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Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume.

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N(2). Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K(+) content and high ratio of Mg(2+) to Ca(2+). Fruit cryopuncture sap became labeled with (14)C following feeding of [(14)C]urea to leaves or adjacent walls of the fruit, of (14)CO(2) to the pod gas space, and of [(14)C] asparagine or [(14)C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of (14)C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of (14)C distribution in phloem sap suggested that [(14)C]allantoin was metabolized to a greater extent in its passage to the fruit than was [(14)C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO(3)-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO(3)-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of (15)NO(3) to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.

Journal Article↗

Transient responses of nitrogenase to acetylene and oxygen in actinorhizal nodules and cultured frankia.

Nitrogenase activity in root nodules of four species of actinorhizal plants showed varying declines in response to exposure to acetylene (10% v/v). Gymnostoma papuanum (S. Moore) L. Johnson. and Casuarina equisetifolia L. nodules showed a small decline (5-15%) with little or no recovery over 15 minutes. Myrica gale L. nodules showed a sharp decline followed by a rapid return to peak activity. Alnus incana ssp. rugosa (Du Roi) Clausen. nodules usually showed varying degrees of decline followed by a slower return to peak or near-peak activity. We call these effects acetylene-induced transients. Rapid increases in oxygen tension also caused dramatic transient decreases in nitrogenase activity in all species. The magnitude of the transient decrease was related to the size of the O(2) partial pressure (pO(2)) rise, to the proximity of the starting and ending oxygen tensions to the pO(2) optimum, and to the time for which the plant was exposed to the lower pO(2). Oxygen-induced transients, induced both by step jumps in pO(2) and by O(2) pulses, were also observed in cultures of Frankia. The effects seen in nodules are purely a response by the bacterium and not a nodule effect per se. Oxygen-induced nitrogenase transients in actinorhizal nodules from the plant genera tested here do not appear to be a result of changes in nodule diffusion resistance.

Journal Article↗

Isolation and characterization of functional insertion sequences of rhizobia.

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.

Computational Biology↗

Rhizobins, a group of peptides in the free-amino-Acid pool of the soybean-Rhizobium system.

Free-living Rhizobium (according to Bergey's Manual of Systematic Bacteriology, [1984, The Williams & Wilkins Co., Baltimore], Bradyrhizobium) japonicum was found to release a peptide into the nutrient media. Soybean nodules contained this peptide and exuded it into the soil. The name "rhizobin A" is suggested for this peptide. Nodules also contained another peptide, rhizobin B, as well as an unidentified, ninhydrin-positive compound, rhizobin C. The three peptides were confined to the free-amino-acid pool of the soluble fraction and eluted consecutively from a cation-exchange column. Rhizobin A was isolated in a highly purified form; its molecular mass was approximately 1,600 daltons as determined by Sephadex gel filtration and mass spectrometry. The amino-acid composition could be determined only approximately, because a long time was necessary for acid hydrolysis, possibly due to unusual linkages. The rhizobin concentration in soybean nodules continually increased during 50 days of growth, from 2 to approximately 400 mug/g (fresh weight). When combined nitrogen was added to nodulated soybean and subsequently removed, nitrogenase activity, nodulation, and nodule growth first decreased and then recovered. The relative amount of rhizobin A followed a similar pattern. Rhizobins were not detected in the roots, stems, and leaves of nodulated soybean plants. They were present in Lupinus nodules, but absent in alder nodules.

Journal Article↗

Identification of Bradyrhizobium nod genes involved in host-specific nodulation.

Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes. Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species. lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product. A mutation in one of the loci was found to result in an alteration of the host range of B. japonicum. This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.

Blotting, Southern↗

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1.

In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII. DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains. B. japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA. The region corresponding to the Frankia sp. strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B. japonicum. The Frankia glnII gene product has a high degree of similarity with both GSII from B. japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins. The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter. GSII produced in E. coli was heat labile, like the enzyme produced in Frankia sp. strain CpI1 but unlike the wild-type E. coli enzyme.

Actinomycetales↗

Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.

Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.

4-Butyrolactone↗

Pesticide side effect on the symbiotic efficiency and nitrogenase activity of Rhizobiaceae bacteria family.

