Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

MAIN CONCLUSION: The complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp. The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Stress-regulated elements in Lotus spp., as a possible starting point to understand signalling networks and stress adaptation in legumes.

Although legumes are of primary economic importance for human and livestock consumption, the information regarding signalling networks during plant stress response in this group is very scarce. Lotus japonicus is a major experimental model within the Leguminosae family, whereas L. corniculatus and L. tenuis are frequent components of natural and agricultural ecosystems worldwide. These species display differences in their perception and response to diverse stresses, even at the genotype level, whereby they have been used in many studies aimed at achieving a better understanding of the plant stress-response mechanisms. However, we are far from the identification of key components of their stress-response signalling network, a previous step for implementing transgenic and editing tools to develop legume stress-resilient genotypes, with higher crop yield and quality. In this review we scope a body of literature, highlighting what is currently known on the stress-regulated signalling elements so far reported in Lotus spp. Our work includes a comprehensive review of transcription factors chaperones, redox signals and proteins of unknown function. In addition, we revised strigolactones and genes regulating phytochelatins and hormone metabolism, due to their involvement as intermediates in several physiological signalling networks. This work was intended for a broad readership in the fields of physiology, metabolism, plant nutrition, genetics and signal transduction. Our results suggest that Lotus species provide a valuable information platform for the study of specific protein-protein (PPI) interactions, as a starting point to unravel signalling networks underlying plant acclimatation to bacterial and abiotic stressors in legumes. Furthermore, some Lotus species may be a source of genes whose regulation improves stress tolerance and growth when introduced ectopically in other plant species.

Metabolic Profiling and Metabolite Correlation Network Analysis Reveal That Fusarium solani Induces Differential Metabolic Responses in Lotus japonicus and Lotus tenuis against Severe Phosphate Starvation.

Root fungal endophytes are essential mediators of plant nutrition under mild stress conditions. However, variations in the rhizosphere environment, such as nutrient depletion, could result in a stressful situation for both partners, shifting mutualistic to nonconvenient interactions. Mycorrhizal fungi and dark septate endophytes (DSEs) have demonstrated their ability to facilitate phosphate (Pi) acquisition. However, few studies have investigated other plant-fungal interactions that take place in the root environment with regard to phosphate nutrition. In the present research work, we aimed to analyze the effect of extreme Pi starvation and the fungal endophyte Fusarium solani on the model Lotus japonicus and the crop L. tenuis. We conducted metabolomics analysis based on gas chromatography-mass spectrometry (GC-MS) on plant tissues under optimal conditions, severe Pi starvation and F.solani presence. By combining statistical and correlation network analysis strategies, we demonstrated the differential outcomes of the two plant species against the combination of treatments. The combination of nutritional stress and Fusarium presence activated significant modifications in the metabolism of L. japonicus affecting the levels of sugars, polyols and some amino acids. Our results display potential markers for further inspection of the factors related to plant nutrition and plant-fungal interactions.

Polyamines and Legumes: Joint Stories of Stress, Nitrogen Fixation and Environment.

Polyamines (PAs) are natural aliphatic amines involved in many physiological processes in almost all living organisms, including responses to abiotic stresses and microbial interactions. On other hand, the family Leguminosae constitutes an economically and ecologically key botanical group for humans, being also regarded as the most important protein source for livestock. This review presents the profuse evidence that relates changes in PAs levels during responses to biotic and abiotic stresses in model and cultivable species within Leguminosae and examines the unreviewed information regarding their potential roles in the functioning of symbiotic interactions with nitrogen-fixing bacteria and arbuscular mycorrhizae in this family. As linking plant physiological behavior with "big data" available in "omics" is an essential step to improve our understanding of legumes responses to global change, we also examined integrative MultiOmics approaches available to decrypt the interface legumes-PAs-abiotic and biotic stress interactions. These approaches are expected to accelerate the identification of stress tolerant phenotypes and the design of new biotechnological strategies to increase their yield and adaptation to marginal environments, making better use of available plant genetic resources.

The fungal endophyte Fusarium solani provokes differential effects on the fitness of two Lotus species.

