Truncatula Plants Research Articles

Genome-wide association of drought-related and biomass traits with HapMap SNPs in Medicago truncatula.

Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought-related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon-13 isotope discrimination (δ(13) C) of well-watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome-wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P-values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase-like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate-cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome-wide association studies analysis.

Long‐term non‐invasive and continuous measurements of legume nodule activity

Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H(+) alongside N2 , and the evolving H2 allows a continuous and non-invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set-up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air-proof separation of a root/nodule and a shoot compartment. H2 evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO2 concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set-up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long-term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.

Mycorrhiza symbiosis increases the surface for sunlight capture in Medicago truncatula for better photosynthetic production.

Arbuscular mycorrhizal (AM) fungi play a prominent role in plant nutrition by supplying mineral nutrients, particularly inorganic phosphate (Pi), and also constitute an important carbon sink. AM stimulates plant growth and development, but the underlying mechanisms are not well understood. In this study, Medicago truncatula plants were grown with Rhizophagus irregularis BEG141 inoculum (AM), mock inoculum (control) or with Pi fertilization. We hypothesized that AM stimulates plant growth through either modifications of leaf anatomy or photosynthetic activity per leaf area. We investigated whether these effects are shared with Pi fertilization, and also assessed the relationship between levels of AM colonization and these effects. We found that increased Pi supply by either mycorrhization or fertilization led to improved shoot growth associated with increased nitrogen uptake and carbon assimilation. Both mycorrhized and Pi-fertilized plants had more and longer branches with larger and thicker leaves than the control plants, resulting in an increased photosynthetically active area. AM-specific effects were earlier appearance of the first growth axes and increased number of chloroplasts per cell section, since they were not induced by Pi fertilization. Photosynthetic activity per leaf area remained the same regardless of type of treatment. In conclusion, the increase in growth of mycorrhized and Pi-fertilized Medicago truncatula plants is linked to an increase in the surface for sunlight capture, hence increasing their photosynthetic production, rather than to an increase in the photosynthetic activity per leaf area.

Maturation of nematode-induced galls in Medicago truncatula is related to water status and primary metabolism modifications

Root-knot nematodes are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, these nematodes induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells (GCs). These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. We analyzed the modifications of water status, ionic content and accumulation of metabolites in development and mature galls induced by Meloidogyne incognita and in uninfected roots of Medicago truncatula plants. Water potential and osmotic pressure are significantly modified in mature galls compared to developing galls and control roots. Ionic content is significantly modified in galls compared to roots. Principal component analyses of metabolite content showed that mature gall metabolism is significantly modified compared to developing gall metabolism. The most striking differences were the three-fold increase of trehalose content associated to the five-fold diminution in glucose concentration in mature galls. Gene expression analysis showed that trehalose accumulation was, at least, partially linked to a significantly lower expression of the trehalase gene in mature galls. Our results point to significant modifications of gall physiology during maturation.

Physiological and proteolytic activities ofMedicago truncatulaunder different cadmic treatments

Medicago truncatula (A17) were cultivated under strictly controlled growth conditions, in a nutrient solution in the absence or presence of Cd (10, 20 and 50 µM CdSO4). After 20 days, decrease on the plant growth was detected and chlorosis appeared on the foliar limbs. Root growth, relative root growth (Cd -treated /untreated seedlings) and tolerance index (difference of root growth between Cd-treated and untreated seedlings) were considered as the indices of tolerance to this toxic metal. The results showed that the internal quantity of cadmium increases essentially in roots level with the elevation of the cadmium concentration in the growth medium. Different cadmium treatments significantly and considerably decreased shoot growth and root growth at Cd concentration 20µM. In addition, cadmium induced the proteolytic activity endopeptidase in roots. The increase of calcium, magnesium and potassium content in plant tissues in response to different Cd treatments reflected a kind of adaptive mineral strategy that apparently failed to give resistance to M. truncatula plant against Cd treatments.

Protected activity of a phytase immobilized in mesoporous silica with benefits to plant phosphorus nutrition

Phosphorus (P) is one of the main nutrients required for plant growth and is absorbed and metabolized in the form of orthophosphate ions (Pi). In agriculture, P is a major component of fertilizers. Forecast shortages in P mine stocks within the coming decades call for alternative sources of this element for agricultural use. In the present study, we explore the possibility of incorporating phytase near the plant root system. Phytase is an enzyme capable of mobilizing the main organic form of P as phytate, which can represent up to 90 % of the total P stock in soils. We report the immobilization of phytase in KIT-6 silica mesoporous materials with large pores of 8.6 nm. The enzymatic activity is localized in the porous network, and the main kinetic features of the free phytase appear to be maintained within the functional material (pHopt = 5–5.5 and T opt = 55 °C). Most importantly, the enzyme immobilized in the pores shows a higher temperature stability and appears to be protected from protease degradation. Finally, phytase-loaded KIT-6 proved efficient at hydrolyzing phytate and delivering inorganic P to growing Medicago truncatula plants, which accumulated this newly available P in both roots and shoots.

