed growth of bacteria. However, bactozol stimulating action on pea rhizobia growth in a pure culture and synthesis of extracellular carbohydrates by them have been established. The trial of three concentrations (0.1%, 0.01%, 0.001%) has shown that bactozol effect has dose-dependent character. The highest effect of stimulation is achieved at concentration 0.1%. Bactozol decreases partially the repressing influence of the mineral nitrogen on growth of rhizobia and their carbohydrate-synthesizing activity under growth of bacteria against a high nitrogen background (20 mM NO3-). Treatment of pea plants by bactozol (0.1%) increases considerably the efficiency of their symbiosis with pea rhizobia, evoking the growth of the overground and root mass of the plants, quantity of nodules and their nitrogenase activity.

Culture Media↗

PubMed1997

Response of higher plants to lead contaminated environment.

Lead concentration is increasing rapidly in the environment due to increased use of its sources by human society. Alarming concentrations of the metal have been reported in dust of densely populated urban areas and, water and land of various areas near the industrial waste disposals. Plants absorb lead and accumulation of the metal have been reported in roots, stems, leaves, root nodules and seeds etc. which increases with the increase in the exogenous lead level. Lead affects plant growth and productivity and the magnitude of the effects depend upon the plant species. Photosynthesis has been found to be one of the most sensitive plant processes and the effect of the metal is multifacial. Nitrate reduction is inhibited drastically in roots by the metal but in the leaves a differential effect is observed in various cultivars. Lead also inhibits nodulation, N-fixation and ammonium assimilation in the root nodules. It appears that the toxic effect of the metal is primarily at physiological level and provision of certain inorganic salts can antagonize the toxic effects to some extent. Further responses of plants to the metal depend on various endogenous, environmental and nutritional factors. Some plants are able to tolerate excess of Pb+2 by involving processes like exclusion, compartmentalization or synthesizing metal detoxifying peptides-the phytochelatins.

Absorption↗

PubMed1987

Effect of nitrate on the organic Acid and amino Acid composition of legume nodules.

Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity.

Journal Article↗

PubMed1990

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria.

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.

Bacteria↗

PubMed1982

Simultaneous Measurement of NH(4) Absorption and N(2) Fixation by Glycine max L. : RESPONSE TO TEMPERATURE, pH, AND EXTERNAL NITROGEN CONCENTRATION.

Ammonium absorption rates by intact nodulated and unnodulated soybean plants (Glycine max [L.] Merr., Amsoy 71) were determined from the liquid phase of a mist assay chamber. From the gas phase, simultaneous measurements of acetylene reduction rates were made from nodulated plants. Ammonium absorption capacity was consistently greater in unnodulated plants.At the beginning of flowering, plant roots were sprayed with an uptake solution ranging from 0.05 to 1.0 millimolar NH(4)Cl, and root nodules concurrently were exposed to 0.12 atmosphere acetylene. The NH(4) (+) absorption system of both nodulated and unnodulated plants were nearly saturated at 0.3 to 0.5 millimolar NH(4) (+).Increasing the pH from 4.0 to 6.8 stimulated NH(4) (+) absorption rate in both flowering and preflowering nodulated and unnodulated plants. Acetylene reduction rate was not altered by short-term exposure to increased acidity. Short-term exposure to lower absorption solution temperatures from 32 degrees C to 18 degrees C did not significantly affect NH(4) (+) absorption rate in flowering nodulated or unnodulated plants. However, acetylene reduction rate increased as the absorption solution temperature increased. The Q(10) value was 1.5 for the reaction rate. Increasing external NO(3) (-) concentration from 0.1 to 5.0 millimolar NO(3) (-) did not significantly influence the kinetics of NH(4) (+) absorption or acetylene reduction rate.

Journal Article↗

PubMed2004

Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula.

In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA-protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA.

Amino Acid Sequence↗

PubMed1990

A biovar-specific signal of Rhizobium leguminosarum bv. viciae induces increased nodulation gene-inducing activity in root exudate of Vicia sativa subsp. nigra.

Flavonoids in root exudate of leguminous plants activate the transcription of Rhizobium genes involved in the formation of root nodules (nod genes). We report that inoculation with the homologous symbiont R. leguminosarum bv. viciae results in an increased nod gene-inducing activity (Ini) in root exudate of V. sativa subsp. nigra, whereas inoculation with heterologous Rhizobium strains results in exudates with nod gene-inducing activity comparable to that of uninfected plants. Ini can be demonstrated by using either of the isogenic indicator strains containing an inducible nod promoter fused to the Escherichia coli lacZ reporter gene and the regulatory nodD gene of R. leguminosarum bv. viciae, R. leguminosarum bv. trifolii, or R. meliloti. The presence of genes nodDABCEL of R. leguminosarum bv. viciae appeared to be essential for induction of Ini. Mutation of the genes nodI and nodJ causes a delay of Ini, whereas gene nodF appears to be required for both the timely appearance and the maximum level of Ini activity. The nodE gene is responsible for the biovar specificity of induction of Ini by Rhizobium spp. Ini is caused by a soluble heat-stable factor of rhizobial origin. This Rhizobium-produced Ini factor has an apparent molecular weight between 1,000 and 10,000 and does not originate from flavonoid precursors.

