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Bio-fertilisation with plant growth promoting rhizobacteria (PGPR) can be extremely beneficial for plant development and growth under harsh environments. PGPR have been recently successfully applied in restoration programmes, but locally adapted strains are needed for successful outcomes. In this study, we evaluated the effect of bio-fertilisation with selected native PGPR strains on the growth and physiological response to drought of Caesalpinia spinosa (Mol.) Kuntze (tara), the main tree species of the Atiquipa forest, a highly valuable and unique ecosystem in the middle of the Atacama Desert in Peru. We compared the growth and physiological status of tara seedlings that were bio-fertilised with three PGPR strains, chemically fertilised and a non-fertilised control, under well-watered and under drought conditions. Seedlings inoculated with Pseudomonas sp. strain RC5.5 showed enhanced tolerance to drought, although under favourable, stress-free conditions it did not promote growth and only significantly increased quantum yield of photosystem II (Fv/Fm). On the other hand, chemical fertilisation highly enhanced plant growth, but led to substantial hydric demand under water limitation. Under drought, RC5.5 bio-fertilised plants showed lower reduction of leaf relative water content, net carbon assimilation rate, stomatal conductance, and a balanced shoot-to-root ratio compared with control plants. Therefore, strain RC5.5 is highlighted as a valuable candidate to be used as inoculant of tara in reforestation and restoration programmes in arid zones, in particular the Atiquipa forest, or in local tara plantations.

The rhizosheath is a belowground area that acts as a communication hub at the root–soil interface to promote water and nutrient acquisition. Certain crops, such as white lupin (Lupinus albus), acquire large amounts of phosphorus (P), owing partially to exudation of acid phosphatases (APases). Plant growth-promoting rhizobacteria also increase soil P availability. However, potential synergistic effects of root APases and rhizosheath-associated microbiota on P acquisition require further research. In this study, we investigated the roles of root purple APases (PAPs) and plant growth-promoting rhizobacteria in rhizosheath formation and P acquisition under conditions of soil drying (SD) and P treatment (+P: soil with P fertilizer; –P: soil without fertilizer). We expressed purple acid phosphatase12 (LaPAP12) in white lupin and rice (Oryza sativa) plants and analyzed the rhizosheath-associated microbiome. Increased or heterologous LaPAP12 expression promoted APase activity and rhizosheath formation, resulting in increased P acquisition mainly under SD–P conditions. It also increased the abundance of members of the genus Bacillus in the rhizosheath-associated microbial communities of white lupin and rice. We isolated a phosphate-solubilizing, auxin-producing Bacillus megaterium strain from the rhizosheath of white lupin and used this to inoculate white lupin and rice plants. Inoculation promoted rhizosheath formation and P acquisition, especially in plants with increased LaPAP12 expression and under SD–P conditions, suggesting a functional role of the bacteria in alleviating P deficit stress via rhizosheath formation. Together, our results suggest a synergistic enhancing effect of LaPAP12 and plant growth-promoting rhizobacteria on rhizosheath formation and P acquisition under SD–P conditions.

The maintenance of intracellular nitrogen-fixing bacteria causes changes in proteins’ location and in gene expression that may be detrimental to the host cell fitness. We hypothesized that the nodule’s high vulnerability toward salt stress might be due to alterations in mechanisms involved in the exclusion of Na+ from the host cytoplasm. Confocal and electron microscopy immunolocalization analyses of Na+/K+ exchangers in the root nodule showed the plasma membrane (MtNHX7) and endosome/tonoplast (MtNHX6) signal in non-infected cells; however, in mature infected cells the proteins were depleted from their target membranes and expelled to vacuoles. This mistargeting suggests partial loss of the exchanger’s functionality in these cells. In the mature part of the nodule 7 of the 20 genes encoding ion transporters, channels, and Na+/K+ exchangers were either not expressed or substantially downregulated. In nodules from plants subjected to salt treatments, low temperature-scanning electron microscopy and X-ray microanalysis revealed the accumulation of 5–6 times more Na+ per infected cell versus non-infected one. Hence, the infected cells’ inability to withstand the salt may be the integral result of preexisting defects in the localization of proteins involved in Na+ exclusion and the reduced expression of key genes of ion homeostasis, resulting in premature senescence and termination of symbiosis.

