Nitrogenase Activity Of Root Nodules Research Articles

Transcriptomic and physiological analyses unravel the effect and mechanism of halosulfuron-methyl on the symbiosis between rhizobium and soybean

Halosulfuron-methyl (HSM) is a new and highly effective sulfonylurea herbicide widely used in weed control, but its residue in the environment poses a potential risk to soybean. Soybean–rhizobium symbiotic nitrogen fixation is crucial for sustainable agricultural development and ecological environment health. However, the impact of HSM on the symbiosis between soybean and rhizobium is unclear. In this study, the effects of HSM on the soybean–rhizobium symbiotic process and nitrogen fixation were investigated by means of transcriptomic and physiological analyses. Treatment with a concentration of HSM less than 0.5 mg L−1 had no effect on rhizobium growth, but significantly reduced nodules number, the biomass of soybean nodules, and nitrogenase activity in root nodules (P < 0.05). Transcriptomic analysis showed that differentially expressed genes (DEGs) involved in NH4+ assimilation were significantly downregulated (P < 0.05). In addition, the activities of NH4+ assimilation enzymes were markedly reduced. This result was further confirmed by the accumulation of NH4+ in root nodules, indicating that the inhibition of nitrogen fixation by HSM may be caused by excessive NH4+ accumulation in root nodules. Furthermore, DEGs involved in flavonoid synthesis, phytohormone biosynthesis, and phytohormone signaling transduction were significantly downregulated (P < 0.05), which was consistent with the decrease in flavonoid and phytohormone contents determined in this study. These results suggested that HSM may inhibit soybean nodulation by inhibiting flavonoid synthesis in soybean roots, disrupting the balance of plant endogenous hormones in roots during symbiosis, and blocking the transmission of hormone signals during the symbiosis. Our findings provide new insights into the effects of HSM on the legume–rhizobium nodule symbiotic process.

大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能

<p id="C3">Plant hormone auxin plays a vital role in the growth and development of plants. Auxin homeostasis and concentration gradient establishment control the polar formation of almost all organs. The synthesis, transportation, perception, and metabolic degradation of auxin in specific cells establish a concentration gradient of auxin in accordance with organ development. In legumes, roots interact with soil microorganisms to form a special organ called nodules, which is used for biological nitrogen fixation. However, the function of auxin homeostasis control of biological nitrogen fixation is unknown. Studies showed that PIN-Like (PILS) proteins in<italic> Arabidopsis</italic> helped to regulate intracellular auxin homeostasis and mediate auxin signal transmission in the downstream nucleus. In this study, 19 PILS family genes (<italic>GmPILSs</italic>) were identified in soybean genome and distributed unevenly on 10 chromosomes of soybean. <italic>GmPILSs</italic> exhibited a variety of expression patterns in nine tissue parts of soybean, and had obvious specificity of tissue expression. <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> were enriched and expressed in the rhizobia region, and the expression of <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> in nodules was down-regulated by artificial microRNA interference (amiRNAi), resulting in the increase of nitrogenase activity in the nodules. However, the overexpression of <italic>GmPILS1f</italic> leaded to the decrease nitrogenase activity in root nodules, <italic>GmPILS1e</italic> and <italic>GmPILS1f</italic> might participate in the regulation of soybean nitrogenase activity. These results lay the foundation for further analysis of the function and mechanism of soybean <italic>GmPILS</italic> family genes, and also provide valuable genetic resources for the application of nodulation and nitrogen fixation in agricultural breeding.

Hendrik Poorter.

Hendrik Poorter.