The laboratory experiments tested the influence of selected pesticides on the symbiotic efficiency and nitrogenase activity of Rhizobium leguminosarumin bv. trifolii KGL, Sinorhizobiuni melilotii Bp and Badyrhizobium sp. Ornithopus B bacteria entering into symbiosis with clover, lucerne and serradella, respectively. The results obtained indicate that the pesticides used in the experiments (Funaben T seed dressing and Pivot 100SL herbicide) caused reduced nitrogenase activity in active strains tested. In addition, a toxic effect of the applied pesticides on the nodulation and root growth of the tested plants was observed.

Benzimidazoles↗

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Cloning, Molecular↗

Effect of some pesticides on the efficiency of the inoculated Rhizobium associated with broad bean plants.

The effect of some pesticides on the efficiency of the inoculated rhizobium in broad bean plants (Vicia faba) was studied in greenhouse experiments. Different pesticides were used in this work, including insecticides, herbicides, nematocides, and a molecide in different concentrations. The study revealed the following conclusions. Application of insecticides in different concentrations generally affected the formation of efficient nodules on the root system of broad bean plants. The inhibitory effect was also reflected to some extent on the dry weight of the plants and total nitrogen fixed by rhizobia in association with the mentioned plants. Birlane (insecticide) was found to be the most inhibitory insecticide on the inoculated rhizobium. Some of the insecticides, such as Temik and Dursban, at lower concentration were found to be a stimulant to symbiotic N-fixation, giving higher amounts of nitrogen fixed in the macrosymbiont.

Antinematodal Agents↗

Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula.

The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts.

Alphaproteobacteria↗

The relationship between foliar injury, nitrogen metabolism, and growth parameters in ozonated soybeans.

A single 12 h ozone exposure peaking at 0.20 ppm proved phytotoxic to greenhouse-grown 'Cutler 71' soybeans at each growth stage tested from V5 to R6. Visible injury occurred within 40 h on the unifoliodate leaves and middle-aged and older trifoliolates while the younger leaves were free from toxicity symptoms. In some instances visible injury was accompanied by a decrease in chlorophyll and an increase in leaf diffusive resistance. Although nitrogen fixation was not significantly altered except at early pod formation (R3), and nitrate reductase activity was significantly reduced only if the ozone exposure occurred at the time of maximal enzyme activity (V5), nitrogen content of the leaves was reduced by ozone treatment. Shoot dry weight was not affected 40 h after ozone treatment, but root dry weight was significantly reduced. Plants grown with supplemental NO(3)(-) were more sensitive to ozone than those dependent on fixed nitrogen. At plant maturity, there was no evidence of an ozone effect on shoot, root, or seed dry weight, NO(3)(-) -grown plants showed a significant increase in growth and yield over N(2)(-) plants; but no ozone effect was observed, despite the increased foliar sensitivity. Multiple ozone exposures at growth stages V3, R1 and R3 exacerbated the effects noted with a single episode and also reduced nitrogenase activity (reflected in specific and total nodule activity) and shoot and root dry weight. At plant maturity, there was again no evidence of a significant effect of multiple ozone treatment on shoot dry weight or seed yield although root weight remained low. The results would tend to support the hypothesis that older leaves of soybean do not make a significant contribution to seed yield. Although they may be injured by ozone during the reproductive phases of growth, seed yield may not be affected if the younger O(3)-tolerant leaves remain functional.

Journal Article↗

A receptor kinase gene regulating symbiotic nodule development.

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified 'nodulation receptor kinase', NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of 'NORK extracellular-sequence-like' (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.

Amino Acid Motifs↗

Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera.

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.

Antigens, Bacterial↗

Inhibitory effects of the herbicide trifluralin on the establishment of the clover root nodule symbiosis.

Trifluralin, a widely used herbicide (Rahman, 1977), may act via interaction with plant microtubule protein (Hess and Bayer, 1974, 1977). In trifluralin treated seedlings of Trifolium repens, root tips increased in diameter and decreased in length, cell elongation was decreased, cell wall deposition was abnormal, root hairs were deformed, and a marked reduction was seen in the number of infection threads induced by the bacterial symbionts. Our observations support the hypothesis that trifluralin (at low concentrations, from 2 x 10(-6) M) interferes with microtubule-based morphogenetic processes in developing seedlings and adversely affects the establishment of the bacteria-legume symbiosis.

Morphogenesis↗

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase.

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).

Journal Article↗

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa.

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.

Amino Acid Sequence↗

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots.

Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M(r) 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.

Journal Article↗