The interactions established between plants and endophytic fungi span a continuum from beneficial to pathogenic associations. The aim of this work was to isolate potentially beneficial fungal endophytes in the legume Lotus tenuis and explore the mechanisms underlying their effects. One of the nine fungal strains isolated was identified as Fusarium solani and shows the highest phosphate-solubilisation activity, and also grows endophytically in roots of L. japonicus and L. tenuis. Interestingly, fungal invasion enhances plant growth in L. japonicus but provokes a contrasting effect in L. tenuis. These differences were also evidenced when the rate of photosynthesis as well as sugars and K contents were assessed. Our results indicate that the differential responses observed are due to distinct mechanisms deployed during the establishment of the interactions that involve the regulation of photosynthesis, potassium homeostasis, and carbohydrate metabolism. These responses are employed by these plant species to maintain fitness during the endophytic interaction.

Interspecific hybridization improves the performance of Lotus spp. under saline stress.

Salinity is one of the most frequent limiting conditions in pasture production for grazing livestock. Legumes, such as Lotus spp. with high forage quality and capable of adapting to different environments, improves pasture performance in restrictive areas. In order to determine potential cultivars with better forage traits, the current study assess the response to salt stress of L. tenuis, L. corniculatus and a novel L. tenuis x L. corniculatus accession. For this purpose, chlorophyll fluorescence, biomass production, ion accumulation and anthocyanins and proanthocyanidins levels have been evaluated in control and salt-treated plants PSII activity was affected by salt in L. tenuis, but not in L. corniculatus or hybrid plants. Analyzed accessions showed similar values of biomass, Na+ and K+ levels after salt treatment. Increasing Cl- concentrations were observed in all accessions. However, hybrid plants accumulate Cl- in stems at higher levels than their parental. At the same time, the levels of anthocyanins considerably increased in L. tenuis x L. corniculatus stems. Chloride and anthocyanin accumulation in stems could explain the best performance of hybrid plants after a long saline treatment. Finally, as proanthocyanidins levels were no affected by salt, L. tenuis x L. corniculatus plants maintained adequate levels to be used as ruminant feed. In conclusion, these results suggest that hybrid plants have a high potential to be used as forage on salt-affected lands. High Cl- and anthocyanins accumulation in Lotus spp. stems seems to be a trait associated to salinity tolerance, with the possibility of being used in legume breeding programs.

The increase of photosynthetic carbon assimilation as a mechanism of adaptation to low temperature in Lotus japonicus.

Low temperature is one of the most important factors affecting plant growth, it causes an stress that directly alters the photosynthetic process and leads to photoinhibition when severe enough. In order to address the photosynthetic acclimation response of Lotus japonicus to cold stress, two ecotypes with contrasting tolerance (MG-1 and MG-20) were studied. Their chloroplast responses were addressed after 7 days under low temperature through different strategies. Proteomic analysis showed changes in photosynthetic and carbon metabolism proteins due to stress, but differentially between ecotypes. In the sensitive MG-1 ecotype acclimation seems to be related to energy dissipation in photosystems, while an increase in photosynthetic carbon assimilation as an electron sink, seems to be preponderant in the tolerant MG-20 ecotype. Chloroplast ROS generation was higher under low temperature conditions only in the MG-1 ecotype. These data are consistent with alterations in the thylakoid membranes in the sensitive ecotype. However, the accumulation of starch granules observed in the tolerant MG-20 ecotype indicates the maintenance of sugar metabolism under cold conditions. Altogether, our data suggest that different acclimation strategies and contrasting chloroplast redox imbalance could account for the differential cold stress response of both L. japonicus ecotypes.

Modulation of plant and bacterial polyamine metabolism during the compatible interaction between tomato and Pseudomonas syringae.

The polyamines putrescine, spermidine and spermine participate in a variety of cellular processes in all organisms. Many studies have shown that these polycations are important for plant immunity, as well as for the virulence of diverse fungal phytopathogens. However, the polyamines' roles in the pathogenesis of phytopathogenic bacteria have not been thoroughly elucidated to date. To obtain more information on this topic, we assessed the changes in polyamine homeostasis during the infection of tomato plants by Pseudomonas syringae. Our results showed that polyamine biosynthesis and catabolism are activated in both tomato and bacteria during the pathogenic interaction. This activation results in the accumulation of putrescine in whole leaf tissues, as well as in the apoplastic fluids, which is explained by the induction of its synthesis in plant cells and also on the basis of its excretion by bacteria. We showed that the excretion of this polyamine by P. syringae is stimulated under virulence-inducing conditions, suggesting that it plays a role in plant colonization. However, no activation of bacterial virulence traits or induction of plant invasion was observed after the exogenous addition of putrescine. In addition, no connection was found between this polyamine and plant defence responses. Although further research is warranted to unravel the biological functions of these molecules during plant-bacterial interactions, this study contributes to a better understanding of the changes associated with the homeostasis of polyamines during plant pathogenesis.