Characterization of the antifungal and plant growth-promoting effects of diffusible and volatile organic compounds produced by Pseudomonas fluorescens strains

We analyzed the antifungal and plant growth-promoting (PGP) effects of diffusible and volatile organic compounds (VOCs) produced by new rhizospheric isolates. The bacterial strains were identified as Pseudomonas fluorescens UM16, UM240, UM256, and UM270, based on their complete 16S ribosomal gene sequencing. These pseudomonads showed a high degree of antagonism against the phytopathogen Botrytis cinerea during confrontation assays. In addition, all strains significantly increased Medicago truncatula biomass and chlorophyll content. Interestingly, these activities were exerted by the emission of either diffusible organic compounds or VOCs. During biocontrol experiments, the four P. fluorescens strains were able to protect M. truncatula plants from B. cinerea infection by reducing stem disease symptoms and root browning. The potential presence of phenazines, cyanogens, and ACC (1-aminocyclopropane-1-carboxylate) deaminase, as well as the production of biofilm, siderophores, proteases, and indole-3-acetic acid were identified in most of the strains. The VOCs emitted by all four Pseudomonas strains were similar, and sulfur-containing compounds were among the most abundant, including dimethyl disulfide. Interestingly, the strain UM270 was the only one that produced dimethylhexadecylamine, a compound with antifungal and PGP activities. In conclusion, the Pseudomonas strains analyzed here exert multiple antagonistic and PGP mechanisms, and represent an excellent option to be used as either biocontrol or biopromoting agents in crops.

From lateral root density to nodule number, the strigolactone analogue GR24 shapes the root architecture of Medicago truncatula.

In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 µM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 µM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

Potential use and mode of action of the new strain Bacillus thuringiensis UM96 for the biological control of the grey mould phytopathogen Botrytis cinerea

The potential use of Bacillus thuringiensis UM96 as a biocontrol agent for the grey mould phytopathogen Botrytis cinerea was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, B. thuringiensis UM96 was characterised based on 16S rRNA and gyrA gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to B. cinerea, the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the chiB gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from B. thuringiensis UM96 showed a strong inhibitory effect of B. cinerea that was not observed with heat-treated supernatant. Interestingly, when the supernatant was supplemented with 100 µM allosamidin, a chitinase specific inhibitor, the antagonistic activity was suppressed significantly. A lack of chitinase activity was also observed in allosamidin-treated supernatants. Our pathogenic B. cinerea strain also exhibited susceptibility to pure Streptomyces griseus chitinase. Finally, the chitinolytic strain B. thuringiensis UM96 was able to protect Medicago truncatula plants in vitro from B. cinerea infection and significantly reduced the necrotic zones and root browning of the plants. Together, these results suggest a potential use of B. thuringiensis UM96 for the biological control of B. cinerea and a role for chitinases during the antagonism displayed.

Nodule carbohydrate catabolism is enhanced in the Medicago truncatula A17-Sinorhizobium medicae WSM419 symbiosis.

The symbiotic association between Medicago truncatula and Sinorhizobium meliloti is a well-established model system in the legume–Rhizobium community. Despite its wide use, the symbiotic efficiency of this model has been recently questioned and an alternative microsymbiont, S. medicae, has been proposed. However, little is known about the physiological mechanisms behind the higher symbiotic efficiency of S. medicae WSM419. In the present study, we inoculated M. truncatula Jemalong A17 with either S. medicae WSM419 or S. meliloti 2011 and compared plant growth, photosynthesis, N2-fixation rates, and plant nodule carbon and nitrogen metabolic activities in the two systems. M. truncatula plants in symbiosis with S. medicae showed increased biomass and photosynthesis rates per plant. Plants grown in symbiosis with S. medicae WSM419 also showed higher N2-fixation rates, which were correlated with a larger nodule biomass, while nodule number was similar in both systems. In terms of plant nodule metabolism, M. truncatula–S. medicae WSM419 nodules showed increased sucrose-catabolic activity, mostly associated with sucrose synthase, accompanied by a reduced starch content, whereas nitrogen-assimilation activities were comparable to those measured in nodules infected with S. meliloti 2011. Taken together, these results suggest that S. medicae WSM419 is able to enhance plant carbon catabolism in M. truncatula nodules, which allows for the maintaining of high symbiotic N2-fixation rates, better growth and improved general plant performance.

Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants

Our study highlights the use of the DNA repair gene MtTdp2α as a tool for improving the plant response to heavy metal stress. Tyrosyl-DNA phosphodiesterase 2 (Tdp2), involved in the removal of DNA topoisomerase II-mediated DNA damage and cell proliferation/differentiation signalling in animal cells, is still poorly characterised in plants. The Medicago truncatula lines Tdp2α-13c and Tdp2α-28 overexpressing the MtTdp2α gene and control (CTRL) line were exposed to 0.2 mM CuCl2. The DNA diffusion assay revealed a significant reduction in the percentage of necrosis caused by copper in the aerial parts of the Tdp2α-13c and Tdp2α-28 plants while neutral single cell gel electrophoresis highlighted a significant decrease in double strand breaks (DSBs), compared to CTRL. In the copper-treated Tdp2α-13c and Tdp2α-28 lines there was up-regulation (up to 4.0-fold) of genes encoding the α and β isoforms of Tyrosyl-DNA phosphodiesterase 1, indicating the requirement for Tdp1 function in the response to heavy metals. As for DSB sensing, the MtMRE11, MtRAD50 and MtNBS1 genes were also significantly up-regulated (up to 2.3-fold) in the MtTdp2α-overexpressing plants grown under physiological conditions, compared to CTRL line, and then further stimulated in response to copper. The basal antioxidant machinery was always activated in all the tested lines, as indicated by the concomitant up-regulation of MtcytSOD and MtcpSOD genes (cytosolic and chloroplastic Superoxide Dismutase), and MtMT2 (type 2 metallothionein) gene. The role of MtTdp2α gene in enhancing the plant response to genotoxic injury under heavy metal stress is discussed.

Cold acclimation-induced freezing tolerance ofMedicago truncatulaseedlings is negatively regulated by ethylene

To evaluate the role of ethylene in cold acclimation and cold stress, freezing tolerance and characteristics associated with cold acclimation were investigated using legume model plant Medicago truncatula Gaertn Jemalong A17. There was a rapid suppression of ethylene production during cold acclimation in A17 plants. Ethylene level was negatively correlated with freezing tolerance as inhibition of ethylene biosynthesis by inhibitors of ethylene biosynthesis enhanced freezing tolerance, while exogenous application of ethylene reduced cold acclimation-induced freezing tolerance. The involvement of ethylene signaling in modulation of freezing tolerance and cold acclimation was further studied using ethylene-insensitive mutant sickle skl. Although skl mutant was more tolerant to freezing than its wild-type counterpart A17 plants, cold acclimation enhanced freezing tolerance in 17 plants, but not in skl mutant. Expression of several ethylene response genes including EIN3, EIN3/EIL and ERFs was suppressed in skl mutant compared to A17 plants under non-cold-acclimated conditions. Cold acclimation downregulated expression of EIN3, EIN3/EIL and ERFs in A17 plants, while expression patterns of these genes were relatively constant in skl mutant during cold acclimation. Cold acclimation-induced increases in transcription of MtCBFs and MtCAS15 were suppressed in skl mutant compared with A17 plants. These results suggest that MtSKL1 is required for perception of the change of ethylene level in M. truncatula plants for the full development of the cold acclimation response by suppressing expression of MtEIN3 and MtEIN3/EIL1, which in turn downregulates expression of MtERFs, leading to the enhanced tolerance of M. truncatula to freezing by upregulating MtCBFs and MtCAS15.

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.

N-feedback regulation is synchronized with nodule carbon alteration in Medicago truncatula under excessive nitrate or low phosphorus conditions

The aim of the present study was to test the hypothesis that the higher nodule amino acid content induced under certain treatments may play a role in the N-feedback regulation of nitrogenase (EC 1.18.6.1) activity by restricting the carbon supply to the functioning nodules. Growing Medicago truncatula plants under sub-optimal phosphorus conditions or upon exposure to large supply of nitrate caused significant asparagine accumulation in nodules of the treated plants. In addition, there was a remarkable decline in the nodule succinate content under phosphorus deprivation while malate was tended to increase. Interestingly, the relative share of succinate in the symbiotic tissues was totally inhibited, i.e. reached zero, by excessive nitrate application. These results provide evidence that succinate might be greatly affected by asparagine content of the nodule fraction, thereby restricting cellular carbon supply to the functioning bacteroids which leads to down-regulation of nodule metabolism and nitrogenase activity.

