Heme catabolism mediated by heme oxygenase in uninfected interstitial cells enables efficient symbiotic nitrogen fixation in Lotus japonicus nodules.

Legume nodules produce large quantities of heme required for the synthesis of leghemoglobin (Lb) and other hemoproteins. Despite the crucial function of Lb in nitrogen fixation and the toxicity of free heme, the mechanisms of heme homeostasis remain elusive. Biochemical, cellular, and genetic approaches were used to study the role of heme oxygenases (HOs) in heme degradation in the model legume Lotus japonicus. Heme and biliverdin were quantified and localized, HOs were characterized, and knockout LORE1 and CRISPR/Cas9 mutants for LjHO1 were generated and phenotyped. We show that LjHO1, but not the LjHO2 isoform, is responsible for heme catabolism in nodules and identify biliverdin as the in vivo product of the enzyme in senescing green nodules. Spatiotemporal expression analysis revealed that LjHO1 expression and biliverdin production are restricted to the plastids of uninfected interstitial cells. The nodules of ho1 mutants showed decreased nitrogen fixation, and the development of brown, rather than green, nodules during senescence. Increased superoxide production was observed in ho1 nodules, underscoring the importance of LjHO1 in antioxidant defense. We conclude that LjHO1 plays an essential role in degradation of Lb heme, uncovering a novel function of nodule plastids and uninfected interstitial cells in nitrogen fixation.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I-Detailed Characteristic of Unusual Apical Senescence.

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.

Using soil nitrogen amendments in mitigating ozone stress in agricultural crops: a case study of cluster beans.

The climate change scenario in the coming years is liable to have serious negative consequences on agricultural productivity. Increasing tropospheric ozone concentration is an important aspect of climate change, which, due to its oxidative nature, is injurious to the plants. Due to the multifarious nature and continuously increasing concentration of tropospheric ozone, it is prerequisite to develop strategies to manage ozone stress in plants. Present study not only evaluates the potential of soil nitrogen amendments in ameliorating ozone stress in plants, but also focuses upon the mechanistic approaches adopted by the different plant cultivars to combat ozone stress. Three doses of nitrogen amendments, recommended (N1), 1.5× recommended (N2) and 2× recommended (N3), were given to two cultivars (S-151 and PUSA-N) of Cymopsis tetragonoloba exposed to ambient ozone stress. Control plants were also maintained in which no nitrogen treatment was given. Nitrogen supplementation reduced the root nodulation frequency and leghaemoglobin content, which subsequently increased the cellular nitrogen metabolism as evident through increase in the activities of nitrate reductase and nitrite reductase in both the test cultivars. The positive effects of nitrogen amendments are clearly evident in the 1D protein profile studies which showed a greater accumulation of larger sub-units of RuBisCO in nitrogen amended plants. The results clearly indicate that N2 treatment effectively enhanced the yield of both the cultivars (84.8% and 76.37%, in S-151 and PUSA-N, respectively); however, the mechanistic approach adopted by the two cultivars was different. Whereas the yield quantity showed higher increments in S-151, the yield quality parameters (carbohydrates and nitrogen contents) responded more positively in PUSA-N.

Role of Nitric Oxide-Derived Metabolites in Reactions of Methylglyoxal with Lysine and Lysine-Rich Protein Leghemoglobin.

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide (•NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other •NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin-leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and •NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology.

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation.

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Expressed Soybean Leghemoglobin: Effect on Escherichia coli at Oxidative and Nitrosative Stress.

Leghemoglobin (Lb) is an oxygen-binding plant hemoglobin of legume nodules, which participates in the symbiotic nitrogen fixation process. Another way to obtain Lb is its expression in bacteria, yeasts, or other organisms. This is promising for both obtaining Lb in the necessary quantity and scrutinizing it in model systems, e.g., its interaction with reactive oxygen (ROS) and nitrogen (RNS) species. The main goal of the work was to study how Lb expression affected the ability of Escherichia coli cells to tolerate oxidative and nitrosative stress. The bacterium E. coli with the embedded gene of soybean leghemoglobin a contains this protein in an active oxygenated state. The interaction of the expressed Lb with oxidative and nitrosative stress inducers (nitrosoglutathione, tert-butyl hydroperoxide, and benzylviologen) was studied by enzymatic methods and spectrophotometry. Lb formed NO complexes with heme-nitrosylLb or nonheme iron-dinitrosyl iron complexes (DNICs). The formation of Lb-bound DNICs was also detected by low-temperature electron paramagnetic resonance spectroscopy. Lb displayed peroxidase activity and catalyzed the reduction of organic peroxides. Despite this, E. coli-synthesized Lb were more sensitive to stress inducers. This might be due to the energy demand required by the Lb synthesis, as an alien protein consumes bacterial resources and thereby decreases adaptive potential of E. coli.

