Comparative GC-MS based nutrients profiling of less explored legume seeds of Melilotus, Medicago, Trifolium, and Ononis analysed using chemometric tools.

Exploring novel sources of plant protein for nutrition of both humans and animals is motivated mainly by its growing demand worldwide, besides identifying healthy alternatives for animal protein. The present study evaluates metabolome diversity within 15 legume seed species. The examined samples comprised three Melilotus, four Medicago, four Trifolium, and four Ononis seed species. A holistic approach for metabolites profiling using gas chromatography-mass spectrometry (GC-MS) led to the annotation and quantification of 87 metabolites comprising alcohols, free amino acids, aromatics, fatty acids/esters, nitrogenous compounds, organic acids, sugar alcohols, sugars, terpenes, and steroids. Fatty acids represented the major metabolite class represented by palmitic, stearic, oleic, linoleic, and linolenic acids. Sucrose and pinitol were the major sugars and sugar alcohols among seeds. Ononis seeds (OR, OS and OA) were the most abundant in fatty acids, sugars, sugar alcohols, and free amino acids, whereas Melilotus species (MO and MS) were least enriched in these key nutrients posing Ononis as potential food source for humans and animals. The examined seeds were generally low in sulfur-containing free amino acids and lacking many of the essential free amino acids. Multivariate data analysis aided in the identification of Ononis metabolite markers belonging to various classes i.e., (alcohol) glycerol, (sugar) allofuranose, and (sugar alcohol) pinitol, although the differentiation between Medicago, Melilotus, and Trifolium genera was not attained suggestive for other analytical platforms for its classification.

Genome-Wide Identification of ERF Transcription Factor Family and Functional Analysis of the Drought Stress-Responsive Genes in Melilotus albus.

As an important forage legume with high values in feed and medicine, Melilotus albus has been widely cultivated. The AP2/ERF transcription factor has been shown to play an important regulatory role in plant drought resistance, but it has not been reported in the legume forage crop M. albus. To digger the genes of M. albus in response to drought stress, we identified and analyzed the ERF gene family of M. albus at the genome-wide level. A total of 100 MaERF genes containing a single AP2 domain sequence were identified in this study, named MaERF001 to MaERF100, and bioinformatics analysis was performed. Collinearity analysis indicated that segmental duplication may play a key role in the expansion of the M. albus ERF gene family. Cis-acting element predictions suggest that MaERF genes are involved in various hormonal responses and abiotic stresses. The expression patterns indicated that MaERFs responded to drought stress to varying degrees. Furthermore, four up-regulated ERFs (MaERF008, MaERF037, MaERF054 and MaERF058) under drought stress were overexpressed in yeast and indicated their biological functions to confer the tolerance to drought. This work will advance the understanding of the molecular mechanisms underlying the drought response in M. albus. Further study of the promising potential candidate genes identified in this study will provide a valuable resource as the next step in functional genomics studies and improve the possibility of improving drought tolerance in M. albus by transgenic approaches.

Genome and systems biology of Melilotus albus provides insights into coumarins biosynthesis.

Melilotus species are used as green manure and rotation crops worldwide and contain abundant pharmacologically active coumarins. However, there is a paucity of information on its genome and coumarin production and function. Here, we reported a chromosome-scale assembly of Melilotus albus genome with 1.04 Gb in eight chromosomes, containing 71.42% repetitive elements. Long terminal repeat retrotransposon bursts coincided with declining of population sizes during the Quaternary glaciation. Resequencing of 94 accessions enabled insights into genetic diversity, population structure, and introgression. Melilotus officinalis had relatively larger genetic diversity than that of M. albus. The introgression existed between M. officinalis group and M. albus group, and gene flows was from M. albus to M. officinalis. Selection sweep analysis identified candidate genes associated with flower colour and coumarin biosynthesis. Combining genomics, BSA, transcriptomics, metabolomics, and biochemistry, we identified a ß-glucosidase (BGLU) gene cluster contributing to coumarin biosynthesis. MaBGLU1 function was verified by overexpression in M. albus, heterologous expression in Escherichia coli, and substrate feeding, revealing its role in scopoletin (coumarin derivative) production and showing that nonsynonymous variation drives BGLU enzyme activity divergence in Melilotus. Our work will accelerate the understanding of biologically active coumarins and their biosynthetic pathways, and contribute to genomics-enabled Melilotus breeding.

