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.

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.

Medicago truncatula natural resistance-associated macrophage Protein1 is required for iron uptake by rhizobia-infected nodule cells.

Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

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.

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.

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.

A light and electron microscopy analysis of the events leading to male sterility in Ogu-INRA CMS of rapeseed (Brassica napus).

Ogura cytoplasmic male sterility (CMS) occurs naturally in radish and has been introduced into rapeseed (Brassica napus) by protoplast fusion. As with all CMS systems, it involves a constitutively expressed mitochondrial gene which induces male sterility to otherwise hermaphroditic plants (so they become females) and a nuclear gene named restorer of fertility that restores pollen production in plants carrying a sterility-inducing cytoplasm. A correlative approach using light and electron microscopy was applied to define what stages throughout development were affected and the subcellular events leading to the abortion of the developing pollen grains upon the expression of the mitochondrial protein. Three central stages of development (tetrad, mid-microspore and vacuolate microspore) were compared between fertile, restored, and sterile plants. At each stage observed, the pollen in fertile and restored plants had similar cellular structures and organization. The deleterious effect of the sterility protein expression started as early as the tetrad stage. No typical mitochondria were identified in the tapetum at any developmental stage and in the vacuolate microspores of the sterile plants. In addition, some striking ultrastructural alterations of the cell's organization were also observed compared with the normal pattern of development. The results showed that Ogu-INRA CMS was due to premature cell death events of the tapetal cells, presumably by an autolysis process rather than a normal PCD, which impairs pollen development at the vacuolate microspore stage, in the absence of functional mitochondria.

A cytokinin receptor homologue is induced during root nodule organogenesis and senescence in Lupinus albus L.

Here we report the isolation of a new cytokinin receptor homologue, LaHK1, from lupin (Lupinus albus) root nodules. LaHK1 transcript accumulation was detected in different plant organs, and expression was analyzed throughout nodule development. We observed notably higher expression in nodule primordia and young nodules compared to the root or to mature nodules. We also detected elevated transcript accumulation in naturally senescent nodules and in senescent nodules subjected to foliar dark stress. The results could be an indication of a putative role of this cytokinin receptor homologue in nodule development, from morphogenesis through senescence.

The Symbiosome: Legume and Rhizobia Co-evolution toward a Nitrogen-Fixing Organelle?

In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle.

Nuclear DNA endoreduplication and expression of the mitotic inhibitor Ccs52 associated to determinate and lupinoid nodule organogenesis.

Lotus japonicus determinate nodules differ greatly from indeterminate nodules in their organogenesis and morphological characteristics, whereas Lupinus albus lupinoid nodules share features of determinate and indeterminate nodules. The mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent cell enlargement during symbiotic cell differentiation in Medicago truncatula indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus nodules; the deduced Ccs52A proteins showed high sequence similarity with other Cdh-1-type activators of the anaphase-promoting complex and were grouped with A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was restricted to the earlier stages of nodule development, whereas ccs52A transcripts accumulated in lotus nodule primordia and, to a lesser extent, in mature nodules. Nodule development in Lupinus albus involved a progressive increase in nuclear and cellular size and ploidy level; similarly, Lotus japonicus nodules contained polyploid nuclei and enlarged cells in the infected zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A expression in the inner cortex cells of the lupin nodule primordium, probably associated to the increased size of these cells in mature nodules. In view of our results, Ccs52A-mediated endoreduplication appears to be a universal mechanism required for nodule cell differentiation during the establishment of nitrogen-fixing symbioses.

Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones.

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.

The future of lupin as a protein crop in Europe.

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.

An unusual infection mechanism and nodule morphogenesis in white lupin (Lupinus albus).

• The infection of white lupin (Lupinus albus) roots and the early stages in organogenesis of the lupinoid nodule are characterized in detail in this work. • Immunolabelling of Bradyrhizobium sp. (Lupinus) ISLU16 and green fluorescent protein labelling of Mesorhizobium loti NZP2037, two strains that induce nodulation in L. albus, allowed us to monitor the infection and morphogenesis process. Light and transmission electron microscopy, low-temperature scanning electron microscopy, fluorescence and confocal microscopy were employed. • Rhizobia penetrated the root intercellularly at the junction between the root hair base and an adjacent epidermal cell. Bacteria invaded the subepidermal cortical cell immediately beneath the root hair through structurally altered cell wall regions. The newly infected cell divided repeatedly to form the central infected zone of the young nodule. Bacteria seemed to be equally distributed between the daughter cells. • A new mode of direct epidermal infection and an unusual morphogenesis for indeterminate nodules lead to the formation of the lupinoid nodule with unique characteristics.