Genome-wide characterization, transcriptome profiling, and functional analysis of the ALMT gene family in Medicago for aluminum resistance.

Aluminum (Al) is the major limiting factor affecting plant productivity in acidic soils. Al3+ ions exhibit increased solubility at a pH below 5, leading to plant root tip toxicity. Alternatively, plants can perceive very low concentrations of Al3+, and Al triggers downstream signaling even at pH 5.7 without causing Al toxicity. The ALUMINUM-ACTIVATED-MALATE-TRANSPORTER (ALMT) family members act as anion channels, with some regulating the secretion of malate from root apices to chelate Al, which is a crucial mechanism for plant Al resistance. To date, the role of the ALMT gene family within the legume Medicago species has not been fully characterized. In this study, we investigated the ALMT gene family in M. sativa and M. truncatula and identified 68 MsALMTs and 18 MtALMTs, respectively. Phylogenetic analysis classified these genes into five clades, and synteny analysis uncovered genuine paralogs and orthologs. The real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that MtALMT8, MtALMT9, and MtALMT15 in clade 2-2b are expressed in both roots and root nodules, and MtALMT8 and MtALMT9 are significantly upregulated by Al in root tips. We also observed that MtALMT8 and MtALMT9 can partially restore the Al sensitivity of Atalmt1 in Arabidopsis. Moreover, transcriptome analysis examined the expression patterns of these genes in M. sativa in response to Al at both pH 5.7 and pH 4.6, as well as to protons, and found that Al and protons can independently induce some Al-resistance genes. Overall, our findings indicate that MtALMT8 and MtALMT9 may play a role in Al resistance, and highlight the resemblance between the ALMT genes in Medicago species and those in Arabidopsis.

Sulfur triggers glutathione and phytochelatin accumulation causing excess Cd bound to the cell wall of roots in alleviating Cd-toxicity in alfalfa.

Although Cd is threatening to the environment, animal, and human, the eco-friendly approach to mitigate the Cd-toxicity in alfalfa was barely studied. Therefore, this study aims at elucidating the role of S, a crucial macroelement, in alleviating Cd toxicity in alfalfa plants. The supplementation of S in Cd-stressed alfalfa reversed the detrimental effect on plant biomass, chlorophyll synthesis, and protein concentration. Interestingly, S surplus restored the photosynthetic kinetics, such as Fv/Fm, Pi_ABS, and Mo values in leaves of Cd-stressed alfalfa. Further, Cd-induced adverse effect on membrane stability, cell viability, and redox status was restored due to S under Cd stress. The exogenous S not only increased S status and the expression of sulfate transporters (MsSULRT1;2 and MsSULTR1;3), but also decreased the Cd concentration in the shoot by retaining elevated Cd in root tissue. Further analysis revealed the upregulation of MsGS (glutathione synthetase) and MsPCS1 (phytochelatin synthase) genes along with the increased concentration of glutathione and phytochelatin, predominantly in roots subjected to S surplus under Cd stress. The subcellular Cd analysis showed elevated Cd in the cell wall but not in the vacuole. It suggests that S-induced elevated glutathione enables the phytochelatin to bind with excess Cd leading to subcellular sequestration in the cell wall of roots. Also, S stimulates the S-metabolites and GR enzyme that coordinately counteracts Cd-induced oxidative damage. These findings can be utilized to popularize the application of S and to perform breeding/transgenic experiments to develop Cd-free forage crops.

Protective roles of salicylic acid in maintaining integrity and functions of photosynthetic photosystems for alfalfa (Medicago sativa L.) tolerance to aluminum toxicity.

Aluminum (Al) can be detrimental to plant growth in areas with Al contamination. The objective of this study was to determine whether salicylic acid (SA) can improve plant tolerance to Al stress by mitigating Al toxicity for chloroplasts and photosynthetic systems in alfalfa (Medicago sativa L.). Plants were treated with Al (100 µM) for 3 d in a hydroponic system. The content of Al increased in leaves treated with Al, resulting in damage and deformation of chloroplasts. In Al-damaged leaves, chloroplast envelopes and starch granules disappeared; the lamellae and stroma lamella were loosely arranged and indistinguishable, and the number of grana was reduced; a large number of small plastoglobules appeared. Foliar spraying of 15 µM SA reduced Al content in roots and leaves and alleviated Al damages in chloroplasts. With 15 µM SA treatments, the chloroplast shape returned to a flat ellipsoid, thylakoids were arranged closely and regularly, chloroplasts had intact starch granules, and small plastoglobules disappeared. SA-treated plants had significantly higher aboveground biomass than the untreated control exposed to Al stress. Photosynthetic index and gene expression analyses demonstrated that SA could alleviate adverse effects of Al toxicity by increasing light capture efficiency, promoting electron transport in the photosynthetic electron transport chain and thylakoid lumen deacidification, and promoting synthesis of aenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). SA played protective roles in maintaining integrity and functions of photosystems in photosynthesis for plant tolerance to Al stress.