Descripción de cambios en la difusibilidad media en resonancia magnética por lesiones del sistema reticular activador ascendente en pacientes adultos con trauma craneoencefálico y alteración del estado de conciencia / Description of changes in mean diffusivity in magnetic resonance of injuries of the ascending reticular system in patients with traumatic brain injury and impaired consciousness

RESUMEN INTRODUCCIÓN: Los pacientes con trauma craneoencefálico severo pueden tener secuelas neurológicas graves que generan discapacidad de rangos diferentes según la escala de Barthel. La alteración del estado de consciencia es la secuela más grave y es causa de dependencia completa de estos pacientes por la pérdida del autocuidado. La relación entre los hallazgos anormales en las neuroimágenes y los diferentes estados de conciencia ha sido objeto de investigación en los últimos años, con la posibilidad de que existan nuevas opciones con el posproceso de imágenes obtenidas por resonancia magnética nuclear. Este estudio pretende describir las alteraciones en la difusibilidad media haciendo posproceso en la secuencia de DWI en 4 pacientes con trauma craneoencefálico severo y alteración del estado de consciencia, quienes en la fase aguda del trauma fueron estudiados con imágenes convencionales de tomografía y resonancia. MATERIALES Y MÉTODOS: Se describe una serie de 4 casos de pacientes con trauma craneoencefálico severo y alteración del estado de consciencia a quienes se realizó resonancia cerebral simple. Las imágenes fueron sometidas a un posproceso de la secuencia de DWI analizando diferentes regiones por donde transcurren las fibras del sistema reticular activador ascendente (SRAA) (cuerpos restiformes, rafe medio del mesencéfalo, tálamo, regiones subinsulares y lóbulos frontales basales). Igualmente se tomaron valores de difusibilidad en regiones similares, en 5 sujetos sin alteración estructural cerebral tomados como controles. RESULTADOS: Se encontró disminución en la difusibilidad media en las diferentes regiones establecidas en el estudio, que tuvieron diferente localización en cada paciente. Los valores disminuyeron aproximadamente en un 50 % respecto a los del grupo control, observando lesiones en áreas que no fueron identificadas en la interpretación de la imagen cuando fue adquirida durante el trauma. CONCLUSIONES: El posproceso de la secuencia DWI muestra disminución en los valores de difusibilidad media en regiones por donde transcurren las vías del sistema reticular activado ascendente, estos cambios que explican las diferentes alteraciones en el estado de conciencia, no fueron visualizados en las imágenes interpretadas bajo las técnicas convencionales. Se requiere nuevos estudios para establecer las características operativas de la prueba que permitan definir su potencial utilidad dentro de los algoritmos de clasificación inicial de los pacientes con trauma craneoencefálico severo.

SUMMARY INTRODUCTION: Patients with severe traumatic brain injury may have severe neurological sequelae that generate disability of different ranges, according to the Barthel scale. The consciousness impairment is the most serious sequela and is a cause of complete dependence of these patients, due to the loss of self-care. The relationship between abnormal findings in neuroimaging and different states of consciousness has been the subject of research in recent years, with the possibility of new options with the post-processing of magnetic resonance imaging (MRI). This study aims to describe the alterations in the mean diffusivity by post-processing of the diffusion weighted imaging (DWI) sequence in 4 patients with severe traumatic brain injury and impairment of consciousness, who were studied with computed tomography (CT) and MRI in the acute phase of the trauma. MATERIALS AND METHODS: We describe a series of 4 cases of patients with severe traumatic brain injury and impaired consciousness in whom a non-enhanced brain MRI was performed. The DWI sequence images were post-processed, analyzing different regions through which the fibers of the Ascending Reticular Activating System (ARAS) (including restiform bodies, midbrain, thalamus, sub-insular regions and basal frontal lobes) pass. Likewise, diffusivity values were taken in similar regions, in 5 subjects without any structural brain abnormality, who were taken as controls. RESULTS: There was a decrease in mean diffusivity in the different regions established in the study, which had different locations in each patient. Values decreased approximately 50 percent from values in normal patients. After post-processing, some lesions were observed in areas that were not identified in the initial MRI interpretation during the trauma. CONCLUSIONS: The post-processing of the DWI sequence shows a decrease in the mean diffusivity values in regions where the ARAS pathways pass through. These changes explaining the different alterations in consciousness were not visualized in the images interpreted under conventional MRI techniques. New studies are required to establish the operative characteristics of this test, that allow to define its potential utility within the algorithms of initial classification of patients with severe traumatic brain injury.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species.

