Increase In Nitrogenase Activity Research Articles

Degradation of Complex Carbon Sources in Organic Fertilizers Facilitates Nitrogen Fixation.

Biological nitrogen fixation is crucial for agriculture and improving fertilizer efficiency, but organic fertilizers in enhancing this process remain debated. Here, we investigate the impact of organic fertilizers on biological nitrogen fixation through experiments and propose a new model where bacterial interactions with complex carbon sources enhance nitrogen fixation. Field experiments showed that adding organic fertilizers increased the nitrogenase activity by 57.85%. Subculture experiments revealed that organic fertilizer addition enriched genes corresponding to complex carbon and energy metabolism, as well as nifJ involved in electron transfer for nitrogenase. It also enhanced bacterial interactions and enhanced connectors associated with complex carbon degradation. Validation experiments demonstrated that combinations increased nitrogenase activity by 2.98 times compared to the single. Our findings suggest that organic fertilizers promoted nitrogen fixation by enhancing microbial cooperation, improved the degradation of complex carbon sources, and thereby provided utilizable carbon sources, energy, and electrons to N-fixers, thus increasing nitrogenase activity and nitrogen fixation.

Investigation of nitrogen fixation activity soybeans plants by mineral fertilizers foliar treatment

The results of the study of nitrogen-fixing activity during foliar treatment of vegetative soybean plants with liquid mineral fertilizers containing macro-microelements in various forms in the foothill zone of Kabardino-Balkaria are presented. The object of research is the early ripe soybean variety SK Veda. The soil of the experimental plot is a leached, medium-thick, low-humus, heavy loamy chernozem on carbonate clays, with a humus content in the arable layer of 3.1 %, pHkcl 5.0, mobile phosphorus 7.5 and exchangeable potassium 8.0 mg/100 g of soil. Encrustation of soybean seeds was carried out using a specific film-forming agent based on soybean phosphatides with the inoculant Nitrofix. The results of studies for 2020-2022 are given. with an assessment of the dynamics of the formation of the symbiotic apparatus by the number of nodules (pcs/m2) and by the mass of nodules (g/m2) in ontogeny of the macrosymbiont. The peak of nodule formation in soybean plants of the SK Veda variety fell on the flowering phase. The highest nodulation rates were noted in the control variant: in 2020 - 306 pcs/m2; in 2021 - 408 pcs/m2; in 2022 - 96 pcs/m2. It has been shown that foliar treatment of soybean plants with nutrient deficiency correctors has an impact on nodulation rates, with differences being noted depending on the treatment time. In the variant of Polydon NPK treatment in the primordial leaf phase, the nodulation parameters were minimal in the experiment over the years of the study (86; 216; 56 pcs/m2). Treatment of SK Veda soybean plants in the primordial leaf phase with molybdenum-containing fertilizers ensures the formation of larger nodules. The values of the nodule weight in the flowering phase in the variant of the treatment of Polydon plants with molybdenum in the primordial leaf phase increased by 1.56; 2.09; 1.11 times, respectively, for 2020, 2021, 2022. Nitrogenase activity averaged 26.4-281.6 µg N2/plant.h over the years of the study hour. In the control (inoculation with nitrogen-fixing microorganisms), the activity of nitrogenase was 208.3 µg N2/plant.h. Inoculation of soybean seeds with nitrogen-fixing microorganisms and foliar treatment of plants with Polydon molybdenum increased nitrogenase activity by 35.2-19.1 % and increased protein content by 5.0-3.9 % relative to control, depending on the treatment phase.

Effect of Phosphorus Application on Groundnut (Arachis hypogaea L.): A Review

The phosphorus in legumes plays an essential role as part of the membrane system of the cell, chloroplast and the mitochondria. Groundnut, being a legume oilseed crop, requires a significant amount of phosphorus. Plants use it for energy transfer in metabolic processes such as photosynthesis (in the form of ATP and ADP), starch and sugar transformation and nutrient movement. In addition to promoting cell division and the development of good root systems phosphorus plays an important role for ensuring timely and uniform ripening of crops, promoting carbohydrate breakdown for energy release, and hastening maturity, it is an essential component for ensuring the growth and maturity of plants. Phosphorus is one of the principal nutrients, which boosts root growth, thus facilitating water and nutrient uptake from deeper layers of the soil. The groundnut crop calls for sensible supply of phosphorus for its regular growth. Phosphorus has been found to improve the crop's effective use of soil nutrients and increase biological N fixation by increasing nitrogenase activity. Various research in this paper clearly indicate that application of phosphorus significantly affects the growth, phenology, yield attributes, yield, quality, nutrient uptake and economics of groundnut.

