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Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules.

Respiration in support of N(2) fixation by rhizobia in legume root nodules depends on an adequate supply of O(2), but excessive O(2) can damage nitrogenase, the key enzyme. The movement of O(2) into and within the nodule is driven by gradients in the concentration of O(2) or in the oxygenation of the O(2)-carrier, leghaemoglobin. Steeper gradients may increase flux to the sites of respiration, but gradients also raise the possibility of inadequate O(2) in some nodule zones and excessive O(2) in others. No detailed study of O(2) gradients in the interior of nodules has been published previously. Spectral changes in leghaemoglobin with oxygenation, previously used to measure the average O(2) status of the nodule interior, were used to map longitudinal gradients in O(2) and in respiratory capacity in the elongated nodules of alfalfa (Medicago sativa L.) and sweetclover (Melilotus officinalis L.). Variability among nodules under air in the magnitude and direction of internal O(2) gradients was seen in both species. Despite consistently higher respiratory capacity near the meristematic tip, a majority of nodules had higher O(2) towards the tip than towards the base. These results contrast with a previous report, apparently based on limited data, but they are consistent with anatomical and tracer studies showing higher gas permeability near the tip.

Algorithms↗

Effect of root death and decay on dissipation of polycyclic aromatic hydrocarbons in the rhizosphere of yellow sweet clover and tall fescue.

A 12-mo greenhouse study was conducted to evaluate the contribution of root death and decay on the dissipation of polycyclic aromatic hydrocarbons (PAHs) in rhizosphere soil. The contaminated soil was previously treated by land-farming, but residual PAHs remained after treatment. Tall fescue (Festuca arundinacea Schreb.) and yellow sweet clover (Melilotus officinalis Lam.) were the target plants. To specifically evaluate the effect of root decay on contaminant dissipation, plants were treated with glyphosate, a broad spectrum herbicide, to induce root decay. Although tall fescue treatments had the highest root and shoot biomass and root surface area, this plant did not result in the highest contaminant degradation rates. Significant differences were noted between treatments for seven PAHs, with the active yellow sweet clover resulting in 60 to 75% degradation of these compounds. Induced root death and decay did not produce a significant enhancement of PAH degradation. The PAH microbial degrader populations in the vegetated treatments were more than 100 times greater than those in the unvegetated control. The phospholipid fatty acid (PLFA) structural group profile shifted over the growing period, indicating a change in the community structure. In conclusion, phytoremediation was shown to be an effective polishing tool for PAH-affected soil previously subjected to biological treatment.

Biodegradation, Environmental↗

[Chronic venous insufficiency: an open trial of FLEBS Crema].

AIM: This study was realized to evaluate the FLEBS CREMA tolerability and its therapeutic efficacy, in patients with functional disease linked to not complicated venous or lymphatic insufficiency. This compound acts as vasoconstrictor thanks to the vegetable extract of Ruscus aculeatus, but it is also able to reduce the edema, thanks to the vegetable extract of Melilotus officinalis. METHODS: All the patients were treated for three weeks with FLEBS CREMA, that was applied 2 or 3 times a day, on both legs. The traditional clinical parameters of chronic venous insufficiency (IVC) (edema, pain, heaviness, itch, cramps and other symptoms) were evaluated during the treatment. RESULTS: The results show an improvement for all the monitored symptoms, also if the positive clinical response was different for each evaluated parameter. The best results were obtained for edema, pain, heaviness and itch: the statistical analysis shows indeed a significant difference between the beginning and the end of the treatment. In this study the dermo-cosmetic qualities and the tolerability of FLEBS CREMA were also evaluated: both these parameters obtained a positive clinical evaluation. CONCLUSIONS: Basing on these data, the final evaluation for this compound was good/very good for the 80-90% both doctor and patients.

Adult↗

Interference of three weed extracts on uptake of nutrient in three different varieties of paddy through radio tracer techniques.