The impact of aqueous and N-hexane extracts of three Fabaceae species on seed germination and seedling growth of some broadleaved weed species.

Weed infestation is a persistent problem for centuries and continues to be major yield reducing issue in modern agriculture. Chemical weed control through herbicides results in numerous ecological, environmental, and health-related issues. Moreover, numerous herbicides have evolved resistance against available herbicides. Plant extracts are regarded as an alternative to herbicides and a good weed management option. The use of plant extracts is environmentally safe and could solve the problem of herbicide resistance. Therefore, laboratory and wire house experiments were conducted to evaluate the phytotoxic potential of three Fabaceae species, i.e., Cassia occidentalis L. (Coffee senna), Sesbania sesban (L.) Merr. (Common sesban) and Melilotus alba Medik. (White sweetclover) against seed germination and seedling growth of some broadleaved weed species. Firstly, N-hexane and aqueous extracts of these species were assessed for their phytotoxic effect against lettuce (Lactuca sativa L.). The extracts found more potent were further tested against germination and seedling growth of four broadleaved weed species, i.e., Parthenium hysterophorus L. (Santa-Maria), Trianthema portulacastrum L. (Pigweed), Melilotus indica L (Indian sweetclover). and Rumex dentatus L. (Toothed dock) in Petri dish and pot experiments. Aqueous extracts of all species were more toxic than their N-hexane forms for seed germination and seedling growth of lettuce; therefore, aqueous extracts were assessed for their phytotoxic potential against four broadleaved weed species. Aqueous extracts of all species proved phytotoxic against T. portulacastrum, P. hysterophorus, M. indica and R. dentatus and retarder their germination by 57, 90, 100 and 58%, respectively. Nevertheless, foliar spray of C. occidentalis extract was the most effective against T. portulacastrum as it reduced its dry biomass by 72%, while M. alba was effective against P. hysterophorus, R. dentatus and M. indica and reduced their dry biomass by 55, 68 and 81%, respectively. It is concluded that aqueous extracts of M. alba, S. sesban and C. occidentalis could be used to retard seed germination of T. portulacastrum, P. hysterophorus, M. indica and R. dentatus. Similarly, aqueous extracts of C. occidentalis can be used to suppress dry biomass of T. portulacastrum, and those of M. alba against P. hysterophorus, R. dentatus. However, use of these extracts needs their thorough testing under field conditions.

Genome-Wide Analysis of the UDP-Glycosyltransferase Family Reveals Its Roles in Coumarin Biosynthesis and Abiotic Stress in Melilotus albus.

Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.

The identification of phytoextraction potential of Melilotus officinalis and Amaranthus retroflexus growing on copper- and molybdenum-polluted soils.

The contamination of soils by heavy metals from the mining industry nowadays is one of the greatest threats to environment and human health. The cleaning of polluted soils using cost-effective and eco-friendly methods such as phytoextraction has wide public recognition. Considering the above-mentioned ones, the objectives of the present study were the identification of Cu and Mo accumulation capability and the phytoextraction potential of Melilotus officinalis and Amaranthus retroflexus as well as the determination of the influence of ammonium nitrate and EDTA on phytoextraction effectiveness. The contaminated soil samples for phytoremediation experiments under ex situ conditions were collected from the surroundings of the Zangezur Copper and Molybdenum Combine, Armenia. During the studies, it was found out that M. officinalis and A. retroflexus are capable of growing in polluted soils. M. officinalis grown in polluted soil had greater ability to accumulate heavy metals in roots, while the ability to transport the copper to aboveground parts was more pronounced in A. retroflexus. During the growing of these plant species for phytoextraction of soils contaminated by copper, it is necessary to use chelates, in particular the EDTA, for the enhancement of the effectiveness of phytoextraction process. EDTA due to chelating influence increased the availability of copper for plants and its mobility in them that lead to greater accumulation of this metal in shoots. The application of chelates did not have a significant impact on molybdenum accumulation intensity in plants; therefore, in case of this metal, it is unreasonable to use additional chelating compounds.

