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Organ regulated expression of Parasponia andersonii haemoglobin gene in transgenic tobacco plants.

Plant haemoglobin genes are known to occur in legume and non-legume families and in both nodulating (e.g., Parasponia andersonii) and non-nodulating species (e.g., Trema tomentosa). Their presence in non-nondulating plants raises the possibility that haemoglobins might serve a function in non-symbiotic tissues distinct from their role in the nitrogen-fixing root nodules induced by micro-organisms. We report here that a P. andersonii haemoglobin promoter can regulate expression of either the P. andersonii haemoglobin gene, or a hybrid construct with the bacterial chloramphenicol acetyltransferase gene (cat), in the non-symbiotic plant, Nicotiana tabacum. Expression is predominantly in the roots, implying that haemoglobins might have a function in roots of non-nodulated plants. We have also observed a low level of haemoglobin protein in non-nodulated P. andersonii roots, but not leaves, supporting this assertion. The expression in transgenic plants will allow further characterization of the promoter sequences essential for the organ-specific expression of haemoglobins in non-symbiotic tissues.

Amino Acid Sequence↗

Nodulation of legumes by members of the beta-subclass of Proteobacteria.

Members of the Leguminosae form the largest plant family on Earth, with around 18,000 species. The success of legumes can largely be attributed to their ability to form a nitrogen-fixing symbiosis with specific bacteria known as rhizobia, manifested by the development of nodules on the plant roots in which the bacteria fix atmospheric nitrogen, a major contributor to the global nitrogen cycle. Rhizobia described so far belong exclusively to the alpha-subclass of Proteobacteria, where they are distributed in four distinct phylogenetic branches. Although nitrogen-fixing bacteria exist in other proteobacterial subclasses, for example Herbaspirillum and Azoarcus from the phylogenetically distant beta-subclass, none has been found to harbour the nod genes essential for establishing rhizobial symbiosis. Here we report the identification of proteobacteria from the beta-subclass that nodulate legumes. This finding shows that the ability to establish a symbiosis with legumes is more widespread in bacteria than anticipated to date.

Betaproteobacteria↗

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures.

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.

Anthranilate Synthase↗

Characterization of a novel gene encoding a phytocyanin-related protein in morning glory (Pharbitis nil).

A cDNA for a novel phytocyanin homolog was cloned from apical buds of morning glory (Pharbitis nil). The predicted protein was most similar to a family of early nodulins, which are expressed during the formation of symbiotic root nodules of legume plants, and less similar to typical phytocyanins such as lacquer tree stellacyanin and cucumber basic protein. The amino acid sequence predicted that it is a secreted protein associated with other components of the extracellular matrix. Hybridization analyses showed that the transcript was expressed specifically in meristems and procambia of apical buds and root tips. The transcript level in the apical buds decreased significantly on flower-inducing treatment. Involvement of this phytocyanin-related protein in plant organ differentiation is discussed.

Amino Acid Sequence↗

New nodulation mutants responsible for infection thread development in Lotus japonicus.

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

Flowers↗

Exopolysaccharides in plant-bacterial interactions.

Rhizobial plant symbionts and bacterial plant pathogens produce exopolysaccharides that often play essential roles in the plant interaction. Many of these exopolysaccharides are acidic heteropolysaccharides that have repeating subunit structures with carbohydrate and noncarbohydrate substituents, while others are homopolysaccharides such as alginate, levan, cellulose, and glucan. While the homopolysaccharides are synthesized by mechanisms that vary with the particular polysaccharide, the heteropolysaccharides as a rule are synthesized by subunit assembly from nucleotide diphosphate-sugar precursors on a membrane-bound lipid carrier followed by polymerization and secretion. Many mutants in exopolysaccharide synthesis have been isolated, and in several cases this has led to the identification of genes that function in particular steps of biosynthesis, as well as in regulation of exopolysaccharide biosynthesis. The genetic regulation of exopolysaccharide synthesis in many plant pathogens is complex, perhaps reflecting the various niches, free living and in planta, in which exopolysaccharides function. In some cases, exopolysaccharide synthesis is regulated coordinately with other virulence factors, and in other cases separately. Regulatory genes that have homology to the two-component sensor and transcriptional effector systems are a common motif. In Rhizobium species, exopolysaccharide synthesis is regulated by transcriptional as well as posttranslational mechanisms. Exopolysaccharides function differently in the root-nodule symbiosis versus plant pathogenesis. Specific Rhizobium exopolysaccharide structures promote nodule development and invasion in legumes that form indeterminate nodules. In plant pathogenesis, less specific mechanisms of pathogenesis occur: exopolysaccharides cause wilting by blocking xylem vessels, are partly responsible for water-soaked lesions, and may also aid in invasion, growth, and survival in plant tissues.