Spectroscopic evidence supporting neutral thiol ligation to ferrous heme iron.

The binding of neutral thiol (ethanethiol, EtSH) or thioether (tetrahydrothiophene, THT) to two types of heme proteins in their ferrous state has been investigated with UV-visible (UV-Vis) absorption and magnetic circular dichroism spectroscopy. For the second GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain from the sensory kinase MsmS (sGAF2), stepwise additions of these respective two sulfur-donor ligands to its dithionite-reduced ferrous form generate homogeneous six-coordinate low-spin ferrous complexes at both pHs 7.0 and 5.4. Similar complexes were partially formed for deoxyferrous soybean leghemoglobin with EtSH or THT within their solubility limits in water. The titrations cause significant UV-Vis spectra changes attributable to a five-coordinate to six-coordinate heme iron coordination change. For sGAF2, the resulting spectra are essentially identical for the both ligands, clearly indicating the direct binding of neutral thiol/thioether to ferrous heme iron as the distal ligand. On the other hand, the thiol EtSH binds to ferric sGAF2 in the anionic thiolate form, while thioether THT forms its ferric sGAF2 complex as a neutral ligand. These observations provide compelling evidence that neutral cysteine is a plausible ligand for ferrous heme proteins.

Safety Evaluation of Soy Leghemoglobin Protein Preparation Derived From Pichia pastoris, Intended for Use as a Flavor Catalyst in Plant-Based Meat.

The leghemoglobin protein (LegH) from soy ( Glycine max) expressed in Pichia pastoris (LegH preparation, LegH Prep) imparts a meat-like flavor profile onto plant-based food products. The safety of LegH Prep was evaluated through a series of in vitro and in vivo tests. The genotoxic potential of LegH Prep was assessed using the bacterial reverse mutation assay (Ames test) and the in vitro chromosome aberration test. LegH Prep was nonmutagenic and nonclastogenic in each test, respectively. Systemic toxicity was assessed in a 28-day dietary study in male and female Sprague Dawley rats. There were no mortalities associated with the administration of LegH Prep. There were no clinical observations, body weight, ophthalmological, clinical pathology, or histopathological changes attributable to LegH Prep administration. There were no observed effects on male reproduction in this study, but the suggestion of a potential estrous cycle distribution effect in female rats prompted a second comprehensive 28-day dietary study in female Sprague Dawley rats. This study demonstrated that female reproductive parameters were comparable between rats treated with LegH Prep and concurrent control rats. These studies establish a no observed adverse effect level of 750 mg/kg/d LegH, which is over 100 times greater than the 90th percentile estimated daily intake. Collectively, the results of the studies presented raise no issues of toxicological concern with regard to LegH Prep under the conditions tested.

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

Molecular dynamics simulations indicate that tyrosineB10 limits motions of distal histidine to regulate CO binding in soybean leghemoglobin.

Myoglobin (Mb) uses strong electrostatic interaction in its distal heme pocket to regulate ligand binding. The mechanism of regulation of ligand binding in soybean leghemoglobin a (Lba) has been enigmatic and more so due to the absence of gaseous ligand bound atomic resolution three-dimensional structure of the plant globin. While the 20-fold higher oxygen affinity of Lba compared with Mb is required for its dual physiological function, the mechanism by which this high affinity is achieved is only emerging. Extensive mutational analysis combined with kinetic and CO-FT-IR spectroscopic investigation led to the hypothesis that Lba depended on weakened electrostatic interaction between distal HisE7 and bound ligand achieved by invoking B10Tyr, which itself hydrogen bonds with HisE7 thus restricting it in a single conformation detrimental to Mb-like strong electrostatic interaction. Such theory has been re-assessed here using CO-Lba in silico model and molecular dynamics simulation. The investigation supports the presence of at least two major conformations of HisE7 in Lba brought about by imidazole ring flip, one of which makes hydrogen bonds effectively with B10Tyr affecting the former's ability to stabilize bound ligand, while the other does not. However, HisE7 in Lba has limited conformational freedom unlike high frequency of imidazole ring flips observed in Mb and in TyrB10Leu mutant of Lba. Thus, it appears that TyrB10 limits the conformational freedom of distal His in Lba, tuning down ligand dissociation rate constant by reducing the strength of hydrogen bonding to bound ligand, which the freedom of distal His of Mb allows.

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 20 days 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 45(th)day 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.

Safety of soy leghemoglobin from genetically modified Komagataella phaffii as a food additive.

