Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Microbial Colonization of the Host Plant: Cellular and Molecular Mechanisms of Symbiosis.

Nitrogen is an essential element for all plants, animals, and microorganisms in the Earth's biosphere [...].

Root acid phosphatases and rhizobacteria synergistically enhance white lupin and rice phosphorus acquisition.

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.

Lupin, a Unique Legume That Is Nodulated by Multiple Microsymbionts: The Role of Horizontal Gene Transfer.

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.

Sodium Accumulation in Infected Cells and Ion Transporters Mistargeting in Nodules of Medicago truncatula: Two Ugly Items That Hinder Coping with Salt Stress Effects.

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.

Genetic basis for denitrification in Ensifer meliloti.

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.

The Beneficial Effects of Inoculation with Selected Nodule-Associated PGPR on White Lupin Are Comparable to Those of Inoculation with Symbiotic Rhizobia.

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.

Effects of Inoculation with Stress-Tolerant Rhizobia on the Response of Alfalfa (Medicago sativa L.) to Combined Salinity and Cadmium Stress.

Agricultural soil salinization, which is often combined with heavy-metal contamination, is an ever-growing problem in the current era of global change. Legumes have a high potential for nitrogen fixation and are ideal crops for the reclamation of degraded soils. Alfalfa (Medicago sativa) is a valuable forage crop cultivated worldwide. Alfalfa plants fertilized with nitrogen or inoculated with a salt- and cadmium-tolerant Sinorhizobium meliloti strain were subjected to combined NaCl and CdCl2 stresses. Our results showed that inoculated plants presented higher aerial biomass than nitrogen-fertilized plants when they were exposed to salinity and cadmium together. To assess the mechanisms involved in the plant response to the combined stresses, superoxide dismutase and catalase antioxidant enzymatic activities were determined. Both increased upon stress; however, the increase in catalase activity was significantly less marked for inoculated plants, suggesting that other tolerance mechanisms might be active. Cd accumulation was lower in inoculated plants than in fertilized plants, which appears to imply that inoculation somehow prevented cadmium uptake by the plant roots. Expression analyses of several involved genes suggested that inoculation stimulated the biosynthesis of proline, phytochelatins, and homophytochelatins, together indicating that inoculated plants might be better suited to withstand combined salinity and cadmium stress effects.

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress.

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate-glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.

Flavonoid Accumulation Varies in Medicago truncatula in Response to Mercury Stress.

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 pattern in roots, associated with the accumulation of aglycones and monoglycosylated flavonoids. The findings are discussed in the context of the flavonoid biosynthetic pathway to provide a better understanding of the role of these secondary metabolites in the response and tolerance to Hg stress in M. truncatula.

Transgenic Medicago truncatula Plants That Accumulate Proline Display Enhanced Tolerance to Cadmium Stress.

Cadmium (Cd) accumulation in agricultural soils constitutes a serious problem for crop yields and food safety. It is known that proline (Pro) can rapidly accumulate in plant tissues in response to abiotic stress. To analyze the potential protective effect of Pro accumulation against Cd toxicity, we compared the response to Cd stress of wild-type (WT) Medicago truncatula and a transgenic line that we had previously obtained and characterized (p18), which expressed the Δ 1-pyrroline-5-carboxylate synthetase gene from Vigna aconitifolia (VaP5CS), and accumulated high Pro levels. Cadmium significantly reduced germination of WT seeds compared to p18 seeds, and seedling relative root growth, a valid indicator of metal tolerance, was significantly higher for p18 than WT seedlings. We analyzed the relative expression of genes related to Pro metabolism, phytochelatin biosynthesis. antioxidant machinery, and NADPH recycling, which are relevant mechanisms in the response to Cd stress. They presented differential expression in the seedlings of both genotypes both under control conditions and under Cd stress, suggesting that the Cd response mechanisms might be constitutively activated in the transgenic line. Pro accumulation promoted higher survival, enhanced growth performance, and minor nutrient imbalance in transgenic p18 plants compared to WT plants. These facts, together with the recorded gluthatione levels, lipid peroxidation and antioxidant enzyme activities strongly suggested that VaP5CS expression and Pro accumulation conferred enhanced Cd tolerance to M. truncatula p18 plants, which was likely mediated by changes in Pro metabolism, increased phytochelatin biosynthesis and a more efficient antioxidant response. Moreover, p18 roots accumulated significantly higher Cd amounts than WT roots, while Cd translocation to the aerial part was similar to WT plants, thus suggesting that high Pro levels increased not only Cd tolerance, but also Cd phytostabilization by rhizosequestration.

Adaptive Mechanisms Make Lupin a Choice Crop for Acidic Soils Affected by Aluminum Toxicity.

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.

Local adaptation optimizes photoprotection strategies in a Neotropical legume tree under drought stress.

