MtZR1, a PRAF protein,is involved in the development of roots and symbiotic root nodules in Medicago truncatula

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen‐fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down‐regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down‐regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.     

A nodule endophytic Bacillus megaterium strain isolated from Medicago polymorpha enhances growth,promotes nodulation by Ensifer medicae and alleviates salt stress in alfalfa plants

A Gram‐positive, fast‐growing, endophytic bacterium was isolated from root nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate, named NMp082, co‐inhabited nodules with the symbiotic rhizobium Ensifer medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100% identical to those of Ensifer meliloti, an unusual event that suggested previous lateral gene transfer from a different rhizobial species. Despite the presence of nodulation and nitrogen fixation genes, the endophyte was not able to form effective nodules; however, it induced nodule‐like unorganised structures in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha, Medicago lupulina, Medicago truncatula and Medicago sativa, and co‐inoculation with E. medicae enhanced growth and nodulation of Medicago spp. plants compared with inoculation with either bacterium alone. B. megaterium NMp082 also induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to produce indole acetic acid (IAA) and the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity displayed by the endophyte in vitro might explain the observed plant growth promotion and salt stress alleviation. The isolate was also highly tolerant to salt stress, water deficit and to the presence of different heavy metals. The newly characterised endophytic bacterium possessed specific characteristics that point at potential applications to sustain plant growth and nodulation under abiotic stress.     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

RNAi Phenotypes and the Localization of a Protein::GUS Fusion Imply a Role for Medicago truncatula PIN Genes in Nodulation

The symbiosis between legumes and rhizobia results in the development of a new plant organ, the nodule. A role for polar auxin transport in nodule development in Medicago truncatula has been demonstrated using molecular genetic tools. The expression of a DR5::GUS auxin-responsive promoter in uninoculated M. truncatula roots mirrored that reported in Arabidopsis, and expression of the construct in nodulating roots confirmed results reported in white clover. The localization of a root-specific PIN protein (MtPIN2) in normal roots, developing lateral roots and nodules provided the first evidence that a PIN protein is expressed in nodules. Reduced levels of MtPIN2, MtPIN3, and MtPIN4 mRNAs via RNA interference demonstrated that plants with reduced expression of various MtPINs display a reduced number of nodules. The reported results show that in M. truncatula, PIN proteins play an important role in nodule development, and that nodules and lateral roots share some early auxin responses in common, but they rapidly differentiate with respect to auxin and MtPIN2 protein distribution.     

Drought stress provokes the down‐regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules

Symbiotic nitrogen fixation is one of the first physiological processes inhibited in legume plants under water‐deficit conditions. Despite the progress made in the last decades, the molecular mechanisms behind this regulation are not fully understood yet. Recent proteomic work carried out in the model legume Medicago truncatula provided the first indications of a possible involvement of nodule methionine (Met) biosynthesis and related pathways in response to water‐deficit conditions. To better understand this involvement, the drought‐induced changes in expression and content of enzymes involved in the biosynthesis of Met, S‐adenosyl‐L‐methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen‐fixing plants were subjected to a progressive water deficit and a subsequent recovery period. Besides the physiological characterization of the plants, the content of total sulphur, sulphate and main S‐containing metabolites was measured. Results presented here show that S availability is not a limiting factor in the drought‐induced decline of nitrogen fixation rates in M. truncatula plants and provide evidences for a down‐regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water‐deficit conditions.     

Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress

To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.     

A novel RNA‐binding peptide regulates the establishment of the Medicago truncatula–Sinorhizobium meliloti nitrogen‐fixing symbiosis

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen‐fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so‐called nodulin genes are induced, including large families that encode small peptides. Using a three‐hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA‐binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single‐stranded RNA‐binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA‐binding peptides.     