Canola is an important temperate oil crop that can be severely affected by drought. Understanding the physiological and molecular mechanisms involved in canola tolerance to water deficit is essential to obtain drought-tolerant productive cultivars. To investigate the role of antioxidant response and the possible involvement of calcium-dependent protein kinases (CDPKs) in canola tolerance to drought, we analyzed four genotypes with different sensitivity to water stress. Leaf relative water content, canopy temperature, PSII efficiency, electrolyte leakage index and lipid peroxidation were used as indicators to classify the cultivars as drought-tolerant or drought-sensitive. Antioxidant enzymes superoxide dismutase, guaiacol peroxidase and catalase displayed significantly higher activities in drought-tolerant than in drought-sensitive cultivars subjected to water deficit, suggesting that the efficiency of the antioxidant response is essential in canola drought tolerance. The increased expression of genes BnaCDPK6 and BnaCDPK14 under drought conditions, their differential expression in drought-tolerant and drought-sensitive genotypes, and the presence of multiple cis-acting stress-related elements in their promoter regions suggest that CDPKs are part of the signaling pathways that regulate drought response in canola. We propose the BnaCDPK genes and their regulator elements as potential molecular targets to obtain drought-tolerant productive canola cultivars through breeding or genetic transformation.

In recent years, salinity-induced soil quality impairment and the misuse of management practices have led to the reduced productivity of agroecosystems. This has prompted a search for simple and effective agricultural management strategies that improve the sustainability of agricultural production through soil quality assessments. In this context, the objective of this study was to establish an integrated soil quality index (SQI) by assessing the influence of different types of abiotic stress in two different seasons, using physical, chemical and biological indicators at three sites in the geothermal zone of “Los Negritos”, Michoacán, Mexico. Thirty-nine indicators related to soil fertility attributes and C, N, P, and S cycling—identified as the total dataset (TDS)—were evaluated. Principal component analysis (PCA) and the Spearman correlation matrix (r2 ≥ 0.6) were used to calculate the SQI using an integrated quality index (IQI) equation, with the indicators total nitrogen (TN), cation exchange capacity (CEC), lithium (Li), and zinc (Zn) identified as the minimum dataset (MDS). Significantly higher SQI values related to the better performance of soil functions were detected during the rainy season.

Lupin is a high-protein legume crop that grows in a wide range of edaphoclimatic conditions where other crops are not viable. Its unique seed nutrient profile can promote health benefits, and it has been proposed as a phytoremediation plant. Most rhizobia nodulating Lupinus species belong to the genus Bradyrhizobium, comprising strains that are phylogenetically related to B. cytisi, B. hipponenese, B. rifense, B. iriomotense/B. stylosanthis, B. diazoefficiens, B. japonicum, B. canariense/B. lupini, and B. retamae/B. valentinum. Lupins are also nodulated by fast-growing bacteria within the genera Microvirga, Ochrobactrum, Devosia, Phyllobacterium, Agrobacterium, Rhizobium, and Neorhizobium. Phylogenetic analyses of the nod and nif genes, involved in microbial colonization and symbiotic nitrogen fixation, respectively, suggest that fast-growing lupin-nodulating bacteria have acquired their symbiotic genes from rhizobial genera other than Bradyrhizobium. Horizontal transfer represents a key mechanism allowing lupin to form symbioses with bacteria that were previously considered as non-symbiotic or unable to nodulate lupin, which might favor lupin’s adaptation to specific habitats. The characterization of yet-unstudied Lupinus species, including microsymbiont whole genome analyses, will most likely expand and modify the current lupin microsymbiont taxonomy, and provide additional knowledge that might help to further increase lupin’s adaptability to marginal soils and climates.

Nodule endophytes and associated bacteria are non-symbiotic bacteria that colonize legume nodules. They accompany nodulating rhizobia and can form beneficial associations, as some of them are plant growth-promoting rhizobacteria (PGPR) that are able to promote germination and plant growth and increase tolerance to biotic and abiotic stress. White lupin (Lupinus albus) is a legume crop that is gaining relevance as a suitable alternative to soybean as a plant protein source. Eleven nodule-associated bacteria were isolated from white lupin nodules grown in a Tunisian soil. They belonged to the genera Rhizobium, Ensifer, Pseudomonas and Bacillus. Their plant growth-promoting (PGP) and enzymatic activities were tested in vitro. Strains Pseudomonas sp., L1 and L12, displayed most PGP activities tested, and were selected for in planta assays. Inoculation with strains L1 or L12 increased seed germination and had the same positive effects on all plant growth parameters as did inoculation with symbiotic Bradyrhizobium canariense, with no significant differences among treatments. Inoculation with efficient nitrogen-fixing rhizobia must compete with rhizobia present in the soil that sometimes nodulate efficiently but fix nitrogen poorly, leading to a low response to inoculation. In such cases, inoculation with highly effective PGPR might represent a feasible alternative to boost crop productivity.