Carbohydrate Sequence↗

Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum.

Nodule-specific root proteins - so called nodulins - were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nodfix and a pop fix) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.

Journal Article↗

Response of purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea to ultraviolet-B radiation.

The effects of elevated UV-B radiation on growth, symbiotic function and concentration of metabolites were assessed in purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea grown outdoors either on tables under supplemental UV-B radiation or in chambers covered with different types of plexi-glass to attenuate solar ultraviolet radiation. Moderately and highly elevated UV-B exposures simulating 15% and 25% ozone depletion as well as sub- ambient UV-B did not alter organ growth, plant total dry matter and N content per plant in both L. luteus and V. atropurpurea. In contrast, elevated UV-B increased (P <0.05) flavonoid and anthocyanin concentrations in roots and leaves of L. luteus, but not of V. atropurpurea. Feeding nodulated plants of L. luteus under elevated UV-B radiation with 2 mM NO3 increased (P <0.05) nodule, leaf and total dry matter, and whole plant N content. With V. atropurpurea, NO3 reduced (P <0.05) nodule activity, root %N and concentrations of flavonoids, anthocyanins in roots and leaves and soluble sugars in roots, in contrast to an observed increase (P <0.05) in nodule dry matter per plant. Similarly, supplying 2 mM NO3 to L. luteus plants exposed to sub-ambient UV-B radiation significantly reduced individual organ growth, plant total biomass, nodule dry matter, nodule %N, and whole plant N content, as well as root concentrations of flavonoids, anthocyanins, soluble sugars, and starch of L. luteus, but not V. atropurpurea plants. These results show no adverse effect of elevated UV-B radiation on growth and symbiotic function of L. luteus and V. atropurpurea plants. However, NO3 supply promoted growth in L. luteus plants exposed to the highly elevated UV-B radiation.

Adaptation, Physiological↗

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Fabaceae↗

enod40 induces dedifferentiation and division of root cortical cells in legumes.

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

Journal Article↗

Nitrogen fixation by white lupin under phosphorus deficiency.

BACKGROUND AND AIMS: White lupin is highly adapted to growth in a low-P environment. The objective of the present study was to evaluate whether white lupin grown under P-stress has adaptations in nodulation and N2 fixation that facilitate continued functioning. METHODS: Nodulated plants were grown in silica sand supplied with N-free nutrient solution containing 0 to 0.5 mm P. At 21 and 37 d after inoculation (DAI) growth, nodulation, P and N concentration, N2 fixation (15N2 uptake and H2 evolution), root/nodule net CO2 evolution and CO2 fixation (14CO2 uptake) were measured. Furthermore, at 21 DAI in-vitro activities and transcript abundance of key enzymes of the C and N metabolism in nodules were determined. Moreover, nodulation in cluster root zones was evaluated. KEY RESULTS: Treatment without P led to a lower P concentration in shoots, roots, and nodules. In both treatments, with or without P, the P concentration in nodules was greater than that in the other organs. At 21 DAI nitrogen fixation rates did not differ between treatments and the plants displayed no symptoms of P or N deficiency on their shoots. Although nodule number at 21 DAI increased in response to P-deficiency, total nodule mass remained constant. Increased nodule number in P-deficient plants was associated with cluster root formation. A higher root/nodule CO2 fixation in the treatment without P led to a lower net CO2 release per unit fixed N, although the total CO2 released per unit fixed N was higher in the treatment without P. The higher CO2 fixation was correlated with increased transcript abundance and enzyme activities of phosphoenolpyruvate carboxylase and malate dehydrogenase in nodules. Between 21 and 37 DAI, shoots of plants grown without P developed symptoms of N- and P-deficiency. By 37 DAI the P concentration had decreased in all organs of the plants treated with no P. At 37 DAI, nitrogen fixation in the treatment without P had almost ceased. CONCLUSIONS: Enhanced nodulation in cluster root zones and increased potential for organic acid production in root nodules appear to contribute to white lupin's resilience to P-deficiency.