Cells, Cultured↗

PubMed1995

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L.

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.

Journal Article↗

PubMed1986

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch).

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with (15)N(2) gas showed asparagine to be the principal nitrogen product exported from N(2)-fixing nodules. Maintaining root systems in an N(2)-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N(2) fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of (15)N-nitrate, and a (15)N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N(2) or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N(2). Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N(2) and nitrate are being used in peanuts.

Journal Article↗

PubMed2004

Nodulin 22 from Phaseolus vulgaris protects Escherichia coli cells from oxidative stress.

Plant genes that are induced during the formation and function of a root nodule are called nodulin genes. Cloning and functional analysis of nodule-specific gene products are of valuable help in establishing the role and requirements of the host plant for the specificity and effectiveness of the symbiosis. A cDNA clone (nod22) was isolated from Phaseolus vulgaris L. (common bean) cDNA library derived from Rhizobium-infected roots. Nodulin 22 (Nod22) transcripts are accumulated from early to late stages in root nodule development. RT-PCR in situ studies indicated that Nod22 transcripts are highly accumulated in cortical, vascular bundle and infected cells. The deduced Nod22 protein contains a highly hydrophobic N-terminus, with signal peptide characteristics, and a C-terminal extension with high identity to the alpha-crystallin domains found in alpha-crystallin lens chaperone, and other small heat-shock proteins. These domains have not been previously described in other known nodulins, but have been observed in small heat-shock proteins found in plant tissues exposed to elevated temperature and oxidative stress. Nod22, when it is over-expressed in Escherichia coli, cells confers protection against oxidative stress suggesting its possible role in plant host protection from oxidative toxicity during the Rhizobium-legume symbiosis.

Amino Acid Sequence↗

PubMed1996

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants.

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5'-Srglb3-uidA-3'nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.

Base Sequence↗

PubMed2001

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula.

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Calcium↗

PubMed2002

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti.

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.

DNA, Complementary↗

PubMed1997

Transient energy coupling between rhizobia and legume cells mediated by the peribacteroid membrane ATPase proton pump.

We present a model for the metabolic coupling between rhizobia and plant cell in the nitrogen-fixing legume root nodules. The symbiosome, an organelle-like structure formed by the modified rhizobia (the bacteroids) enclosed by a plant cell derived peribacteroid membrane, is an unique structure in which two energized membranes are closely packed: the inner bacteroid membrane and the peribacteroid membrane that possesses an ATPase proton pump. The model is based on the following points: (i) The permeability for hydrogen ions of the outer membrane of the rhizobia. (ii) The reversibility of the ATPase proton pump of the peribacteroid membrane [Szafran, M.M. and Haaker, H. (1995) Plant Physiol. 108, 1227-1232]. (iii) The relative affinites for oxygen of the bacteroid and plant mitochondria terminal oxidases, and the prevailing oxygen concentration inside the nodule, which results in aerobic metabolism for the bacteroid, but in quite fermentative catabolism for the host plant cell. We propose that the bacteroid can transiently supply free energy to the plant cell in the form of protonmotive force by the movement of hydrogen ions from the bacteroid periplasmic space to the plant cytoplasm through the peribacteroid membrane ATPase. The proposed hydrogen ion flux could be dependent on the phosphorylation potential in both the plant cell cytoplasm and the bacteroid, and the simultaneous ion movements to avoid the development of opposite delta psi. It could be important in situations of transient ATP depletion inside plant cell, which involves the block of ammonia assimilation and, subsequently, the inhibition of bacteroid nitrogenase.

Adenosine Triphosphatases↗

PubMed1990

Identification of Frankia strains in nodules by hybridization of polymerase chain reaction products with strain-specific oligonucleotide probes.

A set of oligonucleotides has been developed to study the competitivity of two Frankia strains in the nodulation of the roots of two host plant species: Alnus glutinosa and Alnus incana. Two 20 mer-oligonucleotides, complementary to highly conserved sequences inside the nifH gene, were used as primers for the polymerase chain reaction (PCR) system in order to amplify microsymbiont DNA extracted from actinorhizae. PCR products were analyzed using two strain-specific 15-mer oligonucleotides identified in the amplified region. Hybridization data indicate that strain ACoN24d is more competitive than strain ArI3 in the nodulation of both hosts.

Actinomycetales↗