Glyphosate effects on symbiotic nitrogen fixation in glyphosate-resistant soybean

Most soybean (Glycine max L. Merr.) cultivars grown in the U.S. are Roundup Ready®, genetically modified for resistance to the herbicide glyphosate. The objectives of this study were to examine the impacts of glyphosate applications on symbiotic N2 fixation in glyphosate-resistant (GR) soybean and to evaluate the responses of rhizobial isolates and nifH gene abundance in the rhizosphere soil to glyphosate. In two repeated greenhouse experiments, GR soybean received foliar applications of glyphosate once or twice during the study period; the untreated GR and near-isogenic conventional cultivar served as controls. Plants were harvested twice, two days after each glyphosate application. In addition to plant growth parameters, the nitrogenase activity of root nodules and the abundance of the nifH gene in the rhizosphere were determined using the acetylene reduction assay and quantitative PCR, respectively. Glyphosate-treated GR soybean had lower chlorophyll content, root mass, nodule mass, total plant N, and nitrogenase activity than the untreated conventional cultivar, especially for the second harvest. Without glyphosate application, few differences were observed between the two cultivars. Glyphosate inhibited the growth of rhizobia isolated from root nodules; however, the nifH gene abundance in the rhizosphere was not different among the treatments. Glyphosate appeared to exert stress to the GR soybean cultivar used in this study. Further research is needed to verify the greenhouse experiment findings and possible yield effects under field conditions.

Medicago truncatula Molybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency.

Molybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters. Members of the molybdate transporter family Molybdate Transporter type 1 (MOT1) were identified in the model legume Medicago truncatula and their expression in nodules was determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of a Transposable Element from Nicotianatabacum (Tnt1) insertional mutant line were carried out in the one M.truncatula MOT1 family member specifically expressed in nodules. Among the five MOT1 members present in the M.truncatula genome, MtMOT1.3 is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. A mot1.3-1 knockout mutant showed impaired growth concomitant with a reduction of nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore, mot1.3-1 plants transformed with a functional copy of MtMOT1.3 showed a wild-type-like phenotype. These data are consistent with a model in which MtMOT1.3 is responsible for introducing molybdate into nodule cells, which is later used to synthesize functional nitrogenase.

The effects of gibberellins and mepiquat chloride on nitrogenase activity in Bradyrhizobium japonicum

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA3) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA3 or PIX. GA3 treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA3-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA3 and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA3 treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA3, respectively, in B. japonicum.

Foliar application of pyraclostrobin fungicide enhances the growth, rhizobial-nodule formation and nitrogenase activity in soybean (var. JS-335)

A field study was conducted to investigate the impact of the fungicide pyraclostrobin (F500 – Headline®; a.i. 20%) on the activity of nitrogenase in soybean (var. JS-335). Pyraclostrobin (F500) was applied on the leaves of soybean plants at 10 and 20days after emergence (DAE) of seedlings at concentrations ranging from 0.05% to 1%. Leghemoglobin (Lb) content and nitrogenase activity in root nodules were analyzed at 45thday after emergence of seedlings indicated a remarkable increase in Lb content and enhanced activity of nitrogenase in the root nodules of pyraclostrobin treated plants. The fungicide also enhanced the number of nodules along with weight of nodules, root biomass and growth of shoot and leaves. Enhanced nitrogen fixation in the root nodules by pyraclostrobin improves the growth of the plant in soybean before flowering and pod formation which ultimately resulted in yield and yield attributes. These results suggest that pyraclostrobin (F500) can be successfully employed as a foliar spray under field conditions to enhance the growth, nitrogen assimilation and hence yield of soybean.

CO-INOCULATION OF SOYBEAN (GLYCINE MAX) WITH ACTINOMYCETES AND BRADYRHIZOBIUM JAPONICUM ENHANCES PLANT GROWTH, NITROGENASE ACTIVITY AND PLANT NUTRITION

The aim of this study was to determine the potential of the endophytic actinomycetes that produce plant growth promoters used as co-inoculants with Bradyrhizobium japonicum to promote the growth of soybean. These endophytes exhibited the potential to enhance plant growth, nitrogenase activity of root nodules and plant nutrient uptake. Co-inoculum of B. japonicum with Nocardia alba conferred the maximum yield of root and shoot dry weight. All single-inoculated actinomycetes strains had the ability to enhance plant growth. Noc. alba and Nonomuraea rubra increased total plant dry weight up to 2.14-fold and 2.11-fold, respectively, when compared to the uninoculated controls. Co-inoculations of B. japonicum with each of Noc. alba, Non. Rubra, and Actinomadura glauciflava increased acetylene reduction activity up to 1.7 to 2.7-fold. For plant mineral composition, all of co-inoculation treatments significantly increased the nutrient levels of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe) and zinc (Zn) within a soybean plant.