The EFSA Panel on Food Additive and Flavourings (FAF Panel) provides a scientific opinion on the safety of soy leghemoglobin from genetically modified Komagataella phaffii as a food additive in accordance with Regulation (EC) No 1331/2008. The proposed food additive, LegH Prep, is intended to be used as a colour in meat analogue products. The yeast Komagataella phaffii strain MXY0541 has been genetically modified to produce soy leghemoglobin; the safety of the genetic modification is under assessment by the EFSA GMO Panel (EFSA-GMO-NL-2019-162). The amount of haem iron provided by soy leghemoglobin from its proposed uses in meat analogue products is comparable to that provided by similar amounts of different types of meat. The exposure to iron from the proposed food additive, both at the mean and 95th percentile exposure, will be below the 'safe levels of intake' established by the NDA Panel for all population groups. Considering that the components of the proposed food additive will be digested to small peptide, amino acids and haem B; the recipient (non GM) strain qualifies for qualified presumption of safety status; no genotoxicity concern has been identified and no adverse effects have been identified at the highest dose tested in the available toxicological studies, the Panel concluded that there was no need to set a numerical acceptable daily intake (ADI) and that the food additive does not raise a safety concern at the proposed use in food category 12.9 and maximum use level. The Panel concluded that the use of soy leghemoglobin from genetically modified Komagataella phaffii MXY0541 as a new food additive does not raise a safety concern at the proposed use and use level. This safety evaluation of the proposed food additive remains provisional subject to the ongoing safety assessment of the genetic modification of the production strain by the GMO Panel (EFSA-GMO-NL-2019-162).

Production and Analytical Aspects of Natural Pigments to Enhance Alternative Meat Product Color.

Color is a major feature that strongly influences the consumer's perception, selection, and acceptance of various foods. An improved understanding regarding bio-safety protocols, health welfare, and the nutritional importance of food colorants has shifted the attention of the scientific community toward natural pigments to replace their toxic synthetic counterparts. However, owing to safety and toxicity concerns, incorporating natural colorants directly from viable sources into plant-based meat (PBM) has many limitations. Nonetheless, over time, safe and cheap extraction techniques have been developed to extract the purified form of coloring agents from raw materials to be incorporated into PBM products. Subsequently, extracted anthocyanin has displayed compounds like Delphinidin-3-mono glucoside (D3G) at 3.1 min and Petunidin-3-mono glucoside (P3G) at 5.1 277, 515, and 546 nm at chromatographic lambda. Fe-pheophytin was successfully generated from chlorophyll through the ion exchange method. Likewise, the optical density (OD) of synthesized leghemoglobin (LegH) indicated that pBHA bacteria grow more rigorously containing ampicillin with a dilution factor of 10 after 1 h of inoculation. The potential LegH sequence was identified at 2500 bp through gel electrophoresis. The color coordinates and absorbance level of natural pigments showed significant differences (p < 0.05) with the control. The development of coloring agents originating from natural sources for PBM can be considered advantageous compared to animal myoglobin in terms of health and functionality. Therefore, the purpose of this study was to produce natural coloring agents for PBM by extracting and developing chlorophyll from spinach, extracting anthocyanins from black beans, and inserting recombinant plasmids into microorganisms to produce LegH.

High-level expression of leghemoglobin in Kluyveromyces marxianus by remodeling the heme metabolism pathway.

Soy leghemoglobin, when bound to heme, imparts a meat-like color and flavor and can serve as a substitute for animal-derived proteins. Enhancing cellular heme synthesis improves the recombinant expression of leghemoglobin in yeast. To achieve high-level expression of leghemoglobin A (LBA) in Kluyveromyces marxianus, a food-safe yeast, large-scale heme synthesis modules were transferred into K. marxianus using yeast artificial chromosomes (KmYACs). These modules contained up to 8 native and heterologous genes to promote the supply of heme precursors and downstream synthesis. Next, eight genes inhibiting heme or LBA synthesis were individually or combinatorially deleted, with the lsc1Δssn3Δ mutant yielding the best results. Subsequently, heme synthesis modules were combined with the lsc1Δssn3Δ mutant. In the resulting strains, the module genes were all actively expressed. Among these module genes, heterologous S. cerevisiae genes in the downstream heme synthesis pathway significantly enhanced the expression of their counterparts in K. marxianus, resulting in high heme content and LBA yield. After optimizing the medium recipe by adjusting the concentrations of glucose, glycine, and FeSO4·7H2O, a heme content of 66.32 mg/L and an intracellular LBA titer of 7.27 g/L were achieved in the engineered strain in a 5 L fermentor. This represents the highest intracellular expression of leghemoglobin in microorganisms to date. The leghemoglobin produced by K. marxianus can be utilized as a safe ingredient for plant-based protein products.

Five questions on how biochemistry can combat climate change.