Coumarin Content, Morphological Variation, and Molecular Phylogenetics of Melilotus.

Melilotus albus and Melilotus officinalis are widely used in forage production and herbal medicine due to the biological activity of their coumarins, which have many biological and pharmacological activities, including anti-HIV and anti-tumor effects. To comprehensively evaluate M. albus and M. officinalis coumarin content (Cou), morphological variation, and molecular phylogeny, we examined the Cou, five morphological traits and the molecular characterization based on the trnL-F spacer and internal transcribed spacer (ITS) regions of 93 accessions. Significant (p < 0.05) variation was observed in the Cou and all five morphological traits in both species. Analysis of population differentiation (Pst) of the phenotypic traits showed that powdery mildew resistance (PMR) had the greatest Pst, meaning that this trait demonstrated the largest genetic differentiation among the accessions. The Pst values of dry matter yield (DMY) and Cou were relatively high. Biplot analysis identified accessions with higher DMY and higher and lower Cou. Analysis of molecular sequence variation identified seven haplotypes of the trnL-F spacer and 13 haplotypes of the ITS region. Based on haplotype and sequence analyses, the genetic variation of M. officinalis was higher than that of M. albus. Additionally, ITS sequence analysis showed that the variation among accessions was larger than that among species across three geographical areas: Asia, Europe, and North America. Similarly, variation among accessions for both the trnL-F and ITS sequences were larger than the differences between the geographical areas. Our results indicate that there has been considerable gene flow between the two Melilotus species. Our characterization of Cou and the morphological and genetic variations of these two Melilotus species may provide useful insights into germplasm improvement to enhance DMY and Cou.

Waterlogging tolerance, tissue nitrogen and oxygen transport in the forage legume Melilotus siculus: a comparison of nodulated and nitrate-fed plants.

Background and Aims: Soil waterlogging adversely impacts most plants. Melilotus siculus is a waterlogging-tolerant annual forage legume, but data were lacking for the effects of root-zone hypoxia on nodulated plants reliant on N2 fixation. The aim was to compare the waterlogging tolerance and physiology of M. siculus reliant on N2 fixation or with access to NO3-. Methods: A factorial experiment imposed treatments of water level (drained or waterlogged), rhizobia (nil or inoculated) and mineral N supply (nil or 11 mm NO3-) for 21 d on plants in pots of vermiculite in a glasshouse. Nodulation, shoot and root growth and tissue N were determined. Porosity (gas volume per unit tissue volume) and respiration rates of root tissues and nodules, and O2 microelectrode profiling across nodules, were measured in a second experiment. Key Results: Plants inoculated with the appropriate rhizobia, Ensifer (syn. Sinorhizobium) medicae, formed nodules. Nodulated plants grew as well as plants fed NO3-, both in drained and waterlogged conditions. The growth and total N content of nodulated plants (without any NO3- supplied) indicated N2 fixation. Respiration rates (mass basis) were highest in nodules and root tips and lowest in basal root tissues. Secondary aerenchyma (phellem) formed along basal root parts and a thin layer of this porous tissue also covered nodules, which together enhanced gas-phase diffusion of O2 to the nodules; O2 was below detection within the infected zone of the nodule interior. Conclusions: Melilotus siculus reliant on N2 fixation grew well both in drained and waterlogged conditions, and had similar tissue N concentrations. In waterlogged conditions the relatively high respiration rates of nodules must rely on O2 movement via the aerenchymatous phellem in hypocotyl, roots and the outer tissue layers of nodules.

Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content.

