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.

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.

Effects of organic acids on the photosynthetic and antioxidant properties and accumulations of heavy metals of Melilotus officinalis grown in Cu tailing.

The effect of citric acid (CA), acetic acid (Ac), and ethylene diamine tetraacetic acid (EDTA) on the photosynthetic and antioxidant properties and the accumulation of some heavy metals (HMs) of Melilotus officinalis seedling growing in Cu mine tailings for 25 days were studied. Results showed that the formation of photosynthesizing cells of M. officinalis was inhibited by EDTA at 2 mmol/kg. Photosynthetic pigment contents under EDTA of 2 mmol/kg were reduced by 26, 40, and 19 %, respectively, compared to the control. The proline contents in aboveground and underground parts increased as the level of EDTA was enhanced. CA and Ac enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in the aboveground parts and EDTA inhibited the activity of POD in the underground parts. The addition of CA promoted significantly the growth of M. officinalis, while the biomass decreased significantly under 2 mmol/kg EDTA. Cu contents in the aboveground parts treated with 0.5 and 2.0 mmol/kg EDTA reached 175.50 and 265.17 µg/g dry weight, respectively. Ac and EDTA treatments promoted Cd to translocate from root to aboveground parts. The result indicated that M. officinalis was a tolerant species of Cu tailing and can be used to remediate Cu contaminated environment, and rationally utilization of organic acids, especially EDTA, in the phytoremediation can improve the growth and metals accumulation of M. officinalis.

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.

Root aeration via aerenchymatous phellem: three-dimensional micro-imaging and radial O2 profiles in Melilotus siculus.

• Internal root aeration enables waterlogging-tolerant species to grow in anoxic soil. Secondary aerenchyma, in the form of aerenchymatous phellem, is of importance to root aeration in some dicotyledonous species. Little is known about this type of aerenchyma in comparison with primary aerenchyma. • Micro-computed tomography was employed to visualize, in three dimensions, the microstructure of the aerenchymatous phellem in roots of Melilotus siculus. Tissue porosity and respiration were also measured for phellem and stelar tissues. A multiscale, three-dimensional, diffusion-respiration model compared the predicted O(2) profiles in roots with those measured using O(2) microelectrodes. • Micro-computed tomography confirmed the measured high porosity of aerenchymatous phellem (44-54%) and the low porosity of stele (2-5%) A network of connected gas spaces existed in the phellem, but not within the stele. O(2) partial pressures were high in the phellem, but fell below the detection limit in the thicker upper part of the stele, consistent with the poorly connected low porosity and high respiratory demand. • The presented model integrates and validates micro-computed tomography with measured radial O(2) profiles for roots with aerenchymatous phellem, confirming the existence of near-anoxic conditions at the centre of the stele in the basal parts of the root, coupled with only hypoxic conditions towards the apex.

Using the pollen viability and morphology for fluoride pollution biomonitoring.

The methods using plants for biomonitoring of air and soil quality are simple, cheap, and fast and can supplement the classical physicochemical methods. In this study, biological pollen characterization of some collected legume species from an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual value of pollen as a bioindicator of the effects of soil and atmospheric pollution. Young buds and flowers of six legumes (Cercis siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants were removed and compared. Studies of light and electron microscopic preparation showed some abnormalities during pollen development in affect of fluoride pollution. The viability of pollen grains estimated by staining with acetocarmine shows sharp differences in smearing advanced pollen grains from abnormal ones. Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R. pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light, partial, or no staining with acetocarmine, whereas almost all of the control ones clearly stained. Observation of the pollen grains by light microscopy and scanning electron microscopy showed the significant effect of fluoride on shapes and sizes of pollen grains. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration. Therefore, pollen grains provide essential information on biological impact of pollutants and they are good candidates for biomonitoring the atmospheric and edaphic pollutions.

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.

Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens.

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.

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.

[Changes of four common plant populations growth and their anti-oxidative enzymatic system in desertification process].

This paper studied the changes of the growth and anti-oxidative enzymatic system of four common plant populations during the desertification process of sandy grassland. The results showed that in the process of desertification, the individual height and density and the density percentage of the populations all had a decreasing trend. The growth of Melilotoides ruthenica was more vigorous before moderate desertification (MD) stage, but restricted after that. In MD stage, the growth of Leymus chinensis was heavily restricted, and its individual height, density and density percentage accounted for 57.19%, 2.50% and 6.22% of those in original vegetation (OV) stage, respectively. The individual height and density of Cleistogenes squarrosa and Artemisia frigida increased in the stages of potential desertification (PD), light desertification (LD) or MD because of their phase status of dominant species and their stronger stress resistance. The SOD and POD activities of the common plant populations increased in PD and MD stages, but decreased in LD and heavy desertification (HD) stages. The CAT activity of Leymus chinensis was higher, whose response to desertification was not significant (P > 0.05), and that of Melilotoides ruthenica increased significantly in PD and HD stages (P < or = 0.01). The activities of the three anti-oxidative enzymes in the common plant populations, except the CAT activity of Melilotoides ruthenica, decreased in HD stage. The MDA content in the common populations increased firstly, then decreased, and finally increased from OV to MD stage, and had a significant difference in different desertification gradients (P < or = 0.05). Based on the integrated analysis of the ecological and physiological changes, it could be concluded that Leymus chinensis was more sensitive to desertification, while Melilotoides ruthenica had a stronger bioenergy.