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Summary 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 β‐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.

Background Melilotus sulcatus (Fabaceae) is an annual weed species prevalent in the Mediterranean Basin and Asia, characterized by a hard seed coat, indicating physical seed dormancy and long seed-bank longevity. This weed exhibits high phenotypic plasticity, thriving across various climatic regions in Israel. This study investigates seed germination of M. sulcatus populations under different temperatures. Moreover, we have studied the phenology, and reproductive biology of M. sulcatus populations in a common garden experiment. Seeds of M. sulcatus were collected from onion fields at Kibbutz Grofit located in the hyper-arid Southern Arava region, with ~ 30 mm average annual rainfall, and from Kibbutz Yifat, in the Jezreel Valley, where annual rainfall is 450–650 mm. Progeny generations were grown in pollen-proof cages at Newe Ya’ar Research Center also located at the Jezreel Valley. Results Differences in seed area and average weight were observed among populations and generations, with field populations producing heavier seeds compared to progeny populations. The optimal temperature ( T 0 ) for seed germination varied among populations, with the highest T 0 recorded for the Grofit field population (23.40℃) collected at the warm site and the lowest for the Yifat field population (17.67℃) collected at the cold site, while the two progeny populations showed similar T 0 values. Phenology and reproductive biology were tested using seeds of the two field populations. Despite the vigorous growth of the Grofit population, there were no significant differences in average final weight (4.59 g for Grofit and 4.23 g for Yifat) and height (51.8 cm for Grofit and 50.3 cm for Yifat) between populations. Grofit progeny plants exhibited a statistically significantly higher number of seeds per pod, combined with larger seed area and average weight. Conclusions Overall, our findings underscore the adaptive strategies employed by M. sulcatus populations in response to their environment. Vigorous plant growth, a higher number of seeds per pod, and larger seed area and average weight were observed in individuals from the Grofit population compared to the Yifat population. The greater fitness of Grofit population, originated from hyper-arid conditions, highlights the significant impact of environmental factors on species adaptation.

Melilotus comprises 19 species, while the phylogenetic relationships between species remain unclear. In the present work, three chloroplast genes, rbcL, matK, trnL-F, and one nuclear region, ITS (internal transcribed spacer) belonging to 48 populations of 18 species of Melilotus were sequenced and phylogenetic trees were constructed to study their interspecific relationships. Based on the phylogenetic tree generated in this study using rbcL analysis, the Melilotus genus is clearly monophyletic in the legume family. Both Bayesian and maximum-parsimony approaches were used to analyze the data. The nrDNA ITS provided more informative characteristics (9.8%) than cpDNA (3.0%). Melilotus contains two closely related groups, clade I and clade II. M. spicatus, M. indicus and M. segetalis have a close relationship. M. infestus, M. siculus and M. sulcatus are closely related. The comparing between molecular phylogeny and flower color classification in Melilotus showed that the flower color is not much informative for phylogenetics of this genus.

Sweet clover, particularly white sweet clover (Melilotus albus), is an underexplored plant that has gained attention in recent years. This study compared the bioactive compounds content in the flowers, leaves, and stems of Melilotus albus Medic. to the well-known health-promoting Melilotus officinalis (L.) Lam. Both fresh and dried plant materials were analysed, with chromatographic assessments preceded by the optimisation of the extraction method (using 50% aqueous ethanol–water and the plant sample ground in a mortar, followed by 30 min of ultrasound-assisted extraction). Flower and leaf extracts were also evaluated for their total phenolic content (TPC) and antioxidant activity using FRAP and DPPH· assays. Both plant species were rich in phenolic compounds, including coumarins, phenolic acids, and flavonoids. HPLC-PDA analysis revealed similar profiles for both species, with quantitative differences in individual compound fractions. The highest coumarin content was found in the fresh flowers, followed by the leaves and stems. No significant species-specific differences in the coumarin content were observed. However, the flowers of M. albus were a richer source of flavonoids, with the highest hyperoside content. The flavonoid profile in the leaf extracts was similar to that of the flowers, but the content was about four times lower in the leaves and even lower than in the stems. Interestingly, the leaf extracts exhibited higher antioxidant activity than the flower extracts. The presented findings suggest that white sweet clover is an equally valuable source of health-promoting compounds as yellow sweet clover.

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-. 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. 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. 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.

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.

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 O2 profiles in roots with those measured using O2 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. O2 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 O2 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.

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 (O₂) transport properties examined using methylene blue dye and root-sleeving O₂ 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 O₂ 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 O₂ 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, O₂ will diffuse via surface gas-films and internally from the shoot system.

Background Melilotus , a member of the Fabaceae family, is a pivotal forage crop that is extensively cultivated in livestock regions globally due to its notable productivity and ability to withstand abiotic stress. However, the genetic attributes of the chloroplast genome and the evolutionary connections among different Melilotus species remain unresolved. Results In this study, we compiled the chloroplast genomes of 18 Melilotus species and performed a comprehensive comparative analysis. Through the examination of protein-coding genes, we successfully established a robust phylogenetic tree for these species. This conclusion is further supported by the phylogeny derived from single-nucleotide polymorphisms (SNPs) across the entire chloroplast genome. Notably, our findings revealed that M. infestus , M. siculus , M. sulcatus , and M. speciosus formed a distinct subgroup within the phylogenetic tree. Additionally, the chloroplast genomes of these four species exhibit two shared inversions. Moreover, inverted repeats were observed to have reemerged in six species within the IRLC. The distribution patterns of single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within protein-coding genes indicated that ycf1 and ycf2 accumulated nonconservative alterations during evolutionary development. Furthermore, an examination of the evolutionary rate of protein-coding genes revealed that rps18 , rps7 , and rpl16 underwent positive selection specifically in Melilotus . Conclusions We present a comparative analysis of the complete chloroplast genomes of Melilotus species. This study represents the most thorough and detailed exploration of the evolution and variability within the genus Melilotus to date. Our study provides valuable chloroplast genomic information for improving phylogenetic reconstructions and making biogeographic inferences about Melilotus and other Papilionoideae species.

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.