Mixed Enterobacter and Klebsiella bacteria enhance soybean biological nitrogen fixation ability when combined with rhizobia inoculation

Biological nitrogen fixation is a key process for nitrogen geochemical cycling and plant‒microbe interactions. At present, little is known about the possible effects of nonnodulating microbes on rhizobia nodulation in soils. Here, we show that Enterobacter sp. Z1 and Klebsiella sp. Z2 significantly improved soybean growth and nitrogen fixation in the presence of Bradyrhizobium diazoefficiens USDA110. Soil metabolomic analysis showed that the application of mixed strains Z1 and Z2 significantly increased the levels of flavonoids, indoleacetic acid (IAA), salicylic acid (SA), and taurine in rhizosphere soil. The addition of IAA, SA, or taurine increased nitrogenase activity, nodule production, and chlorophyll content in soybean. Flavonoids were the determinant of the interaction between soybeans and rhizobia. Moreover, strains Z1 and Z2 produced an abundance of IAA and SA, thus increasing the expression of flavonoid synthesis-related genes and eventually increasing the accumulation of flavonoids. Both strains Z1 and Z2 exhibited two metabolic pathways of taurine, and they catalyzed taurine to produce acetyl-CoA, a key flavonoid synthesis substrate in soybeans. This study revealed the mechanism by which rhizosphere microorganisms mediated soybean-rhizobium interactions, thus promoting soybean nitrogen fixation.

Increased Nitrogenase Activity in Solar‐Driven Biohybrids Containing Non‐photosynthetic Bacteria and Conducting Polymers

AbstractA conductive polymer‐based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non‐photosynthetic bacterium Azotobacter Chroococcum (A. Chroococcum). The light‐harvesting cationic poly(fluorene‐alt‐phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4+‐N and L‐amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum‐iron (MoFe) protein and relevant nitrogen‐fixing proteins are up‐regulated. These photoactive conductive polymer‐bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non‐photosynthetic nitrogen‐fixing bacteria.

Increased Nitrogenase Activity in Solar-Driven Biohybrids Containing Non-photosynthetic Bacteria and Conducting Polymers.

A conductive polymer-based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non-photosynthetic bacterium Azotobacter Chroococcum (A. Chroococcum). The light-harvesting cationic poly(fluorene-alt-phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4 + -N and L-amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum-iron (MoFe) protein and relevant nitrogen-fixing proteins are up-regulated. These photoactive conductive polymer-bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non-photosynthetic nitrogen-fixing bacteria.

GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.

Chemical Stimulation of Heterocyte Differentiation by the Feather Moss Hylocomium splendens: a Potential New Step in Plant-Cyanobacteria Symbioses.

Cyanobacteria associated with mosses play a key role in the nitrogen (N) cycle in unpolluted ecosystems. Mosses have been found to release molecules that induce morphophysiological changes in epiphytic cyanobionts. Nevertheless, the extent of moss influence on these microorganisms remains unknown. To evaluate how mosses or their metabolites influence N2 fixation rates by cyanobacteria, we assessed the nitrogenase activity, heterocyte frequency and biomass of a cyanobacterial strain isolated from the feather moss Hylocomium splendensand a non-symbiotic strain when they were either growing by themselves, together with H. splendens or exposed to H. splendens water, acetone, ethanol, or isopropanol extracts. The same cyanobacterial strains were added to another moss (Taxiphyllum barbieri) and a liverwort (Monosolenium tenerum) to assess if these bryophytes affect N2 fixation differently. Although no significant increases in nitrogenase activity by the cyanobacteria were observed when in contact with H. splendens shoots, both the symbiotic and non-symbiotic cyanobacteria increased nitrogenase activity as well as heterocyte frequency significantly upon exposure to H. splendens ethanol extracts. Contact with T. barbieri shoots, on the other hand, did lead to increases in nitrogenase activity, indicating low host-specificity to cyanobacterial activity. These findings suggest that H. splendens produces heterocyte-differentiating factors (HDFs) that are capable of stimulating cyanobacterial N2 fixation regardless of symbiotic competency. Based on previous knowledge about the chemical ecology and dynamics of moss-cyanobacteria interactions, we speculate that HDF expression by the host takes place in a hypothetical new step occurring after plant colonization and the repression of hormogonia.

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

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.