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selecti

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selecti

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selecti

Mercury (Hg) contamination is increasing worldwide in both wild ecosystems and agricultural soils due to natural processes, but mostly to anthropic activities. The molecular mechanisms involved in Hg toxicity and tolerance in plants have been extensively studied; however, the role of flavonoids in response to Hg stress remains to be investigated. We conducted a metabolomic study to analyze the changes induced at the secondary metabolite level in three Hg-tolerant and one Hg-sensitive Medicago truncatula cultivars. A total of 46 flavonoid compounds, classified into five different flavonoid families: anthocyanidins, flavones, isoflavones, pterocarpan flavonoids, and flavanones, along with their respective glycoconjugate derivatives, were identified in leaf and root tissues. The synthesis of free isoflavones, followed by monoglycosylation and further malonylation was shown to be characteristic of root samples, whereas higher glycosylation, followed by further acylation with coumaric and ferulic acid was characteristic of leaf tissues. While minor changes were observed in leaves, significant quantitative changes could be observed in roots upon Hg treatment. Some flavonoids were strongly upregulated in roots, including malonylglucosides of biochanin A, formononetin and medicarpin, and aglycones biochanin, daidzein, and irisolidone. Hg tolerance appeared to be mainly associated to the accumulation of formononetin MalGlc, tricin GlcAGlcA, and afrormosin Glc II in leaves, whereas aglycone accumulation was associated with tolerance to Hg stress in roots. The results evidence the alteration of the flavonoid metabolic profile and their glycosylation processes in response to Hg stress. However, notable differences existed between varieties, both in the basal metabolic profile and in the response to treatment with Hg. Overall, we observed an increase in flavonoid production in response to Hg stress, and Hg tolerance appeared to be associated to a characteristic glycosylation pat

Nitrogen (N) and phosphorus (P) are two major plant nutrients, and their deficiencies often limit plant growth and crop yield. The uptakes of N or P affect each other, and consequently, understanding N–P interactions is fundamental. Their signaling mechanisms have been studied mostly separately, and integrating N–P interactive regulation is becoming the aim of some recent works. Lupins are singular plants, as, under N and P deficiencies, they are capable to develop new organs, the N2-fixing symbiotic nodules, and some species can also transform their root architecture to form cluster roots, hundreds of short rootlets that alter their metabolism to induce a high-affinity P transport system and enhance synthesis and secretion of organic acids, flavonoids, proteases, acid phosphatases, and proton efflux. These modifications lead to mobilization in the soil of, otherwise unavailable, P. White lupin (Lupinus albus) represents a model plant to study cluster roots and for understanding plant acclimation to nutrient deficiency. It tolerates simultaneous P and N deficiencies and also enhances uptake of additional nutrients. Here, we present the structural and functional modifications that occur in conditions of P and N deficiencies and lead to the organogenesis and altered metabolism of nodules and cluster roots. Some known N and P signaling mechanisms include different factors, including phytohormones and miRNAs. The combination of the individual N and P mechanisms uncovers interactive regulation pathways that concur in nodules and cluster roots. L. albus interlinks N and P recycling processes both in the plant itself and in nature.

Almost half of the world’s agricultural soils are acidic, and most of them present significant levels of aluminum (Al) contamination, with Al3+ as the prevailing phytotoxic species. Lupin is a protein crop that is considered as an optimal alternative to soybean cultivation in cold climates. Lupins establish symbiosis with certain soil bacteria, collectively known as rhizobia, which are capable of fixing atmospheric nitrogen. Moreover, some lupin species, especially white lupin, form cluster roots, bottlebrush-like structures specialized in the mobilization and uptake of nutrients in poor soils. Cluster roots are also induced by Al toxicity. They exude phenolic compounds and organic acids that chelate Al to form non-phytotoxic complexes in the rhizosphere and inside the root cells, where Al complexes are accumulated in the vacuole. Lupins flourish in highly acidic soils where most crops, including other legumes, are unable to grow. Some lupin response mechanisms to Al toxicity are common to other plants, but lupin presents specific tolerance mechanisms, partly as a result of the formation of cluster roots. Al-induced lupin organic acid secretion differs from P-induced secretion, and organic acid transporters functions differ from those in other legumes. Additionally, symbiotic rhizobia can contribute to Al detoxification. After revising the existing knowledge on lupin distinct Al tolerance mechanisms, we conclude that further research is required to elucidate the specific organic acid secretion and Al accumulation mechanisms in this unique legume, but definitely, white lupin arises as a choice crop for cultivation in Al-rich acidic soils in temperate climate regions.