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.

Effect of Hairy Vetch Incorporated as Green Manure on Growth and N Uptake of Sorghum Crop

Hairy vetch (Vicia villosa Roth) has the potentials for preventing soil erosion and suppressing weed growth as a winter cover crop. We evaluated the additional N supplied by this crop harvested at different growing stages to the succeeding sorghum in pot experiments. Hairy vetch was grown in 1/5000a Wagner pot, and shoots (S) and roots (R) were mixed separately or together (S+R) into the soil on 30 March, 18 April and 2 May. After incorporation of the plants, seeds of sorghum were sown in the pots. Dry weight and N content of hairy vetch increased throughout the growing period. The value of nitrogenase activity in root nodules peaked on 18 April and then drastically declined. When hairy vetch was harvested on 30 March, N content of sorghum in the S pots was definitely less than that in the R and S+R pots. When hairy vetch harvested on 2 May, however, the N content in the S pots was similar to that in the S+R pots, and it was significantly higher than that in the R pots. Although N input from hairy vetch was higher in the S pots than in the R pots, N uptake by sorghum was not reflected in those values. The belowground parts of hairy vetch may have a considerable effect on N uptake of sorghum when using this plant species as green manures.

Effect of Indole Acetic Acid on the Synergistic Interactions of Bradyrhizobium and Glomus fasciculatum on Growth, Nodulation, and Nitrogen Fixation of Dalbergia sissoo Roxb

The legume Dalbergia sissoo Roxb is an important forest tree of the arid and semi-arid regions. The efficiency of Bradyrhizobium (Ds Rhz-9) and Glomus fasciculatum (Thaxter sensu Gerd.) Gerd. and Trappe to promote growth of D. sissoo and the effect of indole acetic acid (IAA) on the tripartite symbiotic association was studied in a pot experiment. Parameters like plant growth, nodulation, total phosphorus, total nitrogen content, nitrogenase activity of root nodules (acetylene reduction assay), and arbuscular mycorrhizal fungi (AMF) root colonization were analyzed. The dual inoculation of Bradyrhizobium along with G. fasciculatum showed synergistic increase in growth, nodulation, and nitrogen fixation of the seedlings. The maximum nitrogen (1.45%) and phosphorus (34.6%) content of dry weight was recorded in the dual inoculated plants when treated with IAA (1.39 × 10−3 mole/liter). The nitrogenase activity of the nodules in dual treatments showed an enhancement of 34.9%, which became further enhanced by 15.7% when treated with IAA. The presence of Bradyrhizobium in the rhizosphere was found to decrease AMF root colonization. In dual inoculated plants, the exogenous application of IAA (1.11 × 10−3 moles/liter) increased AMF root colonization by 60%. The tripartite synergistic interaction of Bradyrhizobium and G. fasciculatum with D. sissoo seems to be promising and can be utilized for its growth promotion in forest nurseries and reforestation of degraded areas. Exogenous application of IAA will be beneficial and recommended.

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO2 exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.