Interactions of microbial inoculants with soil and plant attributes for enhancing Fe and Zn biofortification in maize genotypes

This study aims to improve the iron and zinc mobilization from soil to maize kernels using cyanobacteria-based formulations. Towards this, focused analyses of plant physiological attributes, nutrient availability in soil and its enrichment in the kernels of maize hybrid, Pusa Vivek QPM 9 Improved (G1) and its parental inbreds Pusa Maize Inbred Provitamin 1 (G2) and Pusa Maize Inbred Provitamin 2 (G3) was undertaken. The treatments included formulations of individual agriculturally beneficial organisms-a cyanobacterium- Anabaena torulosa, fungus-Trichoderma viride, and a bacterium Providencia sp. and their laboratory-developed biofilms - A. torulosa – Trichoderma viride (An-Tr) and A. torulosa – Providencia sp. (An-PW5). Application of An-Tr biofilm formulation led to 14- and 3.3-fold increase in nitrogenase activity and chlorophyll of soil respectively, over control, at the seedling stage. Hybrid (G1) recorded a significant increase of 1.2–1.5-fold in terms of nitrogenase activity over the parent genotypes. The highest zinc content in maize kernels, with a 1.34-fold increase over control, was recorded with An-Tr biofilm treatment, while T. viride increased kernel Fe content by 2.1-fold. Multivariate analyses illustrated a significant correlation of soil biological parameters-nitrogenase activity, chlorophyll, polysaccharides, Fe, Zn, with kernel Fe and Zn content in maize genotypes. Linear discriminant analyses validated the significant effects of individual microbial inoculation and biofilms. An-Tr biofilm- G1 (hybrid) combination can be a model system for in-depth analyses of beneficial effects of microbe-mediated biofortification of maize, leading to improved kernel quality and 25 % N savings.

Promoting photo-fermentative hydrogen production performance by substituting the rnf promoter in Rhodobacter capsulatus

The Rnf (Rhodobacter nitrogen fixation) complex encoded by rnfABCDGEH genes is a crucial enzyme for the functioning of nitrogenase. In this work, we studied the effect of Rnf complex expression level on hydrogen production in the hydrogen-producing bacterium Rhodobacter capsulatus SB 1003 by expressing rnf genes with three autologous promoters. Substitution of the rnf promoter with that of pucB resulted in a 1.59-fold increase in rnf transcription level and 1.3-fold increase in nitrogenase activity. Under various test conditions, maximum H2 yields and production rates were increased by 12.4%–24.7% and 6.5%–22.9%, respectively. There was a further increase in H2 production when the Prnf::PpucB::rnf fusion construct was expressed in a background strain with both the alternative Fe nitrogenase and the Mo nitrogenase co-expressed. The results demonstrate that the Rnf complex catalyzes a rate-limiting step in photo-fermentative H2 production. The mutants with overexpressed Rnf complex are more competitive than naturally isolated strains under low light condition or in scale-up bioreactors.

Fe3O4 nanoparticles and Rhizobium inoculation enhance nodulation, nitrogen fixation and growth of common bean plants grown in soil

The effects of Fe3O4 nanoparticles (NPs) and Rhizobium inoculation on nodulation, nitrogen fixation and plant growth of common bean (cv. Red Guama, Phaseolus vulgaris) plants were investigated in growth chambers. Plants were exposed to: Fe3O4 NPs (2000 mg/L) (T1), Rhizobium inoculation (T2) and Fe3O4 NPs + Rhizobium inoculation (T3); non-treated plants were considered as controls. Harvested 35-day-old treated plants showed improved symbiotic performance including increased nitrogenase activity (51.2–90.7%), nodule leghaemoglobin (44.8–80.9%) and iron content (83.4–84.2%), number of active nodules per plant (58.7–122%) and nodule dry weight (40.2–70.6%). This resulted in enhanced symbiotic nitrogen fixation,and increased shoot (26.5–50.2%) and root (24.1–48.2%) total nitrogen content in treated plants in comparison with the controls. The best result was obtained using treatment T3. Furthermore, Fe3O4 NPs were taken up by bean plants in treatments T1 and T3, and these accumulated in their organs, including in nodules. All treatments led to an increase in root (51.9–79.8%) and shoot (27.5–52.7%) lengths, in leaf area (10.9–16.8%) and in root (10.1–17.8%), stem (9.8–12.7%) and leaf dry weight (8–17.3%) compared to control plants. Thus applied treatments have the potential to improve common bean plant growth through enhancement of nodulation and nitrogen fixation during vegetative growth.This study also provides strong evidence that the presence Fe3O4 NPs in nodules improves the symbiotic performance between Rhizobium (leguminosarum CF1 strain) and the common bean plant, due to enhanced nodulation and nitrogen fixation.