MAIN CONCLUSION: By exploiting interspecific hybrids and their progeny, we identified key regulatory and transporter genes intimately related to proanthocyanidin biosynthesis in leaves of Lotus spp. Proanthocyanidins (PAs), known as condensed tannins, are polymeric flavonoids enriching forage legumes of key nutritional value to prevent bloating in ruminant animals. Unfortunately, major forage legumes such as alfalfa and clovers lack PAs in edible tissues. Therefore, engineering the PA trait in herbage of forage legumes is paramount to improve both ecological and economical sustainability of cattle production system. Progresses on the understanding of genetic determinants controlling PA biosynthesis and accumulation have been mainly made studying mutants of Arabidopsis, Medicago truncatula and Lotus japonicus, model species unable to synthesize PAs in the leaves. Here, we exploited interspecific hybrids between Lotus corniculatus, with high levels of PAs in the leaves, and Lotus tenuis, with no PAs in these organs, and relative F2 progeny, to identify among candidate PA regulators and transporters the genes mainly affecting this trait. We found that the levels of leaf PAs significantly correlate with the expression of MATE1, the putative transporter of glycosylated PA monomers, and, among the candidate regulatory genes, with the expression of the MYB genes TT2a, TT2b and MYB14 and the bHLH gene TT8. The expression levels of TT2b and TT8 also correlated with those of all key structural genes of the PA pathways investigated, MATE1 included. Our study unveils a different involvement of the three Lotus TT2 paralogs to the PA trait and highlights differences in the regulation of this trait in our Lotus genotypes with respect to model species. This information opens new avenues for breeding bloat safe forage legumes.

The alkaline tolerance in Lotus japonicus is associated with mechanisms of iron acquisition and modification of the architectural pattern of the root.

The response of fifty-four Lotus japonicus ecotypes, and of six selected ecotypes was investigated under alkaline conditions. Sensitive, but not tolerant ecotypes, showed interveinal chlorosis under all alkalinity conditions and high mortality under extreme alkalinity. Interveinal chlorosis was associated with Fe deficiency, as a reduced Fe2+ shoot content was observed in all sensitive ecotypes. In addition, some showed a decline in photosynthesis rate and PSII performance compared to the control. In contrast, some tolerant ecotypes did not change these parameters between treatments. Alkaline tolerance could be explained by a mechanism of Fe acquisition and a root structural modification. This conclusion was based on the fact that all tolerant, but not the sensitive ecotypes, presented high ferric reductase oxidase activity under alkaline stress compared to the control, and a Herringbone root pattern modification. On this basis, the analysis of these mechanisms of alkaline tolerance could be used in screening programs for the selection of new tolerant genotypes in the Lotus genus.

Photosynthetic responses mediate the adaptation of two Lotus japonicus ecotypes to low temperature.

Lotus species are important forage legumes due to their high nutritional value and adaptability to marginal conditions. However, the dry matter production and regrowth rate of cultivable Lotus spp. is drastically reduced during colder seasons. In this work, we evaluated the chilling response of Lotus japonicus ecotypes MG-1 and MG-20. No significant increases were observed in reactive oxygen species and nitric oxide production or in lipid peroxidation, although a chilling-induced redox imbalance was suggested through NADPH/NADP(+) ratio alterations. Antioxidant enzyme catalase, ascorbate peroxidase, and superoxide dismutase activities were also measured. Superoxide dismutase, in particular the chloroplastic isoform, showed different activity for different ecotypes and treatments. Stress-induced photoinhibition also differentially influenced both ecotypes, with MG-1 more affected than MG-20. Data showed that the D2 PSII subunit was more affected than D1 after 1 d of low temperature exposure, although its protein levels recovered over the course of the experiment. Interestingly, D2 recovery was accompanied by improvements in photosynthetic parameters (Asat and Fv/Fm) and the NADPH/NADP(+) ratio. Our results suggest that the D2 protein is involved in the acclimation response of L. japonicus to low temperature. This may provide a deeper insight into the chilling tolerance mechanisms of the Lotus genus.