Global warming is caused by human activity, such as the burning of fossil fuels, which produces high levels of greenhouse gasses. As a consequence, climate change impacts all organisms and the greater ecosystem through changing conditions from weather patterns to the temperature, pH and salt concentrations found in waterways and soil. These environmental changes fundamentally alter many parameters of the living world, from the kinetics of chemical reactions and cellular signaling pathways to the accumulation of unforeseen chemicals in the environment, the appearance and dispersal of new diseases, and the availability of traditional foods. Some organisms adapt to extremes, while others cannot. This article asks five questions that prompt us to consider the foundational knowledge that biochemistry can bring to the table as we meet the challenge of climate change. We approach climate change from the molecular point of view, identifying how cells and organisms - from microbes to plants and animals - respond to changing environmental conditions. To embrace the concept of "one health" for all life on the planet, we argue that we must leverage biochemistry, cell biology, molecular biophysics and genetics to fully understand the impact of climate change on the living world and to bring positive change.

High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis.

Soy leghemoglobin is a key food additive that imparts meaty flavor and color to meat analogs. Here, a Pichia pastoris strain capable of high-yield secretory production of functional leghemoglobin was developed through gene dosage optimization and heme pathway consolidation. First, multi-copy integration of LegH expression cassette was achieved via both post-transformational vector amplification and CRISPR/Cas9 mediated genome editing methods. A combination of inducible expression and constitutive expression resulted in the highest production of leghemoglobin. Then, heme biosynthetic pathway was engineered to address challenges in heme depletion and leghemoglobin secretion. Finally, the disruption of ku70 was complemented in engineered P. pastoris strain to enable high-density fermentation in a 10-L bioreactor. These engineering strategies increased the secretion of leghemoglobin by more than 83-fold, whose maximal leghemoglobin titer and heme binding ratio reached as high as 3.5 g/L and 93 %, respectively. This represents the highest secretory production of heme-containing proteins ever reported.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215-351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Corrigendum: A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

[This corrects the article DOI: 10.3389/fpls.2020.600336.].

Identification and characterization of a recombinant cognate hemoglobin reductase from Synechocystis sp. PCC 6803.

One popular and relevant proposed function for cyanobacterial hemoglobin (Synechocystis Hb) is anaerobic nitrite reductase in vivo. During such reduction reactions, the hexacoordinated heme iron atom of SynHb is oxidized from the ferrous (Fe+2) to ferric (Fe+3) state and prevent damage by limiting the concentration of toxic metabolites such as nitrite. In order to perform these functions in vivo, there must be a mechanism that converts inactive Fe+3-SynHb back to the active Fe+2-SynHb to accomplish the nitrite reductase function. Here, we report a cognate reductase protein for Synechocystis hemoglobin which can reduce the Fe+3-SynHb to Fe+2-SynHb, thus lending a support to the proposed nitrite reductase function. This reductase is also able to reduce pentacoordinate Hbs such as myoglobin but with lower affinity compared to hexacoordinate SynHb. Insilico model of reductase protein-cyanobacterial hemoglobin complex revealed that the heme active site of Hb faces the catalytic center of the reductase protein and several amino acids in the interface interacts non-covalently thus favoring their interaction. Overall, our in vitro study provides the basic foundation for the understanding of the specific molecular mechanism of action and interaction of the SynHb reductase protein, which need to be investigated in further detail.

The Role of Nitric Oxide in Nitrogen Fixation by Legumes.

The legume-rhizobia symbiosis is an important process in agriculture because it allows the biological nitrogen fixation (BNF) which contributes to increasing the levels of nitrogen in the soil. Nitric oxide (⋅NO) is a small free radical molecule having diverse signaling roles in plants. Here we present and discuss evidence showing the role of ⋅NO during different stages of the legume-rhizobia interaction such as recognition, infection, nodule development, and nodule senescence. Although the mechanisms by which ⋅NO modulates this interaction are not fully understood, we discuss potential mechanisms including its interaction with cytokinin, auxin, and abscisic acid signaling pathways. In matures nodules, a more active metabolism of ⋅NO has been reported and both the plant and rhizobia participate in ⋅NO production and scavenging. Although ⋅NO has been shown to induce the expression of genes coding for NITROGENASE, controlling the levels of ⋅NO in mature nodules seems to be crucial as ⋅NO was shown to be a potent inhibitor of NITROGENASE activity, to induce nodule senescence, and reduce nitrogen assimilation. In this sense, LEGHEMOGLOBINS (Lbs) were shown to play an important role in the scavenging of ⋅NO and reactive nitrogen species (RNS), potentially more relevant in senescent nodules. Even though ⋅NO can reduce NITROGENASE activity, most reports have linked ⋅NO to positive effects on BNF. This can relate mainly to the regulation of the spatiotemporal distribution of ⋅NO which favors some effects over others. Another plausible explanation for this observation is that the negative effect of ⋅NO requires its direct interaction with NITROGENASE, whereas the positive effect of ⋅NO is related to its signaling function, which results in an amplifier effect. In the near future, it would be interesting to explore the role of environmental stress-induced ⋅NO in BNF.