Interference of three dominant weed extracts viz., Ageratum conyzoides L., Melilotus indica All. and Parthenium hysterophorus L. were examined on seed germination, seedling growth, and nutrient uptake (32P and 65Zn) in three different varieties (PD-10, PD-12 and PB) of paddy (Oryza sativa L.). Among the three different varieties irrespective of weed extracts, PD-10 and PD-12 were resistant and PB was susceptible in terms of seed germination, radicle length and plumule dry weight; and PD-12 and PB were resistant and susceptible, respectively, in terms of plumule length and total seedling dry weight. A. conyzoides caused maximum reduction in seed germination and M. indica in seedling growth in different varieties of paddy. The weed extracts interfered in uptake of both 32P and 65Zn and there was a gradual decrease in uptake of both nutrients with increasing concentration of extracts in both root and shoot. The uptake of 32P and 65Zn was more inhibitory with the extracts of A. conyzoides and M. indica, respectively in different varieties. The inhibition in seed germination, seedling growth and nutrient uptake may be due to the presence of phenolics and other secondary metabolities. The phenolics such as gallic, vanillic, protocatechuic and p-hydroxybenzoic acids were identified from these weed extracts.

Analysis of Variance↗

[Application of wastewater land treatment technique to the construction of ecological engineering in sand land].

In this paper studies on the feasibility of harmlessness and resource of wastewater, which was discharged from a thermal power plant, by using slow rate filtration of land treatment technique for the fast recovery of vegetation in the Kubuqi sand land were carried out. The selected arbor, shrub and herbage in the land treatment system were poplar (Populus alba Var. Pyramidalis bunge), seabuckthorn (Hippophae rhamnoides) and sweet clover (Melilotus suaveolens) respectively. Three levels of wastewater hydraulic loading were designed in the field pilot experiment. They were high plot with 3000 mm/a irrigation (H), medium plot with 1500 mm/a irrigation (M) and low plot (L) with small volume of irrigation only used in the period of transplant seedlings. The performance indicate that the purification function of power plant wastewater by pre-treatment through combination of precipitation pool with storage ponds is effective and therefore the effluent after pretreatment can be used to irrigation vegetation. The experiment results show that the volume of tree crown for poplar in H plot and M plot was up to 1.07 and 2.21 times comparing with L plot respectively. The annual yield (dry weight) of sweet clover in H plot and M plot was up to 2.33 and 3.0 times comparing with L plot respectively. The height of seabuckthorn in H zone and M plot was up to 1.08 and 1.32 times comparing with L plot respectively. There is direct proportion between growth status of vegetation and hydraulic loading of irrigation. The contents of heavy metals for sweet clover (Cd 0.021 mg/kg, Pb<0.001 mg/kg, Cr <0.01 mg/kg, As 0.043 mg/kg) are much lower than the food standards of grain and vegetables, therefore the sweet clover for raising livestock is safe. Wastewater in this area is valuable source. Its reasonable utilization can contribute important benefits in economy and ecology in the ecological construction and developing effective agriculture and animal husbandry.

Biodegradation, Environmental↗

Profiling isoflavonoids found in legume root extracts using capillary electrophoresis.

Legumes such as alfalfa (Medicago sativa L.), barrel medic (Medicago truncatula), white sweet clover (Melilotus alba) and fenugreek (Trigonella graecum), normally accumulate (-)-medicarpin and its malonated glucose conjugate as natural inhibitors of fungal pathogens. These plants also accumulate the biosynthetic precursor formononetin as well as the malonated glycoside. We were interested in developing a robust high-throughput method to quantitate the levels of these two isoflavonoids, both free and conjugated, in legume root extracts, for use in screening for mutant plants accumulating altered levels of these compounds. Capillary electrophoresis was examined as an alternative to current high-performance liquid chromatography (HPLC) methods to generate isoflavonoid profiles. The developed assay used micellar electrokinetic capillary chromatography (MEKC) to provide the required selectivity in complex root extracts. The addition of 1,2-hexanediol to the sodium dodecyl sulfate (SDS) electrolyte provided improved resolution of adjacent isoflavonoids. We examined the role of several factors including sample preparation, buffer composition, buffer pH, and organic component in the injected sample. The use of capillaries with longer path lengths were also examined to increase sensitivity. A comparison of results obtained using MEKC and HPLC showed good correlation in the relative amounts of the isoflavonoids studied.