Lotus japonicus plants of the Gifu B-129 ecotype subjected to alkaline stress improve their Fe(2+) bio-availability through inoculation with Pantoea eucalypti M91.

Inoculation assays with Pantoea eucalypti M91 were performed on Lotus japonicus ecotype Gifu. Under alkaline conditions, this ecotype is characterized by the development of interveinal chlorosis of the apical leaves due to low mobilization of Fe(2+). Inoculation with P. eucalypti M91, a plant growth-promoting bacterial strain capable of producing pyoverdine-like and pyochelin-like siderophores under alkaline growth conditions, alters the root, resulting in a herringbone pattern of root branching. Additional features include improvement in Fe(2+) transport to the shoots, acidification of the hydroponic solution of the plant cultures, and an accompanying increase in the efficiency of the PSII parameters. In addition, there was an increase in the expression of the FRO1 and IRT1 genes, accompanied by a significant increase in FRO activity. Results showed that P. eucalypti M91 has a beneficial effect on the Fe acquisition machinery of Strategy I, as described for non-graminaceous monocots and dicots, suggesting its potential as an inoculant for legume crops cultivated in alkaline soils.

Identification of differentially expressed genes potentially involved in the tolerance of Lotus tenuis to long-term alkaline stress.

Soil alkalinity is one of the most serious agricultural problems limiting crop yields. The legume Lotus tenuis is an important forage acknowledged by its ability to naturally grow in alkaline soils. To gain insight into the molecular responses that are activated by alkalinity in L. tenuis plants, subtractive cDNA libraries were generated from leaves and roots of these plants. Total RNAs of non-stressed plants (pH 5.8; E.C. 1.2), and plants stressed by the addition of 10 mM of NaHCO3 (pH 9.0; E.C. 1.9), were used as source of the driver and the tester samples, respectively. RNA samples were collected after 14 and 28 days of treatment. A total of 158 unigenes from leaves and 92 unigenes from roots were obtained and classified into 11 functional categories. Unigenes from these categories (4 for leaves and 8 for roots), that were related with nutrient metabolism and oxidative stress relief were selected, and their differential expression analyzed by qRT-PCR. These genes were found to be differentially expressed in a time dependent manner in L. tenuis during the alkaline stress application. Data generated from this study will contribute to the understanding of the general molecular mechanisms associated to plant tolerance under long-term alkaline stress in plants.

Perturbation of spermine synthase gene expression and transcript profiling provide new insights on the role of the tetraamine spermine in Arabidopsis defense against Pseudomonas viridiflava.

The role of the tetraamine spermine in plant defense against pathogens was investigated by using the Arabidopsis (Arabidopsis thaliana)-Pseudomonas viridiflava pathosystem. The effects of perturbations of plant spermine levels on susceptibility to bacterial infection were evaluated in transgenic plants (35S::spermine synthase [SPMS]) that overexpressed the SPMS gene and accumulated spermine, as well as in spms mutants with low spermine levels. The former exhibited higher resistance to P. viridiflava than wild-type plants, while the latter were more susceptible. Exogenous supply of spermine to wild-type plants also increased disease resistance. Increased resistance provided by spermine was partly counteracted by the polyamine oxidase inhibitor SL-11061, demonstrating that the protective effect of spermine partly depends on its oxidation. In addition, global changes in gene expression resulting from perturbations of spermine levels were analyzed by transcript profiling 35S::SPMS-9 and spms-2 plants. Overexpression of 602 genes was detected in 35S::SPMS-9 plants, while 312 genes were down-regulated, as compared to the wild type. In the spms-2 line, 211 and 158 genes were up- and down-regulated, respectively. Analysis of gene ontology term enrichment demonstrated that many genes overexpressed only in 35S::SPMS-9 participate in pathogen perception and defense responses. Notably, several families of disease resistance genes, transcription factors, kinases, and nucleotide- and DNA/RNA-binding proteins were overexpressed in this line. Thus, a number of spermine-responsive genes potentially involved in resistance to P. viridiflava were identified. The obtained results support the idea that spermine contributes to plant resistance to P. viridiflava.