Nitrogen Fixation in Seabuckthorn (Hippophae rhamnoides L.) Root Nodules and Effect of Nitrate on Nitrogenase Activity

Seabuckthorn (Hippophae rhamnoides L.) is a small fruit tree belonging to the family Elaeagnaceae. Because seabuckthorn fruit is rich in unsaturated fatty acids and vitamins, this plant has potential as a food and medicinal crop. Here, we focused on symbiotic nitrogen fixation that could aid in the cultivation of this species. Microscopic observations showed that seabuckthorn root nodules have a standard morphology characteristic of Frankia-actinorhizal root nodules. Under nitrogen-free conditions, seabuckthorn seedlings inoculated with a homogenate of root nodules grew normally, and the fresh weight of root nodules was positively correlated with plant growth. In the field, nitrogenase activity in root nodules was high from May to September, when air temperatures were high and photosynthesis was active. Inhibition of nitrogen fixation by nitrate has been well documented in the root nodules of legumes. Therefore, we investigated the effect of nitrate on nitrogenase activity in seabuckthorn root nodules. Nitrogenase activity in seabuckthorn root nodules was not inhibited by the addition of high concentrations (up to 30 mM) of nitrate over a short term (5 days), but was apparently inhibited by long-term (20–30 days) treatments with 5 and 10 mM of nitrate.

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes.

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.

The Effect of Seed Vernalization and Irradiation on Growth and Photosynthesis of Field Bean Plants (Vicia faba L. minor) and on Nitrogenase Activity of Root Nodules

The effect of seed vernalization (2–3°C; 36 days) and irradiation intensity during vegetation of field bean upon growth and maximum leaf photosynthesis (Pmax) as well as specific (SNA) and total nitrogenase activity (TNA) of root nodules was investigated. The measurements were performed at the initial stage of pod development of varieties with undetermined (cv. Nadwiśański) and determined (cv. Tibo) growth habit. In comparison with plants in shading, those which grew in full irradiation produced larger leaf area and more dry weight, achieving higher Pmax values. Stimulation of leaf area enlargement and dry weight increase, especially in respect to the dry weight of underground parts had a positive and significant effect upon TNA value. Accumulation of dry weight was higher in vernalized than non‐vernalized plants, but only in the case of cv. Nadwiślański, in full irradiation conditions. In the case of plants grown in poorer irradiation conditions TNA values were about 68 % (cv. Nadwiślański) and 54 % (cv. Tibo) lower, due to significant limitation of root nodules. TNA value of vernalized cv. Nadwiślański plants was 66 % higher in comparison with non‐vernalized plants. However, the effect was visible only in full irradiation conditions, due to simultaneous greater intensity of root nodules production and higher SNA value. A positive effect of vernalization could also be observed in the decrease of a negative correlation between dry weight of root nodules and SNA in both varieties.

Effects of long‐term nitrogen dioxide fumigation and nitrate supply on nitrite and Leghemoglobin concentrations, pH, and nitrogenase activity of soybean root nodules

Exposing 12‐day‐old soybean plants to 0.2 ppm nitrogen dioxide (NO2) for four weeks increased the nitrite concentration and acidity, and decreased the Leghemoglobin (LHb) concentration and the nitrogenase activity of root nodules. The supply of 1 mol.m‐3 nitrate to the roots intensified the nitrite accumulation, decreased the acidity of the nodules, and alleviated the inhibition of nitrogenase activity by NO2 fumigation. These results suggested that the inhibition of nitrogen (N2) fixation by N fertilizer supply might relate to the acid‐alkali balance in nodules.

The acetylene-induced decline in nitrogenase activity in root nodules of Elaeagnus angustifolia

The rate of C2H2 reduction by nodulated seedlings of Elaeagnus angustifolia (Russian olive) was followed as a function of time. Our goals were to: 1) determine whether there is an C2H2-induced decline in nitrogenase activity; and 2) investigate the mechanism of any decline. We found a peak rate of C2H2 reduction at 1.5 min after the introduction of C2H2 that was followed by a rapid decline in activity to 56% of the peak value. After the decline there was a partial recovery to 67% of the peak value at 60 min. When the pO2 was decreased during the decline there was no significant effect (p≤0.05) on nitrogenase activity. When the C2H2 reduction assay was preceded by an incubation in a gas mixture (20 kPa O2) with Ar substituted for N2, there was little decline in nitrogenase activity as a function of time, but the rate of C2H2 reduction per gram nodule was reduced by approximately 50%. From these results we conclude that t Elaeagnus angustifolia exhibits a pronounced C2H2-induced decline and consequently the initial peak rate C2H2 reduction must be determined to obtain a valid measure of nitrogenase activity. We further suggest that cessation of NH3 formation initiates the decline and that the decline is not caused by a change in nodule permeability to gases.