Enhanced H2 production with efficient N2-fixation by fructose mixotrophically grown Anabaena sp. PCC 7120 strain disrupted in uptake hydrogenase

To explore the potential of a model filamentous cyanobacterium Anabaena sp. PCC 7120 for solar based biohydrogen production, an inactivated uptake hydrogenase ΔHupL strain was used in combination with fructose induced N2-fixing ability of the cells. The results revealed that fructose supports N2-fixing and H2-metabolism in ΔHupL strain. Cells showed an increase in growth rate, pigment content, and the formation of mature heterocysts within 6 h after growing under light-fructose mixotrophic condition. Upon supplementation of optimum fructose concentration at 60 mM, the cells grown for 24 h showed a 3.1-fold increase of nitrogenase activity in conjunction with an increase of nifD expression, with increased electron flow from utilization of fructose through the oxidative pentose phosphate pathway. Nitrogenase rather than bidirectional hydrogenase was likely responsible for the fructose mediated increase of H2-production. The maximum H2-production rate of ΔHupL strain under fructose mixotrophic growth was 101.33 μmolH2 mg chl a−1 h−1, accounting for a 4.7-fold increase compared to wild type under the same condition. Overall results indicated that fructose supplementation enabled Anabaena sp. PCC 7120 strain ΔHupL to increase N2-fixation leading to an enhanced H2-production.

Microbial Nitrogen Fixation in the Intestine of Tipulidae Tipula maxima Larvae

Nitrogen-fixing activity of microorganisms in the intestines of Crane fly (Tipulidae) larvae and their influence on the processes of microbial nitrogen fixation in soil have been studied. Two independent methods have shown high rates of nitrogenase activity in the digestive tract of larvae, which is determined mainly by transit nitrogen-fixing microorganisms. Nitrogen fixers actively developing in the intestines of larvae stimulate a sharp increase in nitrogenase activity in the soil: after 3 months of incubation, the activity increases eightfold; it can contribute to the accumulation of nitrogen in the habitats of the larvae.

CHARACTERISTICS OF THE PROPERTIES OF CHICKPEA NODULE BACTERIA EXTENDED IN THE FOREST-STEPPE AND STEPPE ZONES UKRAINE

Objective. To compare morphological, cultural, physiological, biochemical, and symbiotic properties of strains of nodule chickpea bacteria isolated from different soil and climatic zones of Ukraine. Methods. Microbiological, microscopic, gas-chromatographic, statistical. Results. From the nodules of chickpea plant of varieties Skarb, Admiral, Odissei, Budzhak, Triumf and Pamiat, grown in the steppe zone of Ukraine (Region of Odessa), where an active rhizobia population was formed due to the continuous cultivation of this crop, 58 isolates of bacteria were isolated; 11 iso-lates were isolated from the nodules of plants of Triumf and Pamiat varieties selected in the forest-steppe zone (Region of Lviv), in the fields where chickpea was grown for the first time. It has been established that the strains of Mesorhizobium sp. 1 and Mesorhizobium sp. 2, which were isolated from the nodules of chickpea plants grown in the fields with an active rhizobia population of this culture, form colonies of 2–4 mm in size at day 3–4 on the mannitol yeast agar and, according to cultural and morphological properties correspond to the characteristics of the species Mesorhizo-bium ciceri. Their use for pre-sowing chickpea inoculation increases the mass of nodules (2– 3 times) and nitrogenase activity (1.4–2 times) compared with the results obtained using the pro-duction strain of Mesorhizobium ciceri H-12. Inoculation with Mesorhizobium sp. 11, isolated from the forest-steppe zone, which on a mannitol yeast agar forms a colony of 1–2 mm in size at day 5–6, contributed to a significant increase in the number of nodules on the roots of plants (by 40.5 %), their mass (by 31 %), but the increase in nitrogenase activity was negligible. Conclusion. Strains of Mesorhizobium sp. isolated from chickpea nodules grown in different soil-climatic zones of Ukraine, have similar cell morphology, but differ in growth rate, cultural, physiological, biochemi-cal, and symbiotic properties.

Bacterial IAA-Delivery into Medicago Root Nodules Triggers a Balanced Stimulation of C and N Metabolism Leading to a Biomass Increase.

