Effects of nitrogen-fixing bacteria on seabuckthorn growth (Hippophae rhamnoides. L)

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.

Isotopic15N2 experiments confirmed nitrogen fixation inParasponia parviflora. The conversion ratio C2H4/N2 was 6.7 under the experimental conditions employed. Measurements of the δ15N in leaves of Parasponia and Trema showed on the basis of these determinations thatParasponia parviflora possesses N2-fixing capacity and can be distinguished in this respect from the non-nitrogen-fixingTrema cannabina tested by the same method. Therefore, δ15N can be used to monitor N2 fixation in natural ecosystems. Hydrogen evolution and the relative efficiency of N2 fixation in this relation have been determined. DetachedParasponia parviflora root nodules grown in soil and tested in an argon/oxygen atmosphere produced appr. 4 μmol H2.h−1.g−1 fresh weight root nodules. The relative efficiency of hydrogen utilization as measured in argon, air, and in the presence of C2H2 10% (v/v) was for both equations\(\left( {1 - \frac{{H_2 (air)}}{{H_2 (Ar)}}} \right) and \left( {1 - \frac{{H_2 (air)}}{{C_2 H_2 }}} \right)\) used for to express this efficiency 0.96 and 0.97, respectively. This indicates that Parasponia like the root nodules of some actinorhizal symbioses (Alnus, Myrica, Elaeagnus) and some tropical legumes (Vigna sinensis) has evolved mechanisms of minimizing net hydrogen production in air, thus increasing the efficiency of electron transfer to nitrogen. The oxygen relation of nitrogen fixation (C2H2) inParasponia parviflora root nodules was determined. The nitrogenase activity of Parasponia root nodules increased at increasing oxygen concentrations up till c. 40% O2. At oxygen levels above 40% O2, the nitrogenase activity of the root nodules was nil or very erratic suggesting that at these oxygen levels the nitrogenase is not longer protected against the harmful effect of oxygen. In this respect Parasponia root nodules differ from actinorhizal root nodules in other nonlegumes, where optimal nitrogenase activity was observed in the range of 12–25% oxygen. Respiration experiments with Parasponia root nodules showed that in the range 10, 20, and 40% oxygen, the respiration rate (CO2 evolution) increased concomitantly with an increase of the acetylene reduction rate. The CO2/C2H4 values obtained varied between 8.1 and 19.2, being therefore 2–3 times higher than similar estimations in some actinorhizal and legume root nodules. The respiratory quotient (RQ) of detachedParasponia parviflora root nodules was in air initially approximately 2.0, but this value dropped to about 1.0 in a 3-hours period.

<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.

Greenhouse experiments were conducted with the objectives (1) to investigate the nitrogenase activity (NA) of cowpea (Vigna unguiculata (L.) Walp.) root nodules during the development of and subsequent recovery from drought stress and (2) to determine whether the changes in NA during and following drought stress are related to nodule water potential. Nitrogenase activity of root nodules decreased by more than 80% within 6–8 d of withholding water and recovered 1 or 2 d after watering. Nodule water potential declined significantly from approximately −0.2 MPa to −0.48 MPa with 8 d of stress and recovered to prestress levels within 24 h after watering. Midday abaxial stomatal conductance decreased significantly with stress but recovered within 24 h following watering. Midday leaf water potential did not change significantly during the experimental period. Nodule NA declined 2 d before that of nodule water potential in apparent response to declining soil water content. This response and the lag in the recovery of NA following drought stress after nodule water potential had returned to prestress levels support the hypothesis that nodule water potential per se is not the primary cause for the decline in NA of cowpea root nodules during drought stress.Key words: Vigna unguiculata (L.) Walp., nitrogenase activity, drought stress, recovery, cowpea

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 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.

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

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.

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H2 evolution) in both species was inhibited by an increase in rhizosphere pO2 from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO2 evolution). These data suggest that the nodules possess a variable barrier to O2 diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O2 or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO2, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O2‐limitation of nitrogenase‐linked respiration. These results indicate that nodules of pea and lupin regulate their internal O2 concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H2 production in Ar:O2) in pea and lupin nodules was monitored while rhizosphere pO2 was increased gradually to 100%, showed that the resistance of the nodules to O2 diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O2‐limitation coefficient of nitrogenase (OLCN= TNA/PNA) declined significantly with prolonged exposure to Ar:O2 or with shoot excision. Together, these results indicate a significant degree of O2‐limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.

Legume root nodules have a high potential to produce activated forms of oxygen. Particulary, in the presence of Fe, enhanced production of oxygen free radicals is responsible for stress-dependent peroxidation of membran lipids (Elstner, 1987). Increased leghemoglobin concentration and nitrogenase activity in root nodules and strongly increased nitrogen accumulation in plants were sensitive responses of N2 fixation in yellow lupin to copper nutrition (Seliga, 1993, 1995). The present paper reports on the effect of copper supply on lipid peroxidation, the level of catechol-like siderophores and polyphenol oxidase (PPO) activity in nodules and on the interaction with iron in yellow lupin plants.