Carbon Dioxide↗

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Anthocyanins↗

The Rhizobium leguminosarum glnB gene is down-regulated during symbiosis.

Symbiotic nitrogen fixation involves the development, on the legume plant root, of specialised organs called nodules, within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. The glnB gene encodes a signal transduction protein (P(II)) which is a component of the bacterial nitrogen regulation (Ntr) system and an essential regulator of ammonium assimilation. We demonstrate that in Rhizobium leguminosarum the glnB promoter is strongly regulated by nitrogen and NtrC, but still shows a significant level of activity in conditions of nitrogen excess. Expression of genes involved in nitrogen assimilation has been shown to be absent in nitrogen-fixing bacteroids, and, in agreement with this, we find that the glnB promoter is down-regulated during bacteroid differentiation at a time coincident with the arrest of bacterial division in the nodule. This pattern is common to other bacterial genes involved in nitrogen assimilation and it is noteworthy that the zone where the glnB promoter is active is coincident with the region in which NtrC is expressed.

Amino Acid Sequence↗

Molecular Mechanisms in Root Nodule Development.

Under nitrogen-limiting conditions, bacteria from the family Rhizobiaceae establish a symbiosis with leguminous plants to form nitrogen-fixing root nodules. These organs require a coordinated control of the spatiotemporal expression of plant and bacterial genes during morphogenesis. Both plant and bacterial signals are involved in this regulation in the plant host. Plant genes induced during nodule development, the so-called nodulin genes, have been extensively characterized. Products of several of these genes show homologies to known regulators of signal transduction pathways in other plant or animal systems. Initial functional analysis of the molecular mechanisms implicated in nodulation have been undertaken using model legumes. Insertion mutagenesis and transgenic technologies to modify nodulin gene expression, as well as pharmacologic approaches, have been used to analyze molecular mechanisms involved in morphologic responses induced by the bacterial symbiont in the plant. G protein-mediated transduction mechanisms have been implicated, and the nin transcription factor appears to be required for early steps in nodule development. ENOD40, a gene coding for an RNA that contains only short ORFs, seems to be closely tied to nodule primordium formation. In addition, a vascular-associated Krüppel-like transcription factor and small Rab type G-proteins affect bacteroid differentiation and the function of the nitrogen-fixing zone. These initial results presage a wealth of information that will be obtained from the application of genomic approaches to legumes.

Journal Article↗

Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis.

The major phytoalexin in alfalfa is the isoflavonoid (-)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (-)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2'-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35,400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

Amino Acid Sequence↗

Effects of gradual increases in o(2) concentration on nodule activity in soybean.

The objectives of this study were to determine whether attached nodules of soybean (Glycine max L. Merr.) could adjust to gradual increases in rhizosphere pO(2) without nitrogenase inhibition and to determine whether the nitrogenase activity of the nodules is limited by pO(2) under ambient conditions. A computer-controlled gas blending apparatus was used to produce linear increases (ramps) in pO(2) around attached nodulated roots of soybean plants in an open gas exchange system. Nitrogenase activity (H(2) production in N(2):O(2) and Ar:O(2)) and respiration (CO(2) evolution) were monitored continuously as pO(2) was ramped from 20 to 30 kilopascals over periods of 0, 5, 10, 15, and 30 minutes. The 0, 5, and 10 minute ramps caused inhibitions of nitrogenase and respiration rates followed by recoveries of these rates to their initial values within 30 minutes. Distinct oscillations in nitrogenase activity and respiration were observed during the recovery period, and the possible basis for these oscillations is discussed. The 15 and 30 minute ramps did not inhibit nitrogenase activity, suggesting that such inhibition is not a factor in the regulation of nodule diffusion resistance. During the 30 minute ramp, a stimulation of nitrogenase activity was observed, indicating that an O(2)-based limitation to nitrogenase activity occurs in soybean nodules under ambient conditions.

Journal Article↗

Shoot control of root development and nodulation is mediated by a receptor-like kinase.

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

Alleles↗