Physiological, biochemical and transcriptomic responses of Medicago sativa to nickel exposure.

Metal accumulation in soil could lead to severe damage to plants, animals, and humans. The present work aims to evaluate the effects of nickel (Ni) exposure on Medicago sativa at physiological, biochemical, and transcriptomic levels. Plants were exposed to five increasing concentrations of Ni (0, 50, 150, 250, and 500 mg/kg) for 60 days. Agronomic parameters (fresh and dry matter) and chlorophyll content (Chl) were determined in an alfalfa plant. Chemical analyses were conducted, involving the determination of Ni loads in plants (roots and shoots). Moreover, malondialdehyde accumulation (MDA), glutathione-S-transferase (GST), and peroxidase activities, termed as oxidative stress biomarkers, were measured. The gene expression levels of Prx1C, GST, and phytochelatins (PCs) were determined at different nickel concentrations. Our results showed that Ni concentration in plants increased significantly along with Ni concentration in the soil. Regarding oxidative stress biomarkers, Ni contamination caused an increase in peroxidase and GST activities, with a remarkable accumulation of MDA, especially for the highest Ni concentration (500 mg/kg of Ni). Our data showed also a significant upregulation of Prx1C and GST genes in shoots and roots. The PCs' gene expression was significantly enhanced in response to the different nickel concentrations, suggesting their important role in Ni detoxification in alfalfa plants. Our data provided evidence about the clear toxicity of Ni, an often-underestimated trace element.

Transcriptome analysis reveals insight into molecular hydrogen-induced cadmium tolerance in alfalfa: the prominent role of sulfur and (homo)glutathione metabolism.

BACKGROUND: Hydrogen gas (H2) is hypothesised to play a role in plants that are coping with stresses by regulating signal transduction and gene expression. Although the beneficial role of H2 in plant tolerance to cadmium (Cd) has been investigated previously, the corresponding mechanism has not been elucidated. In this report, the transcriptomes of alfalfa seedling roots under Cd and/or hydrogen-rich water (HRW) treatment were first analysed. Then, the sulfur metabolism pathways were focused on and further investigated by pharmacological and genetic approaches. RESULTS: A total of 1968 differentially expressed genes (DEGs) in alfalfa seedling roots under Cd and/or HRW treatment were identified by RNA-Seq. The DEGs were classified into many clusters, including glutathione (GSH) metabolism, oxidative stress, and ATP-binding cassette (ABC) transporters. The results validated by RT-qPCR showed that the levels of relevant genes involved in sulfur metabolism were enhanced by HRW under Cd treatment, especially the genes involved in (homo)glutathione metabolism. Additional experiments carried out with a glutathione synthesis inhibitor and Arabidopsis thaliana cad2-1 mutant plants suggested the prominent role of glutathione in HRW-induced Cd tolerance. These results were in accordance with the effects of HRW on the contents of (homo)glutathione and (homo)phytochelatins and in alleviating oxidative stress under Cd stress. In addition, the HRW-induced alleviation of Cd toxicity might also be caused by a decrease in available Cd in seedling roots, achieved through ABC transporter-mediated secretion. CONCLUSIONS: Taken together, the results of our study indicate that H2 regulated the expression of genes relevant to sulfur and glutathione metabolism and enhanced glutathione metabolism which resulted in Cd tolerance by activating antioxidation and Cd chelation. These results may help to elucidate the mechanism governing H2-induced Cd tolerance in alfalfa.

Physiological effects of zero-valent iron nanoparticles in rhizosphere on edible crop, Medicago sativa (Alfalfa), grown in soil.