Annual CO2 evolution, H2 evolution, and C2H2 reduction were measured in root nodules from a vigorous Myrica gale stand in a Massachusetts peatland at 3-week intervals in 1980. Nodule activity was approximately the same under the experimental conditions (excised nodules reducing C2H2) as in nature (attached nodules reducing N2) and the CO2 evolution to O2 uptake ratio averaged 1.07. Nitrogenase activity was first detectable in late May, reached its maximum [Formula: see text] in mid-July, and disappeared in late October. The seasonal pattern of CO2 evolution was similar except that it continued at low rates when nitrogenase activity was absent. Hydrogen evolution was barely detectable. The energy cost of nitrogen fixation, expressed as the molar CO2:C2H4 ratio, was relatively low [Formula: see text] throughout the period of substantial nitrogenase activity and had a mean annual value of 4.9. Annual N2 fixation was estimated to be 2.8 g N m−2year−1, contributing about 33% of the annual N requirement measured in 1979. Annual C use by nodules was about 21.0 g C m−2 year−1. If this C were available for additional net production, it would increase it by about 5.5%.

Although infected cell O2 concentration (Oi) is known to limit respiration and nitrogenase activity in legume nodules, techniques have not been available to measure both processes simultaneously in an individual legume nodule. Consequently, details of the relationship between nitrogenase activity and Oi are not fully appreciated. For the present study, a probe was designed that allowed open circuit measurements of H2 evolution (nitrogenase activity) and CO2 evolution (respiration rate) in a single attached soybean nodule while simultaneously monitoring fractional oxygenation of leghemoglobin (and thereby Oi) with a nodule oximeter. Compared to measurements of whole nodulated roots, use of the probe led to inhibition of nitrogenase activity in the single nodules. During oximetry measurements, total nitrogenase activity (TNA; peak H2 evolution in Ar/O2) in the single nodules was 16% of that in whole nodulated roots and 48% of nodulated root activity when Oi was not being measured simultaneously. This inhibition did not affect the nodules' ability to regulate Oi, because exposure to Ar/O2 (80:20, v/v) caused nitrogenase activity and respiration rate to decline, and this decline was linearly correlated with a concurrent decrease in Oi. When the nodules were subsequently exposed to a linear increase in external pO2 from 20 to 100% O2 at 2.7% O2/min, fractional leghemoglobin oxygenation first increased gradually and then more rapidly, reaching saturation at a pO2 between 76 and 100% O2. Plots of nitrogenase activity and respiration rate against Oi showed that rates increased with Oi up to a value of 57 nM, with half-maximal rates being attained at Oi values between 10 and 14 nM O2. The maximum nitrogenase activity achieved during the increase in pO2 (potential nitrogenase activity) was 30 to 57% of that measured in intact nodulated roots, showing that O2 limitation of nitrogenase activity could account for a significant proportion of the inhibition of TNA associated with the use of the probe. However, some factor(s) in addition to O2 must have limited the activity of single nodules at both subsaturating and saturating Oi. At Oi values greater than about 57 nM, nitrogenase activity and nodule respiration were inhibited, but, because this inhibition has been shown previously to be readily reversible when the Oi was lowered, it was not attributed to direct O2 inactivation of the nitrogenase protein. These results indicate that maximum nitrogenase activity in legume nodules is supported by a narrow range of Oi values. Possible biochemical mechanisms are discussed for both O2 limitation of nitrogenase activity at low Oi and inhibition of nitrogenase activity at high Oi.

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.

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

Mung beans (Vigna radiata) were grown in the field, and measurement of leghaemoglobin and nitrogenase activity of root nodules during plant growth showed a maximum 30 days after sowing, i.e., just when flowering began. Mung beans were also grown in hydroponic cultures with varying amounts of nitrate nitrogen and harvested at 30 day s. The extent of root nodulation, leghaemoglobin accumulation and nitrogenase activity in the nodules, and plant dry weights were found to be maximal at a low concentration (0.5 mM) of nitrate. Dry weights of shoots and roots (exclusive of nodules) and total chlorophyll content of leaves were also increased more by this low concentration than at higher concentrations of 1–3.5 mM. Nodule formation, including leghaemoglobin content and nitrogenase activity, declined with increased nitrate levels. At the highest nitrate levels studied (4.5 and 5 mM) no nodules were formed, but the shoot and root dry matter increased markedly. It is evident from the results that a minimal amount of nitrate nitrogen is essential for efficient symbiotic nitrogen fixation.