Chromatography, High Pressure Liquid↗

Evaluation of proteome reference maps for cross-species identification of proteins by peptide mass fingerprinting.

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.

Electrophoresis, Gel, Two-Dimensional↗

Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective.

The proteome of the model symbiotic bacterium, Sinorhizobium meliloti was examined to determine the enzymatic reactions and cell processes that occur when S. meliloti occupies the root nodules of Medicago truncatula and Melilotus alba. The proteomes of the nodule bacteria were compared to that of S. meliloti grown under laboratory cultured conditions as an additional control. All the detectable protein spots on the two-dimensional (2-D) gels between pH 4-7 were analyzed. In total, the identity of proteins in 1545 spots from 2-D gels was determined using peptide mass fingerprinting. There were clear differences in the proteome of nodule bacteria and cultured bacteria and putative nodule-specific and nodule suppressed proteins were identified. The data were analyzed using metabolic pathway prediction programs and used to review the biochemical and genetic studies that had been done previously on S. meliloti over several decades. There was a broad congruency between the proteomic and biochemical data when the overall pathways of central carbon and nitrogen metabolism were considered. A selective suite of ABC-type transporters was present in nodule bacteria that were biased towards the transport of amino acids and inorganic ions (P and Fe) suggesting that a highly specialized nutrient exchange was occurring between the nodule bacteria and the host. Proteins prominent in nodule bacteria were those involved in the pathways for vitamin synthesis and stress-related processes (chaperoning, heat shock, detoxification of reactive oxygen species, regulation of stress and osmo-regulation). Some of these proteins were found only in nodule bacteria. These results show the extent of the shift in metabolism that occurs when S. meliloti invades legume plants and establishes a nitrogen fixing symbiosis.

Biological Transport↗

Natural establishment and selenium accumulation of herbaceous plant species in soils with elevated concentrations of selenium and salinity under irrigation and tillage practices.

The effects of irrigation and tillage practices were studied on species richness, biomass, and selenium accumulation of naturally established herbaceous plants in soils with elevated levels of selenium (Se) and salinity at Kesterson Reservoir, Merced County, California. The four different irrigation-tillage practice combinations were (1) no irrigation, no tillage; (2) irrigation, no tillage; (3) no irrigation, tillage; and (4) irrigation, tillage. The fields were allowed to become colonized naturally by herbaceous plant species. For the Mediterranean climate in the study site, irrigation was conducted biweekly through the summer months, and tillage was done in 3-month intervals. Biomass and Se accumulation of Atriplex patula L, Bassia hyssopifolia Kuntze, Rev. Gen. Pl., Melilotus indica (L.) All., and Salsola kali L. were substantially affected by irrigation. The degree and direction of the effects were found to be species dependent. The field plots which were tilled at 3-month intervals remained bare throughout the experiment. The total soil Se concentrations in the top 15 cm soil horizon were found to be in the range of 40 to 70 mg kg-1 dry wt. Soil Se concentrations below 25 cm soil depth were much lower and within a range of 2 to 4 mg kg-1. Less than 1/10th of the total soil Se inventory in the top soil horizon was water extractable, and the distribution of the Se inventory did not change significantly over the period of 1990 and 1991 despite the irrigation and tillage practices suggesting that a large portion of the Se inventory was not remobilized. The water-extractable soil Se concentration was found to be significantly lower in soils with the greatest biomass production suggesting an effective bioextraction of soil selenium by the native herbaceous plants.

Agriculture↗

Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for D-glucosamine synthetase.

The nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the D-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS- mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied D-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. melioti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.

Amino Acid Sequence↗

Plant bioassays for an in situ monitoring of air near an industrial area and a municipal solid waste: Zilina (Slovakia).