We investigated the effects of nanoscale zero-valent iron (nZVI) that has been widely used for groundwater remediation on a terrestrial crop, Medicago sativa (Alfalfa), and comprehensively addressed its development and growth in soil culture. Root lengths, chlorophyll, carbohydrate and lignin contents were compared, and no physiological phytotoxicity was observed in the plants. In the roots, using an omics-based analytical, we found evidence of OH radical-induced cell wall loosening from exposure to nZVI, resulting in increased root lengths that were approximately 1.5 times greater than those of the control. Moreover, germination index (GI) was employed to physiologically evaluate the impact of nZVI on germination and root length. In regard to chlorophyll concentration, nZVI-treated alfalfa exhibited a higher value in 20-day-old seedlings, whereas the carbohydrate and lignin contents were slightly decreased in nZVI-treated alfalfa. Additionally, evidence for translocation of nZVI into plant tissues was also found. Vibrating sample magnetometry on shoots revealed the translocation of nZVI from the root to shoot. In this study, using an edible crop as a representative model, the potential impact of reactive engineered nanomaterials that can be exposed to the ecosystem on plant is discussed.

Metagenomic analysis of rhizosphere microflora of oil-contaminated soil planted with barley and alfalfa.

The role of rhizosphere microbial communities in the degradation of hydrocarbons remains poorly understood and is a field of active study. We used high throughput sequencing to explore the rhizosphere microbial diversity in the alfalfa and barley planted oil contaminated soil samples. The analysis of 16s rRNA sequences showed Proteobacteria to be the most enriched (45.9%) followed by Bacteriodetes (21.4%) and Actinobacteria (10.4%) phyla. The results also indicated differences in the microbial diversity among the oil contaminated planted soil samples. The oil contaminated planted soil samples showed a higher richness in the microbial flora when compared to that of untreated samples, as indicated by the Chao1 indices. However, the trend was different for the diversity measure, where oil contaminated barley planted soil samples showed slightly lower diversity indices. While the clustering of soil samples grouped the oil contaminated samples within and across the plant types, the clean sandy soil samples formed a separate group. The oil contaminated rhizosphere soil showed an enrichment of known oil-degrading genera, such as Alcanivorax and Aequorivita, later being specifically enriched in the contaminated soil samples planted with barley. Overall, we found a few well known oil-degrading bacterial groups to be enriched in the oil contaminated planted soil samples compared to the untreated samples. Further, phyla such as Thermi and Gemmatimonadetes showed an enrichment in the oil contaminated soil samples, indicating their potential role in hydrocarbon degradation. The findings of the current study will be useful in understanding the rhizosphere microflora responsible for oil degradation and thus can help in designing appropriate phytoremediation strategies for oil contaminated lands.

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium.

Proteomic analysis provides insights into the molecular bases of hydrogen gas-induced cadmium resistance in Medicago sativa.

Recently, molecular hydrogen (H2) has emerged as a bio-regulator both in animals and plants. Normally, functions of endogenous generated H2 could be mimicked by exogenously applied hydrogen-rich water (HRW) or hydrogen-rich saline (particularly in animals). Although alfalfa seedlings showed more cadmium (Cd) resistance after the administration with HRW, corresponding molecular mechanism is still elusive. To address this gap, iTRAQ-based quantitative proteomics was used. The results showed that a total of 2377 proteins were identified with <1% FDR, and 1254 protein abundance perturbations were confidently assessed. Total of 248 significant differential proteins were identified in Cd- and/or HRW-treated samples. Furthermore, 92 proteins from the 248 proteins were selected for further bioinformatics analysis. Interestingly, results indicated that they were classified into seven categories: defense and response to stress, sulfur compound metabolic process, amino acid and protein metabolic process, carbohydrate and energy metabolic process, secondary metabolic process, oxidation-reduction process, and metal ion homeostasis. In addition, the protein expression patterns were consistent with the results of decreased lipid peroxidation, increased non-protein thiols abundance, as well as iron and zinc content. These suggest that HRW alleviates Cd toxicity mainly by decreasing oxidative damage, enhancing sulfur compound metabolic process, and maintaining nutrient element homeostasis. BIOLOGICAL SIGNIFICANCE: Contamination of soils by Cd has become a potential concern to crops. Medicago sativa is a widely used forage around the world. Recently, hydrogen gas (H2) was suggested as a candidate of signal molecule, and found to effectively attenuate Cd-induced damage in alfalfa seedlings. However, the underlying molecular mechanism still needs to be further elucidated. In the present work, an iTRAQ-based quantitative proteomics was firstly carried out, and the results revealed the main molecular targets and metabolic processes associated with Cd resistance conferred by H2. This study may expand our understanding of hydrogen gas-medicated heavy metal tolerance in plants.

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids.

KEY MESSAGE: A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.

Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa.