Salt stress has become a major menace to plant growth and productivity. The main goal of this study was to investigate the effect of inoculation with the arbuscular mycorrhizal fungi (AMF; Rhizophagus intraradices) in combination or not with plant growth‐promoting rhizobacteria (PGPR; Pseudomonas sp. (Ps) and Bacillus subtilis) on the establishment and growth of Sulla coronaria plants under saline conditions. Pot experiments were conducted in a greenhouse and S. coronaria seedlings were stressed with NaCl (100 mM) for 4 weeks. Plant biomass, mineral nutrition of shoots and activities of rhizosphere soil enzymes were assessed. Salt stress significantly reduced plant growth while increasing sodium accumulation and electrolyte leakage from leaves. However, inoculation with AMF, whether alone or combined with the PGPR Pseudomonas sp. alleviated the salt‐induced reduction of dry weight. Inoculation with only AMF increased shoot nutrient concentrations resulting in higher K+: Na+, Ca2+: Na+, and Ca2+: Mg2+ ratios compared to the non‐inoculated plants under saline conditions. The co‐inoculation with AMF and Pseudomonas sp. under saline conditions lowered shoot sodium accumulation, electrolyte leakage and malondialdehyde (MDA) levels compared to non‐inoculated plants and plants inoculated only with AMF. The findings strongly suggest that inoculation with AMF alone or co‐inoculation with AMF and Pseudomonas sp. can alleviate salt stress of plants likely through mitigation of NaCl‐induced ionic imbalance, thereby improving the nutrient profile.

The symbiotic association between certain soil bacteria (rhizobia) and most legumes leads to the formation of root nodules, symbiotic organs in which the fixation of atmospheric nitrogen takes place. This symbiosis is significantly important for the nitrogen input in both agricultural and natural ecosystems, but it can be affected by environmental stresses. Nodulation is a complex process not completely understood; some features differ depending on the legume, and they lead to distinct nodule types. After a specific recognition between the symbionts through the exchange of bacterial and plant signalling molecules, the nodule primordium is induced and rhizobia enter the root cortex and infect cells of this primordium. Bacteria are released into the host cell surrounded by a plantderived membrane and form a new cellular compartment, the symbiosome, which is a nitrogen-fixing microfactory. Based on several characteristics of the symbiosome including its metabolic behaviour, the composite origin of the membrane, the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells in certain legume nodules, symbiosome can be considered an organelle-like 1 . Legume nodules are probably one of the most fascinating plant organs. They have been studied for more than a century, and microscopy techniques have been and will be key tools to enhance our understanding of this symbiosis. We will present examples of how microscopy techniques (light, fluorescence, laser confocal, TEM, LTSEM and X-Ray microanalysis, etc.) have been essential to elucidate different aspects of nodule development, structure and functioning, as well as its response to abiotic stress.

Pollution of soils with toxic heavy metals is increasing over the world due mainly to anthropogenic activities. Natural soils containing high levels of heavy metals also exist. In polluted soils, plant production for food and feed is a risk for human and animal health. To overcome this problem two strategies have been proposed: soil bioremediation with plants and their associated microorganisms, and culture of plants with low capability to translocate heavy metals to the edible parts. Legumes are promising candidates for these proposals; in fact, legumes inoculated with rhizobia isolated from heavy metal-polluted soils have been successfully used in polluted soils. Plants and microorganisms display mechanisms of response to toxic heavy metal stress, which included uptake, translocation and accumulation of the metal to avoid toxic effects of the metals. Structural and ultrastructural alterations of cells and tissues are also responses to heavy metals. In bacteria and plants upon heavy metal stress, common responses to another abiotic stresses (imbalance of the redox homeostasis, hormonal alteration, etc.) and more specific responses (transporters, detoxification by proteins and enzymes, etc.) can be found. Studies of transcriptomic, proteomic, metabolomic, ionomic, etc. are revealing new factors involved in these responses. These new approaches might help to draw the network of molecular, biochemical and physiological changes that allow the maintenance of the cellular homeostasis and the metabolic reprogramming. So far, the responses of legumes, rhizobia and their symbiosis to toxic heavy metals have not been extensively studied. A better knowledge of these responses will allow the selection of more tolerant rhizobialegume partners. We will present an overview of the responses of legumes and their microsymbionts to heavy metals, mainly focused on cadmium and mercury. We will present and discuss our results on Medicago, Lupinus, Ensifer and Bradyrhizobium and on their

