Transport of phosphate from leaves to leguminous root nodules

Summary When P³²-orthophosphate was applied to leaves ofLotus pedunculatus radioactivity was detected in the root nodules. About 50 per cent of the radioactivity was in ATP, ADP and AMP. More than 90 per cent of the P³² compounds were localized in the soluble part of the plant tissue of the nodules. Rhizobial bacteroids contained only 1 per cent of P³² incorporated in the nodules.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Monoxenic nodulation process of Acacia mangium (Mimosoideae, Phyllodineae) by Bradyrhizobium sp

Acacia mangium Willd., a native tree in Australia and Papua New Guinea, has been introduced to countries in Asia and South America where plantations have been established that cover several hundred thousand ha. The present study investigated the early stages of the nodulation process in A. mangium using an homologous Australian Bradyrhizobium strain. After optimizing the axenic nodulation, histological and cytological studies were conducted using light and electron microscopy. These documented the proliferation of Bradyrhizobium, the lysis of mucilage at the root surface, root hair deformation and initiation, as well as the development and growth of multiple infection threads. A belt of tannin-filled cells was shown to surround the central nodular fixation zone. The nodules were of the indeterminate type and the bacteroids had a rod shape, without size modification and with few polyhydroxybutyrate (PHB) granules. Several bacteroids can share the same symbiosome. A. mangium exhibits both classical and novel features in its nodulation.     

Differential response of Trifolium species to 4-(2,4-dichlorophenoxy)butyric acid treatments

The differential response of white clover ( Trifolium repens L. cv. Regal Ladino) and berseem clover ( Trifolium alexandrinum L. cv. Mississippi ecotype) was investigated by treating greenhouse cultured plants with 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Berseem clover plants were significantly injured by a treatment concentration of 0.6 kg ha^-1 of 2,4-DB, whereas white clover plants were not injured by treatment levels below 2.4 kg ha^-1. The metabolism of 2,4-DB in cell suspension cultures of white clover and berseem clover was investigated using [ring-¹⁴C]-2,4-DB and non-labeled 2,4-DB. White clover cell cultures metabolized ca 4-fold more 2,4-DB than berseem cultures over a 44-h treatment period. The decrease in berseem cell population was 4-fold greater than the decrease in white clover cell population in response to the 8 μ M 2,4-DB treatment. The herbicide and its [ring-¹⁴C]-labeled metabolites were isolated from treated cells and medium after 44 h by partition and thin-layer chromatography. White clover cells metabolized 90% of the [¹⁴C]-2,4-DB and berseem clover cells metabolized 22% of the herbicide. The major portion of the radiolabel was in the glycoside fractions from extracts of both species. The differential response of Trifolium species to 2,4-DB is implied to be due to the differential rate of 2,4-DB metabolism to a glycoside by the clover plants.     

Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by ¹⁴CO₂-labeling studies and analysis of nodule enzymes. Plants were exposed to ¹⁴CO₂ for 30 minutes, followed by ¹²CO₂ for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).

The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.

In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in α,α-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

     

Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid

The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB). Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities. The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members. Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids. During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown. This was also verified using an indicator medium. Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members. First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types. The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.     

Effect of herbicide residues in a sandy loam on the growth,nodulation and nitrogenase activity (C2H2/C2H4) of Trifolium subterraneum

The herbicides 2,4-D, amitrole, atrazine, diclofop-methyl, diquat, paraquat and trifiluralin were applied at rates of 0, 2, 5 and 10 μg ai. g⁻¹ to a sandy loam soil and allowed to degrade for 120 days. After this period, subterranean clover seedlings were transplanted into treated soil and the effect of herbicide residues on plant growth, number of nodules formed and nitrogenase activity was investigated. At all rates of atrazine and chlorsulfuron, and at all rates of amitrole in excess of 2 mg ai g⁻¹ of soil, sufficient herbicide remained to be lethal to the seedlings. When amitrole was applied at the rate of 2 mg ai g⁻¹ of soil, plant growth, nodulation and nitrogenase activity of plants were reduced. Residues of diquat reduced all plant parameters studied while, residues of 2,4-D reduced plant growth and nodule formation, but plant nitrogenase activity was unaffected. Residues of trifluralin had no effect on plant growth parameters but the number of nodules formed per plant was reduced. Residues of paraquat and diclofop-methyl had no effect on any of the plant parameters studied.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

An improved large-scale isolation procedure for bacteroids of Rhizobium leguminosarum Frank. from Pisum savitum L.