The process of a bioindication of genotoxic effects of complex mixtures on the environment using higher plants is very appropriate and effective. We present the results of an in situ indication of the genotoxic effects of polluted environment near Zilina city. For a more complex monitoring we used: the Tradescantia micronucleus (Trad-MCN) assay, the Tradescantia microspore test and an evaluation of the abortivity of the pollen grains of native plant species. We found significant differences in the frequency of the micronuclei when using the Trad-MCN test in local of Duben. The Tradescantia pollen abortivity test showed significant differences in the frequency of the abortive pollen grains between the exposed groups and the control group. By using native plant species in the pollen abortivity test we found significant differences in both of the two locations for the four following species during two consecutive years: Artemisia vulgaris, Melilotus albus, Trifolium pratense, Typha latifolia.

Air Pollutants↗

Structure and development of P-protein in phloem parenchyma and companion cells of legumes.

Phloem parenchyma and companion cells from four species of legumes, Phaseolus vulgaris L., Melilotus alba Desr., Desmodium canadense, L. and Dolichos lablab L., were examined electron microscopically to estabish the presence, structure and development of P-protein. P-protein components consisting of granular, fibrillar, tubular or crystalline structures were found in parenchyma cells of all species and in companion cells of M. alba. The earliest stages of P-protein formation were closely associated with dictyosome cisternae, dictyosome vesicles and/or spiny vesicles. After their formation, the P-protein bodies were frequently transformed into one or more structurally different components. Although these components appeared to be develop-mentally related, their specific associations and transformations differed in each species examined.

Inclusion Bodies↗

Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

Bacterial Proteins↗

Species richness and selenium accumulation of plants in soils with elevated concentration of selenium and salinity.

Field studies were conducted in soils with elevated concentrations of Se and salinity at Kesterson, California. Biomass distribution, species richness, and selenium accumulation of plants were examined for two sites where 15 cm of surface soil was removed and replaced with fill dirt in the fall of 1989, and two sites were native soil cover. The Se concentrations in the top 15 cm of fill dirt ranged from undetectable to 36 ng g-1. For the native soil sites, Se levels ranged from 75 to 550 ng g-1. Soil Se concentrations below 15 cm ranged from 300 to 700 ng g-1 and were comparable between the fill dirt and the native soil sites. At least 20 different plant species were brought into the two fill dirt sites with the top soil. Avena fatua L., Bassia hyssopifolia Kuntze Rev. Gen. Pl., Centaurea solstitialis L., Erysimum officianale L., Franseria acanthicarpa Cav. Icon., and Melilotus indica (L.) All. contributed over 60% of the total biomass. Only 5 species were found in the native soil sites, and salt grass (Distichlis spicata L.) was the predominant species and accounted for over 80% of the total biomass. Between 1989 and 1990, two years after the surface soil replacement, the two fill dirt sites had a 70% reduction in species richness. Plant tissue selenium concentrations were found to be quite variable between plant species and between sites of sampling. At the fill dirt sites, the plant species with deep root systems accumulated greater amounts of selenium than the shallow-rooted species. The soil selenium concentration of the field soil had no negative effect on pollen fertility, seed set, and seed germination for the plant species examined. However, seedling growth was impaired by the soil selenium concentrations. This suggests that a selection pressure of soil Se concentration may have been imposed on plant species such as M. indica in an early stage of its life cycle.

Plant Development↗

Antiviral activities of some Ethiopian medicinal plants used for the treatment of dermatological disorders.