Competition and facilitation in synthetic communities of arbuscular mycorrhizal fungi

Interactions between arbuscular mycorrhizal fungal (AMF) species cocolonizing the same host plant are still little understood in spite of major ecological significance of mycorrhizal symbiosis and widespread occurrence of these fungi in communities rather than alone. Furthermore, shifting the composition of AMF communities has demonstrated consequences for the provision of symbiotic benefits to the host as well as for the qualities of ecosystem services. Therefore, here we addressed the nature and strength of interactions between three different AMF species in all possible two-species combinations on a gradient of inoculation densities. Fungal communities were established in pots with Medicago truncatula plants, and their composition was assessed with taxon-specific real-time PCR markers. Nature of interactions between the fungi was varying from competition to facilitation and was influenced by both the identity and relative abundance of the coinoculated fungi. Plants coinoculated with Claroideoglomus and Rhizophagus grew bigger and contained more phosphorus than with any of these two fungi separately, although these fungi obviously competed for root colonization. On the other hand, plants coinoculated with Gigaspora and Rhizophagus, which facilitated each other's root colonization, grew smaller than with any of these fungi separately. Our results point to as yet little understood complexity of interactions in plant-associated symbiotic fungal communities, which, depending on their composition, can induce significant changes in plant host growth and/or phosphorus acquisition in either direction.

Isolation and characterization of genetic variability in bacteria with β-hemolytic and antifungal activity isolated from the rhizosphere of Medicago truncatula plants.

In the present study, we analyzed the frequency of hemolytic and antifungal activities in bacterial isolates from the rhizosphere of Medicago truncatula plants. Of the 2000 bacterial colonies, 96 showed β-hemolytic activities (frequency, 4.8 x 10(-2)). Hemolytic isolates were analyzed for their genetic diversity by using random amplification of polymorphic DNA, yielding 88 haplotypes. The similarity coefficient of Nei and Li showed a polymorphic diversity ranging from 0.3 to 1. Additionally, 8 of the hemolytic isolates showed antifungal activity toward plant pathogens, Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The 16S ribosomal sequencing analysis showed that antagonistic bacterial isolates corresponded to Bacillus subtilis (UM15, UM33, UM42, UM49, UM52, and UM91), Bacillus pumilus (UM24), and Bacillus licheniformis (UM88). The present results revealed a higher genetic diversity among hemolytic isolates compared to that of isolates with antifungal action.

Fate of Polyfluoroalkyl Phosphate Diesters and Their Metabolites in Biosolids-Applied Soil: Biodegradation and Plant Uptake in Greenhouse and Field Experiments

Significant contamination of perfluoroalkyl acids (PFAAs) in wastewater treatment plant (WWTP) sludge implicates the practice of applying treated sludge or biosolids as a potential source of these chemicals onto agricultural farmlands. Recent efforts to characterize the sources of PFAAs in the environment have unveiled a number of fluorotelomer-based materials that are capable of degrading to the perfluoroalkyl carboxylates (PFCAs), such as the polyfluoroalkyl phosphate diesters (diPAPs), which have been detected in WWTP and paper fiber biosolids. Here, a greenhouse microcosm was used to investigate the fate of endogenous diPAPs and PFCAs present in WWTP and paper fiber biosolids upon amendment of these materials with soil that had been sown with Medicago truncatula plants. Biodegradation pathways and plant uptake were further elucidated in a separate greenhouse microcosm supplemented with high concentrations of 6:2 diPAP. Biosolid-amended soil exhibited increased concentrations of diPAPs (4-83 ng/g dry weight (dw)) and PFCAs (0.1-19 ng/g dw), as compared to control soils (nd-1.4 ng/g dw). Both plant uptake and biotransformation contributed to the observed decline in diPAP soil concentrations over time. Biotransformation was further evidenced by the degradation of 6:2 diPAP to its corresponding fluorotelomer intermediates and C4-C7 PFCAs. Substantial plant accumulation of endogenous PFCAs present in the biosolids (0.1-138 ng/g wet weight (ww)) and those produced from 6:2 diPAP degradation (100-58 000 ng/g ww) were observed within 1.5 months of application, with the congener profile dominated by the short-chain PFCAs (C4-C6). This pattern was corroborated by the inverse relationship observed between the plant-soil accumulation factor (PSAF, Cplant/Csoil) and carbon chain length (p < 0.05, r = 0.90-0.97). These results were complemented by a field study in which the fate of diPAPs and PFCAs was investigated upon application of compost and paper fiber biosolids to two farm fields. Together, these studies provide the first evidence of soil biodegradation of diPAPs and the subsequent uptake of these chemicals and their metabolites into plants.