Summary By manipulation of the growth medium level we have been able to concentrate the bulk of root nodules on a small proximal part of the main root. Measurements of several plant parameters on plants grown in a specially developed cultivation bin, show how changes in growth medium level effect nodulation.An isolation procedure is described for aerobic and anaerobic isolation of bacteroids of up to 1200 plants within 1 hour. Specific activities of nitrogenase preparations in these were found to be upto 21 n moles C₂H₂ red. per min per mg protein. re]19750724     

Degradation of 2,4-DB in Argentinean agricultural soils with high humic matter content

The dissipation of 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) in high-humic-matter-containing soils from agricultural fields of the Argentinean Humid Pampa region was studied, employing soil microcosms under different experimental conditions. The added herbicide was dissipated almost completely by soils with and without history of herbicide use by day 28. At 500 ppm, both soils showed the same degradation rates; but at 5-ppm concentration, the chronically exposed soil demonstrated a faster degradation of the herbicide. 2,4-DB addition produced increases in herbicide-degrading bacteria of three and 1.5 orders of magnitude in soils with and without history of herbicide use, respectively, in microcosms with 5 ppm. At 500-ppm concentration, the increase in 2,4-DB degraders was five orders of magnitude after 14 days, independent of the history of herbicide use. No differences were observed in either 2,4-DB degradation rates or in degrader bacteria numbers in the presence and absence of alfalfa plants, in spite of some differential characteristics in patterns of 2,4-DB metabolite accumulation. The main factor affecting 2,4-DB degradation rate would be the history of herbicide use, as a consequence of the adaptation of the indigenous microflora to the presence of herbicides in the field.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Arabidopsis thaliana mutants disrupted in lipid mobilization

To isolate mutants in the process of lipid mobilization during post-germinative growth we employed a screen using the pro-herbicide 2,4-dichlorophenoxybutyric acid (2,4-DB). The phenotypes of a number of 2,4-DB-resistant mutants are compared with previously characterized mutants disrupted in beta-oxidation or the glyoxylate cycle. We conclude that the strength of 2,4-DB resistance and the ability of the seedlings to grow in the absence of exogenous sugar are inversely correlated. Sugar dependence of 2,4-DB-resistant seedlings is a consequence of impaired storage-lipid mobilization.     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

Cytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineered Sinorhizobium strains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed the Agrobacterium ipt gene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although the ipt gene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineered Sinorhizobium strains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.     

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules

Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS-) formed mixed infected nodules that were capable of fixing atmospheric nitrogen. The formation of infected nodules was dependent on close contact between the inoculation partners. When the partners were separated by a filter, empty Fix- nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes. In mixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids. The formation of bacteroids was not dependent on cell-to-cell contact between the mutants. Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation. The introduction of an additional nif/fix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules. The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.     

Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions

The pasture legumes Lotus uliginosus (Schk.) and Lotus corniculatus (L.), known to differ in their tolerance to flooding, were inoculated with Rhizobium loti and flooded for 60 d while subjected to two levels of dissolved pO2: 0.241 and 0.094 mol ml^-1. L. uliginosus showed significantly greater growth (shoot and root) and N2 fixation under both pO2s, compared to L. corniculatus, although growth and N2 fixation by L. corniculatus was not affected by the low pO2. Surprisingly, in L. uliginosus., growth, nodulation and N2 fixation were all increased by low pO2 while nodulation of L. corniculatus where low pO2 plants showed a significant increase over that of the higher pO2 plants while L. uliginosus plants showed a decline. Root porosity of L. uliginosus doubled in the low pO2-treatment from a mean of 14.5% in high pO2 roots to 28.5%, whereas that of L. corniculatus was relatively unaffected by pO2, being 7% and 9% for high and low pO2 plants, respectively. The structure of nodules differed little between species and treatments, although nodules/nodulated roots from the L. uliginosus plants had particularly profuse lenticels and aerenchyma. However, L. corniculatus nodules, especially those grown in the lower pO2 showed signs of early senescence with vacuolation of infected cells and green coloration when cut open. Leghaemoglobin (Lb) concentrations in nodules from both species were unaffected by low pO2, although that of L. corniculatus nodules, regardless of pO2, was significantly greater than L. uliginosus. Concentrations of the intercellular glycoprotein recognized by the monoclonal antibody MAC265 were significantly reduced in nodules from the low pO2 treatment in both species. Immunogold labelling showed that the MAC265 antigen was localized primarily within intercellular spaces within nodule cortices from both Lotus species. A marked decrease in deposition of the MAC265 antigen within the cortices of L. uliginosus nodules grown in the lower pO2, is discussed in terms of the relative abilities of the two Lotus spp. to maintain an O2 supply to the N2-fixing bacteroids within submerged nodules.Keywords: Lotus uliginosus, Lotus corniculatus, N2 fixation, flooding, oxygen.      

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.