Phytoremediation efficiency of a pcp-contaminated soil using four plant species as mono- and mixed cultures.

Bioremediation of soil polluted by pentachlorophenol (PCP) is of great importance due to the persistence and carcinogenic properties of PCP. Phytoremediation has long been recognized as a promising approach for removal of PCP from soil. The present study was conducted to investigate the capability of four plant species; white clover, ryegrass, alfalfa, and rapeseed grown alone and in combination to remediate pentachlorophenol contaminated soil. After 60 days cultivation, white clover, raygrass, alfalfa, and rapeseed all significantly enhanced the degradation of PCP in soils. Alfalfa showed highest efficiency for the removal of PCP in single cropping flowed by rapeseed and ryegrass. Mixed cropping significantly enhanced the remediation efficiencies as compared to single cropping; about 89.84% of PCP was removed by mixed cropping of rapeseed and alfalfa, and 72.01% of PCP by mixed cropping of rape and white clover. Mixed cropping of rapeseed with alfalfa was however far better for the remediation of soil PCP than single cropping. An evaluation of soil biological activities as a monitoring mechanism for the bioremediation process of a PCP-contaminated soil was made using measurements of microbial counts and dehydrogenase activity.

Involvement of abscisic acid in the response of Medicago sativa plants in symbiosis with Sinorhizobium meliloti to salinity.

Legumes are classified as salt-sensitive crops with their productivity particularly affected by salinity. Abcisic acid (ABA) plays an important role in the response to environmental stresses as signal molecule which led us to study its role in the response of nitrogen fixation and antioxidant metabolism in root nodules of Medicago sativa under salt stress conditions. Adult plants inoculated with Sinorhizobium meliloti were treated with 1 µM and 10 µM ABA two days before 200 mM salt addition. Exogenous ABA together with the salt treatment provoked a strong induction of the ABA content in the nodular tissue which alleviated the inhibition induced by salinity in the plant growth and nitrogen fixation. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were induced by ABA pre-treatments under salt stress conditions which together with the reduction of the lipid peroxidation, suggest a role for ABA as signal molecule in the activation of the nodular antioxidant metabolism. Interaction between ABA and polyamines (PAs), described as anti-stress molecules, was studied being detected an induction of the common polyamines spermidine (Spd) and spermine (Spm) levels by ABA under salt stress conditions. In conclusion, ABA pre-treatment improved the nitrogen fixation capacity under salt stress conditions by the induction of the nodular antioxidant defenses which may be mediated by the common PAs Spd and Spm that seems to be involved in the anti-stress response induced by ABA.

Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation.

S-adenosylmethionine synthetase (SAMS) is the key enzyme catalysing the formation of S-adenosylmethionine (SAM), a precursor of polyamines and ethylene. To investigate the potential role of SAMS in cold tolerance, we isolated MfSAMS1 from the cold-tolerant germplasm Medicago sativa subsp. falcata and analysed the association of SAM-derived polyamines with cold tolerance. The expression of MfSAMS1 in leaves was greatly induced by cold, abscisic acid (ABA), H2O2 and nitric oxide (NO). Our data revealed that ABA, H2O2 and NO interactions mediated the cold-induced MfSAMS1 expression and cold acclimation in falcata. SAM, putrescine, spermidine and spermine levels, ethylene production and polyamine oxidation were sequentially altered in response to cold, indicating that SAMS-derived SAM is preferentially used in polyamine synthesis and homeostasis during cold acclimation. Antioxidant enzyme activities were also induced in response to cold and showed correlation with polyamine oxidation. Overexpression of MfSAMS1 in tobacco resulted in elevated SAM levels, but polyamine levels and ethylene production in the transgenic plants were not significantly changed. Compared to the wild type, transgenic plants had increased levels of apoplastic H2O2, higher transcript levels of genes involved in polyamine synthesis and oxidation, and higher activities of polyamine oxidation and antioxidant enzymes. The results showed that overexpression of MfSAMS1 promoted polyamine synthesis and oxidation, which in turn improved H2 O2 -induced antioxidant protection, as a result enhanced tolerance to freezing and chilling stress in transgenic plants. This is the first report demonstrating that SAMS plays an important role in plant tolerance to cold via up-regulating polyamine oxidation.

Effect of elevated CO2 and temperature on the oxidative stress response to drought in Lolium perenne L. and Medicago sativa L.

