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Nickel oxide nanoparticles (NiO-NPs) pose potential threats to agricultural production. Bacillus subtilis has emerged as a stress-mitigating microbe that alleviates the phytotoxicity caused by NiO-NPs. However, the mechanisms underlying its effectiveness, particularly in root-nodule symbiosis and biological N2-fixation (BNF), remain unclear. Here, we tested the combined exposure of NiO-NPs (50 mg kg-1) and B. subtilis on soybean (Glycine max L.) growth and BNF. Combined exposure increased root length, shoot length, root biomass, and shoot biomass by 19% to 26%, while Ni (200 mg kg-1) reduced them by 38% to 53% compared to the control. NiO-NPs at 100 and 200 mg kg-1 significantly (P < 0.05) reduced nodule formation by 16% and 58% and Nitrogen assimilation enzyme activities levels (urease, nitrate reductase, glutamine synthetase, and glutamate synthetase) by 13% to 57%. However, co-exposure with B. subtilis improved nodule formation by 22% to 44%. Co-exposure of NiO-NPs (200 mg kg-1) with B. subtilis increased peroxidase, catalase, and glutathione peroxidase activity levels by 20%, 16%, and 14% while reducing malondialdehyde (14%) and hydrogen peroxide (12%) levels compared to NiO-NPs alone. Additionally, co-exposure of NiO-NPs (100 and 200 mg kg-1) with B. subtilis enhanced the relative abundance of Stenotrophomonas, Gemmatimonas, and B. subtilis, is associated with N2-cycling and N2-fixation potential. This study confirms that B. subtilis effectively mitigates NiO-NP toxicity in soybean, offering a sustainable method to enhance BNF and crop growth and contribute to addressing global food insecurity., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

N-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with soil bacteria, rhizobia. The nodule functioning is still a poorly characterized step of the symbiotic interaction, as only a few of the genes induced in N-fixing nodules have been functionally characterized. We present here the characterization of a member of the Lotus japonicus nitrate transporter1/peptide transporter family, LjNPF8.6 The phenotypic characterization carried out in independent L. japonicus LORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning, as knockout mutants display N-fixation deficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two striking phenotypes: anthocyanin accumulation already displayed 4 weeks after inoculation and shoot biomass deficiency, which is detected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes at both 0.5 and 30 mm external concentrations, and a possible role as a nitrate transporter in the control of N-fixing nodule activity is discussed., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO(2) partial pressure (pCO(2)) with higher N(2) fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO(2) (150 and 900 microatm) and light (50 and 200 micromol photons m(-2) s(-1)) on Trichodesmium IMS101. We expand on a complementary study that demonstrated that while elevated pCO(2) enhanced N(2) fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO(2) and light controlled the operation of the CO(2)-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO(2) and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N(2) fixation and growth at elevated pCO(2) and light. We suggest that changes in the redox state of the photosynthetic electron transport chain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enable Trichodesmium to flourish in future surface oceans characterized by elevated pCO(2), higher temperatures, and high light.

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum (IMS101) showed that increasing CO(2) partial pressure (pCO(2)) enhances N(2) fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO(2), its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses of Trichodesmium IMS101 grown under a matrix of low and high levels of pCO(2) (150 and 900 microatm) and irradiance (50 and 200 micromol photons m(-2) s(-1)). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO(2) and light levels in the cultures. The pCO(2)-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO(2) stimulated rates of N(2) fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO(2). HCO(3)(-) was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO(2) uptake was enhanced under high pCO(2). A comparison between carbon fluxes in vivo and those derived from (13)C fractionation indicates high internal carbon cycling, especially in the low-pCO(2) treatment under high light. Light-dependent oxygen uptake was only detected under low pCO(2) combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO(2) effect on N(2) fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO(2). These findings are supported by a complementary study looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 microM ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant's endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.

Decreased N2 fixation in soybean (Glycine max) L. Merr. during water deficits has been associated with increases in ureides and free amino acids in plant tissues, indicating a potential feedback inhibition by these compounds in response to drought. We evaluated concentrations of ureides and amino acids in leaf and nodule tissue and the concurrent change in N2 fixation in response to exogenous ureides and soil-water treatments for the cultivars Jackson and KS4895. Exogenous ureides applied to the soil and water-deficit treatments inhibited N2 fixation by 85% to 90%. Mn fertilization increased the apparent catabolism of ureides in leaves and hastened the recovery of N2 fixation following exogenous ureide application for both cultivars. Ureides and total free amino acids in leaves and nodules increased during water deficits and coincided with a decline in N2 fixation for both cultivars. N2 fixation recovered to 74% to 90% of control levels 2 d after rewatering drought-stressed plants, but leaf ureides and total nodule amino acids remained elevated in KS4895. Asparagine accounted for 82% of the increase in nodule amino acids relative to well-watered plants at 2 d after rewatering. These results indicate that leaf ureides and nodule asparagine do not feedback inhibit N2 fixation. Compounds whose increase and decrease in concentration mirrored the decline and recovery of N2 fixation included nodule ureides, nodule aspartate, and several amino acids in leaves, indicating that these are potential candidate molecules for feedback inhibition of N2 fixation.

In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation. Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants. We have identified three symbiotically regulated plant genes encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor. The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes. The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M. truncatula by S. meliloti.