Application of sodium nitroprusside results in distinct antioxidant gene expression patterns in leaves of mature and senescing Medicago truncatula plants

Sodium nitroprusside (SNP) represents one of the most commonly used NO donors in biological sciences, which acts as a signal molecule in plants responsible for the regulation of the expression of many defense-related enzymes. This study attempts to provide novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on antioxidant gene expression (cAPX, GST, FeSOD, CAT, and AOX) in mature (40 day) and senescing (65 day) Medicago truncatula plants. Quantitative real-time RT-PCR suggests that low concentration of SNP applied in mature leaves leads to an overall induction of antioxidant gene expression, while increasing concentration results in suppression of these genes. Conversely, older plants demonstrate a much more variable regulation which appears to be time dependent. The observed transcriptional regulation pattern in mature M. truncatula plants comes in support of the previously documented protective or damaging effect of SNP depending on concentration applied, whereas senescing M. truncatula plants demonstrated a general suppression in antioxidant gene expression levels regardless of SNP concentration, indicative of reduced overall plant defense capacity against free radicals.

Systemic regulation of sulfur homeostasis in Medicago truncatula

Sulfur (S) is an essential macronutrient for plants, and deficiency in soil S availability limits plant growth. Adaptive strategies have been evolved by plants to respond to S deficiency by coordinating systemic regulatory mechanism. A split-root experiment using legume model plant Medicago truncatula Gaertn. was conducted to investigate the systemic response to S deficiency. Plant growth, root morphology and S contents under varying conditions of S supply were determined, and the expression of genes encoding sulfate transporter (MtSULTRs) and MtAPR1 encoding an enzyme involved in S assimilation was monitored. Our results demonstrated that there was an apparent systemic response of M. truncatula to heterogeneous S supply in terms of root length, S contents, and S uptake and assimilation at the transcriptional level. When exposed to heterogeneous S supply, M. truncatula plants showed proliferation of lateral roots in S-rich medium and reduction in investment to S-depleted roots. Growth was stimulated with half-part of roots exposed to S-deficient medium. There were different expression patterns of MtSULTRs and MtAPR1 in response to heterogeneous S supply both in roots and shoots of M. truncatula. Expression of MtSULTR1.1 and MtSULTR1.3 was systemically responsive to S deficiency, leading to an enhancement of S uptake in roots exposed to S-sufficient medium. In addition, the response of S-deprived seedlings to re-supply of sulfate and Cys was also analyzed. It was shown that sulfate, but not Cys, may serve as a systemic signal to regulate the expression of genes associated with S absorption and assimilation in M. truncatula. These findings provide a comprehensive picture of systemic responses to S deficiency in leguminous species.

Developmental stage- and concentration-specific sodium nitroprusside application results in nitrate reductase regulation and the modification of nitrate metabolism in leaves of Medicago truncatula plants

Nitric oxide (NO) is a bioactive molecule involved in numerous biological events that has been reported to display both pro-oxidant and antioxidant properties in plants. Several reports exist which demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the expression regulation of many antioxidant enzymes. This study attempts to provide a novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on the nitrosative status and nitrate metabolism of mature (40 d) and senescing (65 d) Medicago truncatula plants. Higher concentrations of SNP resulted in increased NO content, cellular damage levels and reactive oxygen species (ROS) concentration, further induced in older tissues. Senescing M. truncatula plants demonstrated greater sensitivity to SNP-induced oxidative and nitrosative damage, suggesting a developmental stage-dependent suppression in the plant’s capacity to cope with free oxygen and nitrogen radicals. In addition, measurements of the activity of nitrate reductase (NR), a key enzyme involved in the generation of NO in plants, indicated a differential regulation in a dose and time-dependent manner. Furthermore, expression levels of NO-responsive genes (NR, nitrate/nitrite transporters) involved in nitrogen assimilation and NO production revealed significant induction of NR and nitrate transporter during long-term 2.5 mM SNP application in mature plants and overall gene suppression in senescing plants, supporting the differential nitrosative response of M. truncatula plants treated with different concentrations of SNP.