Coumarin and its derivatives are widely used as fragrances in industrial products and have medical value. The goal of the present study was to discover genes and pathways related to coumarin biosynthesis in Melilotus albus using transcriptome analysis. The genes of five M. albus near-isogenic lines (NILs) that had different coumarin content and ß-glucosidase activity according to the investigation of pedigree were quantified and then analysed by RNA-Seq. Using transcriptome analysis, differentially expressed genes (DEGs) were identified in two pairwise comparisons that differed in coumarin content as well as in two pairwise comparisons that differed in ß-glucosidase activity. Gene expression pattern analysis suggested similar transcriptional trends in the genotypes with the same coumarin levels. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database of DEGs was used to identify functional pathways associated with coumarin biosynthesis. We identified 111 unigenes, with several DEGs among them possibly being related to coumarin synthesis pathways. Unigenes encoding a hexokinase, an abscisic acid receptor, a phenylalanine ammonia-lyase (PAL) and two peroxidases particularly showed correspondence with the coumarin content of different genotypes. These results will contribute to a better understanding of the coumarin biosynthesis in M. albus.

Hydrogen peroxide-dependent antibacterial action of Melilotus albus honey.

Honey originating from different floral sources exhibits the broad spectrum of antibacterial activity as a result of the presence of hydrogen peroxide as well as nonperoxide bioactive compounds. The mechanisms of antibacterial activity of Polish melilot honey were investigated for the first time. Polish melilot honey samples (Melilotus albus biennial = 3 and annual = 5, Melilotus officinalis = 1) were collected directly from beekeepers and analysed for pollen profile, basic physicochemical parameters, antioxidant capacity, radical scavenging activity, total phenolic contents as well as antibacterial properties against pathogenic bacteria Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella spp. The physicochemical properties of melilot honey were specific for light-coloured unifloral honey samples and were not dependent on its botanical and geographical origin (P > 0·05). All tested honey samples exhibited inhibitory activity (above 90%) against Gram-positive bacteria at the concentration of 12·5-25%. Above 30-50% of antibacterial activity of melilot honey was connected with glucose oxidase enzyme action and was destroyed in the presence of catalase. Hydrogen peroxide-dependent antibacterial activity of honey was inversely correlated with its radical scavenging activity (r = -0·67) and phenolic compounds (r = -0·61). Antibacterial action of melilot honey depends not only on hydrogen peroxide produced by glucose oxidase, but also on other nonperoxide bioactive components of honey. SIGNIFICANCE AND IMPACT OF THE STUDY: Melilot honey is used in traditional medicine as an anticoagulant agent due to the possibility of the presence of the coumarin compounds which are specific for Melilotus plant. Melilotus albus is rarely used to produce honey, and antibacterial properties of this variety of honey had not been studied yet. Nine samples of melilot honey produced in different regions of Poland were analysed according to their antibacterial activity which was correlated with physiochemical parameters and antioxidant activity. It was shown that antibacterial activity of melilot honey is created by hydrogen peroxide and other bioactive compounds.

The single evolutionary origin of chlorinated auxin provides a phylogenetically informative trait in the Fabaceae.

Chlorinated auxin (4-chloroindole-3-acetic acid, 4-Cl-IAA), a highly potent plant hormone, was once thought to be restricted to species of the tribe Fabeae within the Fabaceae, until we recently detected this hormone in the seeds of Medicago, Melilotus and Trifolium species. The absence of 4-Cl-IAA in the seeds of the cultivated species Cicer aeritinum from the Cicerae tribe, immediately basal to the Fabeae and Trifolieae tribes, suggested a single evolutionary origin of 4-Cl-IAA. Here, we provide a more robust phylogenetic placement of the ability to produce chlorinated auxin by screening key species spanning this evolutionary transition. We report no detectable level of 4-Cl-IAA in Cicer echinospermum (a wild relative of C. aeritinum) and 4 species (Galega officinalis, Parochetus communis, Astragalus propinquus and A. sinicus) from tribes or clades more basal or sister to the Cicerae tribe. We did detect 4-Cl-IAA in the dry seeds of 4 species from the genus Ononis that are either basal to the genera Medicago, Melilotus and Trigonella or basal to, but still within, the Fabeae and Trifolieae (ex. Parochetus) clades. We conclude that the single evolutionary origin of this hormone in seeds can be used as a phylogenetically informative trait within the Fabaceae.