MtMOT1.2 is responsible for molybdate supply to Medicago truncatula nodules

Symbiotic nitrogen fixation in legume root nodules requires a steady supply of molybdenum for synthesis of the iron‐molybdenum cofactor of nitrogenase. This nutrient has to be provided by the host plant from the soil, crossing several symplastically disconnected compartments through molybdate transporters, including members of the MOT1 family. Medicago truncatula Molybdate Transporter (MtMOT) 1.2 is a Medicago truncatula MOT1 family member located in the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 indicates that it is associated to the plasma membrane and to intracellular membrane systems, where it would be transporting molybdate towards the cytosol, as indicated in yeast transport assays. Loss‐of‐function mot1.2‐1 mutant showed reduced growth compared with wild‐type plants when nitrogen fixation was required but not when nitrogen was provided as nitrate. While no effect on molybdenum‐dependent nitrate reductase activity was observed, nitrogenase activity was severely affected, explaining the observed difference of growth depending on nitrogen source. This phenotype was the result of molybdate not reaching the nitrogen‐fixing nodules, since genetic complementation with a wild‐type MtMOT1.2 gene or molybdate‐fortification of the nutrient solution, both restored wild‐type levels of growth and nitrogenase activity. These results support a model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into the nodules.     

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N₂ for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N₂ in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N₂‐fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species‐dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N₂, RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N₂‐fixing ability of the rhizobia strains.     

Salt stress‐induced seedling growth inhibition coincides with differential distribution of serotonin and melatonin in sunflower seedling roots and cotyledons

Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress‐induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt‐induced long distance signaling from roots to cotyledons. Salt stress‐induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt‐treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress‐induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N‐acetylserotonin O‐methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N‐acetylserotonin.     

Salinity tolerance is related to cyanide‐resistant alternative respiration in Medicago truncatula under sudden severe stress

Salt respiration is defined as the increase of respiration under early salt stress. However, the response of respiration varies depending on the degree of salt tolerance and salt stress. It has been hypothesized that the activity of the alternative pathway may increase preventing over‐reduction of the ubiquinone pool in response to salinity, which in turn can increase respiration. Three genotypes of Medicago truncatula are reputed as differently responsive to salinity: TN1.11, A17 and TN6.18. We used the oxygen‐isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in leaves and roots of these genotypes treated with severe salt stress (300 mM) during 1 and 3 days. In parallel, AOX capacity, gas exchange measurements, relative water content and metabolomics were determined in control and treated plants. Our study shows for first time that salt respiration is induced by the triggered AOP in response to salinity. Moreover, this phenomenon coincides with increased levels of metabolites such as amino and organic acids, and is shown to be related with higher photosynthetic rate and water content in TN6.18.     

Changes in fatty acids composition,hydrogen peroxide generation and lipid peroxidation of salt-stressed corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) roots

Comparative study about the salt-induced oxidative stress and lipid composition has been realised in primary root tissues for two varieties of maize (Zea mays L.) in order to evaluate their responses to salt stress. The root growth, root water content (WC), hydrogen peroxide (H₂O₂) generation, lipid peroxidation, membrane stability index and the changes in the profile of fatty acids composition were investigated. Salinity impacts in term of root growth, water content, H₂O₂ generation, lipid peroxidation and membrane destabilisation were more pronounced in primary roots of Aristo than in those of Arper indicating more sensitivity of the first variety. It was confirmed by gas chromatography that the composition of fatty acids in roots of both varieties was constituted mainly by 16:0 and 18:0 as major saturated fatty acids and 18:1ω9, 18:2ω6 and 18:3ω3 as major unsaturated fatty acids. Total lipid extracts from the roots of both varieties showed that the lipid saturation level increased under salt stress, notwithstanding the increased proportion of polyunsaturated fatty acids. The changes in lipid saturation being predominantly due to decreases in oleic acid (18:1ω9) and increases in palmitic acid (16:0). However, Arper root extracts contained a lower proportion of saturated lipids than Aristo. The enhanced proportion of highly polyunsaturated fatty acids especially linolenic and eicosapentaenoic acids was considered to be the characteristic of the relatively salt tolerance in Arper roots.