Relationship between Activities of Enzymes for the Removal of O2 ·- and H2O2 and Nitrogenase in Root Nodules of Phaseolus vulgaris L.

Abstract Nodules of Rhizobium leguminosarum bv. phaseoli in symbiosis with Phaseolus vulgaris were compared with regard to their nitrogenase activity and activities of enzymes involved in the removal of O2·- and H2O2 as well as total ascorbate content. Activities of catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and total ascorbate content were consist­ently higher in nodules inhabited by bacterial strains with higher nitrogenase activity. Values for superoxide dismutase (EC 1.15.11), and guaiacol peroxidase activity did not differ for the bacterial strains compared. On the other hand, when different plant cultivars were inoculated with the same bacterial strain, high nitrogenase activity did not correlate with a higher activ­ity of the oxygen scavenging enzyms or a higher content of total ascorbate. In this case, values for guaiacol peroxidase activity were greatly enhanced in nodules with lower nitrogen­ ase activity. This may be part of a hypersensitive reaction of the plant cultivar against the bacterial symbiotic partner. Inhibition of catalase activity in the nodules by addition of triazole to the nutrient solution did not alter nitrogenase activity within the first nine hours after addition. It can be concluded that the activity of catalase, ascorbate peroxidase, and superoxide dismutase is not generally coupled to nitrogenase activity in root nodules of P. vulgaris.

Physiological changes in soybean treated with ozone and molybdenum

Abstract An open‐top field chamber experiment was conducted to evaluate the impact of Molybdenum (Mo) addition to soil on the physiological changes in soybean (Glycine max L. Merrill) exposed to ozone (O3). Plants grown with Mo (0, 1.0, or 2.0 mg kg1 soil dry weight) were exposed to O3 (O, 0.06, or 0.12 μmol mol‐1) in open‐top field chambers for 12 h d‐1 for 21 d with a N‐free fertilizer, during the sensitive growth stage (R2). The rate of photosynthesis (PN), specific root nodule nitrogenase activity (SNA), leaf nitrogen (N), chlorophyll (chl‐a, chl‐b) and biomass of soybean were measured. The increase in O3 levels significantly reduced PN, SNA, leaf‐N, chl‐a, chl‐b, and biomass. Addition of Mo increased leaf‐N, shoot, root, and nodule dry weights but did not change PN, SNA, or chlorophyll. The addition of Mo (2 mg kg ‐1) helped in significantly increasing PN and chlorophyll in the presence of 0.06 umol mol‐1 O3 but no change was observed in the presence of 0.12 μmol mol‐1 O3.

Soybean response to carbon dioxide and molybdenum

Abstract The objective of this experiment was to measure the impact of molybdenum (Mo) addition to the soil on changes in rate of photosynthesis (PN), specific root nodule nitrogenase activity (SNA), chlorophyll ‐ (Chl‐a) and (Chl‐b) and biomass of soybean (Glycine max L. Merrill), exposed to carbon dioxide (CO2) (350, 400, or 450 μL/L) for 5 d during its sensitive growth stage (R2). Plants grown in soil with Mo (0, 0.5, or 1.0 mg/kg soil dry weight) were exposed to carbon dioxide (CO2) in open‐top field chambers for 12 h/d for 5 d with a nitrogen (N)‐free fertilizer. Increasing CO2 levels reduced SNA and increased both Chl‐a and Chl‐b. Addition of 1 mg/kg Mo significantiy reduced SNA for plants grown with 350 μL/L CO2 with no change in PN.