Acokanthera schimperi (Apocynaceae), Euclea schimperi (Ebenaceae), Inula confertiflora (Asteraceae), Melilotus elegans (Leguminosae), and Plumbago zeylanica (Plumbaginaceae), are some of the medicinal plants used in Ethiopia for treatment of various skin disorders. In this study, the antiviral activities of the 80% methanolic extracts of these plants have been examined against coxsackievirus B3 (CVB3), influenza A virus and herpes simplex virus type1 Kupka (HSV-1) using cytopathic effect (CPE) inhibitory assays in HeLa, MDCK, and GMK cells, respectively. In parallel, the cytotoxicity was quantified using a crystal violet uptake assay. The antiviral activity of the most active compound was confirmed with plaque reduction assays. The results revealed that the extracts of Acokanthera schimperi and Euclea schimperi showed antiviral activity against all three tested viruses albeit with unequal efficacy. Whereas the Acokanthera schimperi extract exhibited the strongest activity against CVB3, the extract of Euclea schimperi inhibited influenzavirus A replication most effectively. A weak anti-influenzavirus A activity was also exhibited by the other plant extracts tested. In addition, CVB3 was inhibited by the extracts of Plumbago zeylanica and HSV-1 by Inula confertiflora. Thus, the extracts of these plants, particularly those of Acokanthera schimperi, Euclea schimperi and Inula confertiflora which showed activity against CVB3 and HSV-1 support their traditional use in the treatment of skin diseases of viral origin.

Animals↗

Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region.

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.

DNA Fingerprinting↗

Rhizobium meliloti has three functional copies of the nodD symbiotic regulatory gene.

We have identified two Rhizobium meliloti genes (nodD2 and nodD3) that are highly homologous and closely linked to the regulatory gene nodD (nodD1). R. meliloti strains containing mutations in the three nodD genes in all possible combinations were constructed and their nodulation phenotypes were assayed on Medicago sativa (alfalfa) and Melilotus alba (sweet clover). A triple nodD1-nodD2-nodD3 mutant exhibited a Nod- phenotype on alfalfa and sweet clover, indicating that nodD is an essential nodulation gene in R. meliloti. A nodD2 mutant exhibited no discernable defect in nodulation and nodD3 mutants exhibited a delayed nodulation phenotype of 2-3 days when inoculated onto either host. Alfalfa nodules elicited by a nodD1 mutant appeared 5-6 days after wild-type nodules, and sweet clover nodules elicited by a nodD1 mutant appeared 2-3 days after wild-type nodules. nodD1-nodD2 double mutants formed nodules with the same delay as single nodD1 mutants on both hosts. nodD2-nodD3 double mutants elicited sweet clover nodules at the same rate as single nodD3 mutants, but this same double mutant was slightly more delayed in alfalfa nodule formation than the nodD3 mutant. The nodD1-nodD3 mutant exhibited an extremely delayed nodulation phenotype on alfalfa and elicited no nodules on sweet clover. These experiments indicate that nodD1 and nodD3 have equivalent roles in nodulating sweet clover but that nodD1 plays a more important role than nodD3 in eliciting nodules on alfalfa. The nodD2 gene appears to have some effect on alfalfa nodulation and none on sweet clover. Our results indicate that R. meliloti has three functional nodD genes that modulate the nodulation process in a host-specific manner.

Chromosome Mapping↗

A second exopolysaccharide of Rhizobium meliloti strain SU47 that can function in root nodule invasion.

Rhizobium meliloti strain SU47 produces the calcofluor-binding exopolysaccharide, succinoglycan, that is required for alfalfa root nodule invasion. Strains derived from R. meliloti SU47 secreted an acidic exopolysaccharide, EPSb, that replaced succinoglycan in nodule invasion. EPSb, which has not formerly been identified among the Rhizobiaceae, consisted of the repeating unit 4,6-O-(1-carboxyethylidene)-alpha-D-Galp1----3(X-O-Ac)-beta-D-G lcp1----3. EPSb synthesis occurred either in strains containing a mutation in a locus designated mucR or in strains with a recombinant cosmid pMuc. mucR mapped slightly counterclockwise from pyr49 on the chromosome, while pMuc contained genes mapping to the megaplasmid pRmeSU47b. In exoA, -F, and -H mutants, which are deficient in normal succinoglycan secretion and nodule invasion, a transposon Tn5 insertion in mucR or the presence of pMuc resulted in EPSb secretion and a restoration of nodule invasion on Medicago sativa and Melilotus alba. Mutants in exoB and exoC were incapable of succinoglycan and EPSb secretion as well as nodule invasion. A mutant that secreted succinoglycan but was incapable of EPSb secretion invaded nodules normally.

Carbohydrate Conformation↗