Indole-3-acetic acid (IAA) is the main auxin acting as a phytohormone in many plant developmental processes. The ability to synthesize IAA is widely associated with plant growth-promoting rhizobacteria (PGPR). Several studies have been published on the potential application of PGPR to improve plant growth through the enhancement of their main metabolic processes. In this study, the IAA-overproducing Ensifer meliloti strain RD64 and its parental strain 1021 were used to inoculate Medicago sativa plants. After verifying that the endogenous biosynthesis of IAA did not lead to genomic changes during the initial phases of the symbiotic process, we analyzed whether the overproduction of bacterial IAA inside root nodules influenced, in a coordinated manner, the activity of the nitrogen-fixing apparatus and the photosynthetic function, which are the two processes playing a key role in legume plant growth and productivity. Higher nitrogen-fixing activity and a greater amount of total nitrogen (N), carbon (C), Rubisco, nitrogen-rich amino acids, soluble sugars, and organic acids were measured for RD64-nodulated plants compared to the plants nodulated by the wild-type strain 1021. Furthermore, the RD64-nodulated plants showed a biomass increase over time, with the highest increment (more than 60%) being reached at six weeks after infection. Our findings show that the RD64-nodulated plants need more substrate derived from photosynthesis to generate the ATP required for their increased nitrogenase activity. This high carbohydrate demand further stimulates the photosynthetic function with the production of molecules that can be used to promote plant growth. We thus speculate that the use of PGPR able to stimulate both C and N metabolism with a balanced C/N ratio represents an efficient strategy to obtain substantial gains in plant productivity.

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner–Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

Ecological and genetic bases for construction of highly effective nitrogen-fixing microbe-plant symbioses

Expression of quantitative traits characterizing the N2-fixing symbiosis of nodule bacteria and leguminous plants is associated with operation of the evolutionary derived polygenic systems controlling the symbiotic efficiency (SE) (impact of inoculation on the plant productivity) and nodulation competitiveness (NC) (formation of nodules by rhizobia under mixed inoculation). Optimization of balance between positive and negative symbiotic regulators aimed at an increase of nitrogenase activity and at a complete allocation of its products into the plant metabolism provides the generation of rhizobia strains with high SE and NC. Inactivation of the negative symbiotic regulators often results in a decreased survival of rhizobia under the edaphic stresses but is responsible for a balanced increase of plant biomass and N accumulation. Improvement of symbiotic activity is to be based on the complementary interactions of microorganisms with the genetically engineered plant cultivars which are able for selection from soil of actively fi xing N2 rhizobia strains and for their preferential multiplication in nodules. Construction of highly effective microbe-plant systems should be based on modifications of mechanisms controlling symbiosis development from the plant and bacterial sides providing the maintenance of N2-fixing zone in nodules and synthesis of NCR proteins activating the bacteroid differentiation.

Mycorrhizae enhance nitrogen fixation and photosynthesis in phosphorus-starved soybean (Glycine max L. Merrill)

Nitrogen (N) and phosphorus (P) are nutrients that frequently limit plant performance and crop yield. Currently, awareness of the importance of plant-microbe interactions in plant nutrient acquisition and growth promotion persist, particularly under stressful conditions, such as those resulting from P scarcity. The aim of the present study was to evaluate the effects of P starvation on the influence of arbuscular mycorrhiza (AM) symbiosis on N2-fixing capacities, plant growth, nutrient uptake and photosynthetic performance of soybean plants at flowering and grain filling stages. To this end, soybean (Glycine max) plants were nodulated with Bradyrhizobium elkanii and inoculated or not with the AM fungus Glomus macrocarpum. The +AM and −AM plants were grown under low P conditions (50μM), and a control treatment with −AM plants under sufficient P concentration (500μM) was also performed (−AM+P). Plants were grown until the beginning of the flowering and another set till the grain filling stage. The results showed that mycorrhization improved soybean plant performance under P scarcity compared with non-AM associated plants under the same conditions. AM positively influenced growth and plant nodulation at both flowering and grain-filling stages, without significant changes in plant P content. AM symbiosis increased nitrogenase activity, P content in nodules and leaf N content, the maximum carboxylation rate of Rubisco and reduced the metabolic limitation of photosynthesis under P starvation, which indicated the AM stimulus to symbiotic N2 fixation (SNF) and photosynthesis. Under these conditions, mycorrhizal symbiosis could not completely meet soybean P demand compared with well-nourished soybean plants, which produced higher plant and nodules biomass. In conclusion, AM association in nodulated soybean plants is likely an important strategy for maintaining SNF functioning, conferring increased plant growth and better N status under P scarcity conditions and significantly attenuating the negative effects of low Pi in nodulated-soybean plants.