Photoprotection is a plant functional mechanism to prevent photooxidative damage by excess light. This is most important when carbon assimilation is limited by drought, and as such, it entails a trade-off between carbon assimilation vs stress avoidance. The ecological adaptation of plants to local water availability can lead to different photoprotective strategies. To test this, we used different provenances of Caesalpinia spinosa (Mol.) Kuntze (commonly known as 'tara') along a precipitation gradient. Tara is a Neotropical legume tree with high ecological and commercial value, found in dry tropical forests, which are increasingly threatened by climate change. Morphological and physiological responses of tara provenances were analysed under three different treatments of drought and leaflet immobilization, i.e., light stress, in a common garden greenhouse experiment. Tara quickly responded to drought by reducing stomatal conductance, evapotranspiration, photochemical efficiency, carbon assimilation and growth, while increasing structural and chemical photoprotection (leaflet angle and pigments for thermal dissipation). Leaflet closure was an efficient photoprotection strategy with overall physiological benefits for seedlings as it diminished the evaporative demand and avoided photodamage, but also entailed costs by reducing net carbon assimilation opportunities. These responses depended on seed origin, with seedlings from the most xeric locations showing the highest dehydration tolerance, suggesting local adaptation and highlighting the value of different strategies under distinct environments. This plasticity in its response to environmental stress allows tara to thrive in locations with contrasting water availability. Our findings increase the understanding of the factors controlling the functional ecology of tara in response to drought, which can be leveraged to improve forecasts of changes in its distribution range, and for planning restoration projects with this keystone tree species.

Cadmium-Tolerant and -Sensitive Cultivars Identified by Screening of Medicago truncatula Germplasm Display Contrasting Responses to Cadmium Stress.

Cadmium (Cd) pollution in soils is an increasing problem worldwide, and it affects crop production and safety. We identified Cd-tolerant and -sensitive cultivars by testing 258 accessions of Medicago truncatula at seedling stage, using the relative root growth (RRG) as an indicator of Cd tolerance. The factorial analysis (principal component analysis method) of the different growth parameters analyzed revealed a clear differentiation between accessions depending on the trait (tolerant or sensitive). We obtained a normalized index of Cd tolerance, which further supported the suitability of RRG to assess Cd tolerance at seedling stage. Cd and elements contents were analyzed, but no correlations with the tolerance trait were found. The responses to Cd stress of two accessions which had similar growth in the absence of Cd, different sensitivity to the metal but similar Cd accumulation capacity, were analyzed during germination, seedling stage, and in mature plants. The results showed that the Cd-tolerant accession (CdT) displayed a higher tolerance than the sensitive cultivar (CdS) in all the studied stages. The increased gene expression of the three main NADPH recycling enzymes in CdT might be key for this tolerance. In CdS, Cd stress produced strong expression of most of the genes that encode enzymes involved in glutathione and phytochelatin biosynthesis (MtCYS, MtγECS, and MtGSHS), as well as GR, but it was not enough to avoid a redox status imbalance and oxidative damages. Our results on gene expression, enzyme activity, antioxidant content, and lipid peroxidation indicate different strategies to cope with Cd stress between CdS and CdT, and provide new insights on Cd tolerance and Cd toxicity mechanisms in M. truncatula.

Nitrogen and Phosphorus Interplay in Lupin Root Nodules and Cluster Roots.

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.

Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula.

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 selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin.

Several recent studies have demonstrated that the expression of a cyanobacterial flavodoxin in plants can provide tolerance to a wide range of environmental stresses. Indeed, this strategy has been proposed as a potentially powerful biotechnological tool to generate multiple-tolerant crops. To determine whether flavodoxin expression specifically increased tolerance to salt stress and whether it might also preserve legume nitrogen fixation under saline conditions, the flavodoxin gene was introduced into the model legume Medicago truncatula. Expression of flavodoxin did not confer saline tolerance to the whole plant, although the sensitive nitrogen-fixing activity was maintained under salt stress in flavodoxin-expressing plants. Our results indicate that flavodoxin induced small but significant changes in the enzymatic activities involved in the nodule redox balance that might be responsible for the positive effect on nitrogen fixation. Expression of flavodoxin can be regarded as a potential tool to improve legume symbiotic performance under salt stress, and possibly other environmental stresses.

Mercury-Tolerant Ensifer medicae Strains Display High Mercuric Reductase Activity and a Protective Effect on Nitrogen Fixation in Medicago truncatula Nodules Under Mercury Stress.

Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.

Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of peanut (Arachis hypogaea L.) grown in Moroccan soils.

The phenotypic and genotypic characterization of sixty-two rhizobial isolates obtained from nodules of Arachis hypogaea in north-western Morocco was performed. Their physiological and biochemical properties revealed a great deal of diversity among them. Isolates were classified into two major groups based on the numerical analysis of their phenotypic and genotypic characteristics. Isolates in the first group were alkali- and salt-sensitive, slow or extra-slow growers; they did not use disaccharides as carbon source and varied in the use of amino acids. ARDRA analysis of the 16S rDNA region grouped them together with reference strains belonging to the genus Bradyrhizobium. In the second group, isolates were fast growers, acid-sensitive, and alkali- and salt-tolerant; they used both mono and disaccharides as carbon sources, and methionine was the only amino acid they could metabolize as a nitrogen source. ARDRA analysis grouped them with fast-growing reference strains. Both groups exhibited a range of variability in tolerance to heavy metals. The Intergenic Spacer (IGS)-PCR fingerprinting analysis confirmed a high genotypic diversity at the strain level. This characterization provides a basis for the selection of peanut-nodulating rhizobia which may have applications in formulating appropriate inocula for improving peanut crop yield on Moroccan soils, including saline and acidic marginal areas.