Biogas production from coumarin-rich plants--inhibition by coumarin and recovery by adaptation of the bacterial community.

Plants like sweet clover (Melilotus spp.) are not suitable as fodder for cattle because of harmful effects of the plant secondary metabolite coumarin. As an alternative usage, the applicability of coumarin-rich plants as substrates for biogas production was investigated. When coumarin was added to continuous fermentation processes codigesting grass silage and cow manure, it caused a strong inhibition noticeable as decrease of biogas production by 19% and increase of metabolite concentrations to an organic acids/alkalinity ratio higher than 0.3(gorganic acids) gCaCO3 (-1). Microbial communities of methanogenic archaea were dominated by the genera Methanosarcina (77%) and Methanoculleus (11%). This community composition was not influenced by coumarin addition. The bacterial community analysis unraveled a divergence caused by coumarin addition correlating with the anaerobic degradation of coumarin and the recovery of the biogas process. As a consequence, biogas production resumed similar to the coumarin-free control with a biogas yield of 0.34 LN g(volatile solids) (-1) and at initial metabolite concentrations (∼ 0.2 g(organic acids) gCaCO3 (-1)). Coumarin acts as inhibitor and as substrate during anaerobic digestion. Hence, coumarin-rich plants might be suitable for biogas production, but should only be used after adaptation of the microbial community to coumarin.

Arbuscular mycorrhizal fungi on growth, nutrient status, and total antioxidant activity of Melilotus albus during phytoremediation of a diesel-contaminated substrate.

This research evaluated the effects of arbuscular mycorrhizal fungi (AMF) on growth, nutritional status, total antioxidant activity (AOX), total soluble phenolics content (TPC), and total nitrate reductase activity (NRA) of leaves and roots of Melilotus albus Medik planted in diesel-contaminated sand (7500 mg kg(-1)). Seedlings of Melilotus either Non inoculated (Non-AMF) or pre-inoculated plants (AMF) with the AMF-inoculum Glomus Zac-19 were transplanted to non-contaminated or contaminated sand. After 60 days, diesel significantly reduced plant growth. AMF- plants had no significant greater (64% and 89%, respectively) shoot and leaf dry weight than Non-AMF plants, but AMF plants had lower specific leaf area. AMF-plants had significantly greater content of microelements than non-AMF plants. Regardless diesel contamination, the total AOX and TPC were significantly higher in leaves when compared to roots; in contrast, NRA was higher in roots than leaves. Diesel increased total AOX of leaves, but AMF-plants had significantly lower AOX than non-AMF plants. In contrast, roots of AMF-plants had significantly higher AOX but lower NRA than non-AMF plants. AMF-colonization in roots detected via the fungal alkaline phosphatase activity was significantly reduced by the presence of diesel. AMF-inoculation alleviated diesel toxicity on M. albus by enhancing plant biomass, nutrient content, and AOX activity. In addition, AMF-plants significantly contributed in higher degradation of total petroleum hydrocarbons when compared to non-AMF-plants.

Aerenchymatous phellem in hypocotyl and roots enables O2 transport in Melilotus siculus.