Studies addressing the combined impact of multiple climate factors on plant abiotic stress responses are still scarce. We investigated physiological and molecular (antioxidant), responses to water deficit, in grassland-model species, Lolium perenne L. and Medicago lupulina L., under future climate conditions, i.e. elevated CO2 (+CO2, +375 ppm) and elevated temperature (+T, +3 °C). Elevated CO2, but not warming, significantly increased biomass (gDW) in L. perenne, but not in M. lupulina. Photosynthesis (A(sat)) and stomatal conductance (g(s)), were differently affected by climate in each species, L. perenne generally being more sensitive. Elevated CO2 increased lipid peroxidation levels in M. lupulina, but not in L. perenne, and had no effect on protein oxidation and little effect on antioxidant levels. Drought stress caused severe inhibition in biomass and photosynthesis, most severely in L. perenne, and strongly increased oxidative damage. Elevated CO2 protected against the drought-induced damage. Decreased activities of APX and POX may indicate lower levels of oxidative challenge (relaxation) at the level of H2O2 production. Polyphenols, tocopherols and antioxidant capacity, increased under drought stress, in all climate conditions. Elevated CO2, increased reduced ascorbate (ASC) and reduced glutathione (GSH), and their redox status, in both species, although to different levels. Changes in activities of key ASC/GSH cycle enzymes, under stress and climate treatments, showed weak correlations with ASC and GSH levels, indicating the complexity of this network. Together this work supports the idea that redox changes are involved in responses to climate changes, in the absence and presence of water-deficit stress.

Overexpression of alfalfa mitochondrial HSP23 in prokaryotic and eukaryotic model systems confers enhanced tolerance to salinity and arsenic stress.

The cloning and characterization of a gene (MsHSP23) coding for a heat shock protein in alfalfa in a prokaryotic and model plant system is described. MsHSP23 contains a 633 bp ORF encoding a polypeptide of 213 amino acids and exhibits greater sequence similarity to mitochondrial sHSPs from dicotyledons than to those from monocotyledons. When expressed in bacteria, recombinant MsHSP23 conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was cloned in a plant expressing vector and transformed into tobacco, a eukaryotic model organism. The transgenic plants exhibited enhanced tolerance to salinity and arsenic stress under ex vitro conditions. In comparison to wild type plants, the transgenic plants exhibited significantly lower electrolyte leakage. Moreover, the transgenic plants had superior germination rates when placed on medium containing arsenic. Taken together, these overexpression results imply that MsHSP23 plays an important role in salinity and arsenic stress tolerance in transgenic tobacco. This approach could be useful to develop stress tolerant crops including forage crops.

From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement.

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa.

Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants were assayed for exclusion of Al from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Overall, only the soil assays yielded consistent results. Based on the soil assays, two transgenic events were identified that were more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance.

Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

Ghrelin enhances glucose-induced insulin secretion in scheduled meal-fed sheep.

The purpose of this study was to investigate the effects of physiologic levels of ghrelin on insulin secretion and insulin sensitivity (glucose disposal) in scheduled fed-sheep, using the hyperglycemic clamp and hyperinsulinemic euglycemic clamp respectively. Twelve castrated Suffolk rams (69.8 +/- 0.6 kg) were conditioned to be fed alfalfa hay cubes (2% of body weight) once a day. Three hours after the feeding, synthetic ovine ghrelin was intravenously administered to the animals at a rate of 0.025 and 0.05 mug/kg body weight (BW) per min for 3 h. Concomitantly, the hyperglycemic clamp or the hyperinsulinemic euglycemic clamp was carried out. In the hyperglycemic clamp, a target glucose concentration was clamped at 100 mg/100 ml above the initial level. In the hyperinsulinemic euglycemic clamp, insulin was intravenously administered to the animals for 3 h at a rate of 2 mU/kg BW per min. Basal glucose concentrations (44+/- 1 mg/dl) were maintained by variably infusing 100 mg/dl glucose solution. In both clamps, plasma ghrelin concentrations were dose-dependently elevated and maintained at a constant level within the physiologic range. Ghrelin infusions induced a significant (ANOVA; P < 0.01) increase in plasma GH concentrations. In the hyperglycemic clamp, plasma insulin levels were increased by glucose infusion and were significantly (P < 0.05) greater in ghrelin-infused animals. In the hyperinsulinemic euglycemic clamp, glucose infusion rate, an index of insulin sensitivity, was not affected by ghrelin infusion. In conclusion, the present study has demonstrated for the first time that ghrelin enhances glucose-induced insulin secretion in the ruminant animal.