Polyamine catabolism is involved in response to salt stress in soybean hypocotyls.

The possible relationship between polyamine catabolism mediated by copper-containing amine oxidase and the elongation of soybean hypocotyls from plants exposed to NaCl has been studied. Salt treatment reduced values of all hypocotyl growth parameters. In vitro, copper-containing amine oxidase activity was up to 77-fold higher than that of polyamine oxidase. This enzyme preferred cadaverine over putrescine and it was active even under the saline condition. On the other hand, saline stress increased spermine and cadaverine levels, and the in vivo copper-containing amine oxidase activity in the elongation zone of hypocotyls. The last effect was negatively modulated by the addition of the copper-containing amine oxidase inhibitor N,N'-diaminoguanidine. In turn, plants treated with the inhibitor showed a significant reduction of reactive oxygen species in the elongation zone, even in the saline situation. In addition, plants grown in cadaverine-amended culture medium showed increased hypocotyl length either in saline or control conditions and this effect was also abolished by N,N'-diaminoguanidine. Taken together, our results suggest that the activity of the copper-containing amine oxidase may be partially contributing to hypocotyl growth under saline stress, through the production of hydrogen peroxide by polyamine catabolism and reinforce the importance of polyamine catabolism and hydrogen peroxide production in the induction of salt tolerance in plants.

Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region.

The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region.

Polyamine oxidase activity contributes to sustain maize leaf elongation under saline stress.

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H(2)O(2), *O(2)(-), and HO* production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 microM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 microM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl(2) (an *O(2)(-) dismutating agent) or the HO* scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25-30% of the maize leaf blade elongation.

Phenotypic plasticity with respect to salt stress response by Lotus glaber: the role of its AM fungal and rhizobial symbionts.

Our hypothesis is that Lotus glaber (a glycophytic species, highly tolerant to saline-alkaline soils) displays a plastic root phenotypic response to soil salinity that may be influenced by mycorrhizal and rhizobial microorganisms. Uninoculated plants and plants colonised by Glomus intraradices or Mesorhizobium loti were exposed to either 150 or 0 mM NaCl. General plant growth and root architectural parameters (morphology and topology) were measured and phenotypic plasticity determined at the end of the salt treatment period. Two genotypes differing in their salt tolerance capacity were used in this study. G. intraradices and M. loti reduced the total biomass of non-salinised, sensitive plants, but they did not affect that of corresponding tolerant ones. Root morphology of sensitive plants was greatly affected by salinity, whereas mycorrhiza establishment counteracted salinity effects. Under both saline conditions, the external link length and the internal link length of mycorrhizal salt-sensitive plants were higher than those of uninoculated control and rhizobial treatments. The topological trend (TT) was strongly influenced by genotype x symbiosis interaction. Under non-saline conditions, nodulated root systems of the sensitive plant genotype had a more herringbone architecture than corresponding uninoculated ones. At 150 mM NaCl, nodulated root systems of tolerant plants were more dichotomous and those of the corresponding sensitive genotype more herringbone in architecture. Notwithstanding the absence of a link between TTs and variations in plant growth, it is possible to predict a dissimilar adaptation of plants with different TTs. Root colonisation by either symbiotic microorganisms reduced the level of root phenotypic plasticity in the sensitive plant genotype. We conclude that root plasticity could be part of the general mechanism of L. glaber salt tolerance only in the case of non-symbiotic plants.

Apoplastic polyamine oxidation plays different roles in local responses of tobacco to infection by the necrotrophic fungus Sclerotinia sclerotiorum and the biotrophic bacterium Pseudomonas viridiflava.

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decarboxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast. Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis due to infection by S. sclerotiorum. Specific inhibition of diamine and PA oxidases attenuated the PA-induced enhancement of leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PA oxidase inhibitor. It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting pathogenesis strategies. This response affects the pathogen's ability to colonize host tissues and results are detrimental for plant defense against necrotrophic pathogens that feed on necrotic tissue; on the contrary, this response plays a beneficial role in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to significant changes in host susceptibility to different kinds of pathogens.