• Aerenchymatous phellem (secondary aerenchyma) has rarely been studied in roots. Its formation and role in internal aeration were evaluated for Melilotus siculus, an annual legume of wet saline land. • Plants were grown for 21 d in aerated or stagnant (deoxygenated) agar solutions. Root porosity and maximum diameters were measured after 0, 7, 14 and 21 d of treatment. Phellem anatomy was studied and oxygen (O(2)) transport properties examined using methylene blue dye and root-sleeving O(2) electrodes. • Interconnecting aerenchymatous phellem developed in hypocotyl, tap root and older laterals (but not in aerial shoots), with radial intercellular connections to steles. Porosity of main roots containing phellem was c. 25%; cross-sectional areas of this phellem were threefold greater for stagnant than for aerated treatments. Root radial O(2) loss was significantly reduced by complete hypocotyl submergence; values approached zero after disruption of hypocotyl phellem below the waterline or, after shoot excision, by covering hypocotyl phellem in nontoxic cream. • Aerenchymatous phellem enables hypocotyl-to-root O(2) transport in M. siculus. Phellem increases radially under stagnant conditions, and will contribute to waterlogging tolerance by enhancing root aeration. It seems likely that with hypocotyl submerged, O(2) will diffuse via surface gas-films and internally from the shoot system.

Dissolved organic carbon in runoff and tile-drain water under corn and forage fertilized with hog manure.

Dissolved organic carbon (DOC) export from soils can play a significant role in soil C cycling and in nutrient and pollutant transport. However, information about DOC losses from agricultural soils as influenced by management practices is scarce. We compared the effects of mineral fertilizer (MF) and liquid hog manure (LHM) applications on the concentration and molecular size of DOC released in runoff and tile-drain water under corn (Zea mays L.) and forage cropping systems. Runoff and tile-drain water samples were collected during a 2-mo period (October to December 1998) and DOC concentration was measured. Characterization of DOC was performed by tangential ultrafiltration with nominal cut-offs at 3 and 100 kDa. Mean concentration of DOC in runoff water (12.7 mg DOC L(-1)) was higher than in tile-drain water (6.5 mg DOC L(-1)). Incorporation of corn residues increased the DOC concentration by 6- to 17-fold in surface runoff, but this effect was short-lived. In runoff water, the relative size of the DOC molecules increased when corn residues and LHM were applied probably due to partial microbial breakdown of these organic materials and to a faster decomposition or preferential adsorption of the small molecules. The DOC concentration in tile-drain water was slightly higher under forage (7.5 mg DOC L(-1)) than under corn (5.4 mg DOC L(-1)) even though the application rates of LHM were higher in corn plots. We suggest that preferential flow facilitated the migration of DOC to tile drains in forage plots. In conclusion, incorporation of corn residues and LHM increased the concentration of DOC and the relative size of the molecules in surface runoff water, whereas DOC in tile-drain water was mostly influenced by the cropping system with relatively more DOC and larger molecules under forage than corn.

[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.

The expression of MaEXP1, a Melilotus alba expansin gene, is upregulated during the sweetclover-Sinorhizobium meliloti interaction.

Expansins are a highly conserved group of cell wall-localized proteins that appear to mediate changes in cell wall plasticity during cell expansion or differentiation. The accumulation of expansin protein or the mRNA for specific expansin gene family members has been correlated with the growth of various plant organs. Because expansin proteins are closely associated with plant cell wall expansion, and as part of a larger study to determine the role of different gene products in the legume-Rhizobium spp. symbiosis, we investigated whether a Melilotus alba (white sweetclover) expansin gene is expressed during nodule development. A cDNA fragment encoding an expansin gene (EXP) was isolated from Sinorhizobium meliloti-inoculated sweetclover root RNA by reverse-transcriptase polymerase chain reaction using degenerate primers, and a full-length sweetclover expansin sequence (MaEXP1) was obtained using 5' and 3' rapid amplification of cDNA end cloning. The predicted amino acid of the sweetclover expansin is highly conserved with the various alpha-expansins in the GenBank database. MaEXP1 contains a series of eight cysteines and four tryptophans that are conserved in the alpha-expansin protein family. Northern analysis and whole-mount in situ hybridization analyses indicate that MaEXP1 mRNA expression is enhanced in roots within hours after inoculation with S. meliloti and in nodules. Western and immunolocalization studies using a cucumber expansin antibody demonstrated that a cross-reacting protein accumulated in the expanding cells of the nodule.

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.