En nuestro grupo se está estudiando el potencial fitorremediador de suelos contaminados por metales pesados de la simbiosis leguminosa-rizobio. Se ha demostrado, en cultivo semi-hidropónico y concentraciones entre 0 y 200 ¿M de HgCl2 en la solución de riego, que la inoculación de plantas de altramuz (L. albus) con la cepa L-7AH de Bradyrhizobium canariense, tolerante al mercurio, confiere a las plantas la capacidad de desarrollarse en presencia de este metal pesado y acumularlo en altas concentraciones, especialmente en raíces y nódulos. Crecimiento, eficiencia fotosintética y actividad nitrogenasa de las plantas inoculadas con L-7AH eran similares a los de las plantas control (sin Hg), mientras que en las inoculadas con la cepa sensible, L-3, todos los parámetros disminuían significativamente y apenas acumulaban Hg. Estos resultados sugerían que la simbiosis L. albus-B. canariense L-7AH podría ser útil para la fitorremediación, mediante fitoestabilización, de suelos contaminados por Hg. Para evaluar la capacidad fitorremediadora de estas plantas, se utilizaron suelos de las localidades de Almadenejos y Las Cuevas (Ciudad Real, España) con diferentes contenidos totales en Hg (22 y 2.6 g kg-1, respectivamente), que se mezclaron con vermiculita (1:1) para evitar su compactación. Se sembraron semillas de altramuz de las variedades G1 y N1 y se inocularon con B. canariense L-7AH, o L-3, o no se inocularon. Tras 6 semanas de riego con agua estéril, todas las plantas mostraban un aspecto similar. Se midieron parámetros de crecimiento, fotosíntesis, actividades enzimáticas del metabolismo antioxidante y nitrogenasa, sin encontrar diferencias significativas. El análisis físico-químico de los suelos, sin embargo, mostró que éstos presentaban una gran diferencia en su pH, siendo 6.9 el de Almadenejos y 5.1 el de Las Cuevas, lo cual afecta al porcentaje de Hg ligado a ácidos húmicos que es del 2.1% y del 13.1%, respectivamente. En esas condiciones, la concentración de Hg biodi

© 2015 Lucas, Stoddard, Annicchiarico, Frías, Martínez-Villaluenga, Sussmann, Duranti, Seger, Zander and Pueyo. Europe has become heavily dependent on soya bean imports, entailing trade agreements and quality standards that do not satisfy the European citizen’s expectations. White, yellow, and narrow-leafed lupins are native European legumes that can become true alternatives to soya bean, given their elevated and high-quality protein content, potential health benefits, suitability for sustainable production, and acceptability to consumers. Nevertheless, lupin cultivation in Europe remains largely insufficient to guarantee a steady supply to the food industry, which in turn must innovate to produce attractive lupin-based protein-rich foods. Here, we address different aspects of the food supply chain that should be considered for lupin exploitation as a high-value protein source. Advanced breeding techniques are needed to provide new lupin varieties for socio-economically and environmentally sustainable cultivation. Novel processes should be optimized to obtain high-quality safe lupin protein ingredients, and marketable foods need to be developed and offered to consumers. With such an integrated strategy, lupins can be established as an alternative protein crop, capable of promoting socio-economic growth and environmental benefits in Europe.

[Background] Denitrification is defined as the dissimilatory reduction of nitrate or nitrite to nitric oxide (NO), nitrous oxide (N2O), or dinitrogen gas (N2). N2O is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Legume crops might contribute to N2O production by providing nitrogen-rich residues for decomposition or by associating with rhizobia that are able to denitrify under free-living and symbiotic conditions. However, there are limited direct empirical data concerning N2O production by endosymbiotic bacteria associated with legume crops. Analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD and nosRZDFYLX denitrification genes. It was recently reported that this bacterium is able to grow using nitrate respiration when cells are incubated with an initial O2 concentration of 2%; however, these cells were unable to use nitrate respiration when initially incubated anoxically. The involvement of the nap, nirK, nor and nos genes in E. meliloti denitrification has not been reported., [Results] E. meliloti nap, nirK and norC mutant strains exhibited defects in their ability to grow using nitrate as a respiratory substrate. However, E meliloti nosZ was not essential for growth under these conditions. The E. meliloti napA, nirK, norC and nosZ genes encode corresponding nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. The NorC component of the E. meliloti nitric oxide reductase has been identified as a c-type cytochrome that is 16 kDa in size. Herein, we also show that maximal expression of the E. meliloti napA, nirK, norC and nosZ genes occurred when cells were initially incubated anoxically with nitrate., [Conclusion] The E. meliloti napA, nirK, norC and nosZ genes are involved in nitrate respiration and in the expression of denitrification enzymes in this bacterium. Our findings expand the short list of rhizobia for which denitrification gene function has been demonstrated. The inability of E. meliloti to grow when cells are initially subjected to anoxic conditions is not attributable to defects in the expression of the napA, nirK, norC and nosZ denitrification genes.