A comparative plastome approach enhances the assessment of genetic variation in the Melilotus genus.

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.

Homeopathic Agents or Vitamins in Reducing Ecchymosis after Oculofacial Surgery: A Report by the American Academy of Ophthalmology.

PURPOSE: To review the published literature to determine the efficacy and safety of homeopathic agents or vitamins in reducing ecchymosis after oculofacial surgery or laser surgery. METHODS: A literature search was conducted in the PubMed database initially in December 2019 and updated in March 2020 to identify all studies in the English language literature on the use of homeopathic agents or vitamins in oculofacial procedures, including laser surgery. The search yielded 124 citations, and 11 articles met all inclusion criteria for this assessment. A panel methodologist then assigned a level of evidence rating for each study. Eleven studies met inclusion criteria; 9 were rated level I, and 2 were rated level III. RESULTS: The agents studied in the articles identified included oral or topical Arnica montana (AM), oral Melilotus extract, topical vitamin K oxide, and topical AM combined with Rhododendron tomentosum. Metrics to describe ecchymosis varied. In 7 controlled studies, perioperative AM provided no or negligible benefit versus placebo. In 2 studies, vitamin K cream was equivalent to placebo. One study of oral Melilotus extract had less ecchymosis compared with controls in paranasal and eyelid ecchymosis at postoperative day (POD) 7, but not at PODs 1 and 4. A lone cohort study of combined topical AM and R. tomentosum lacked objective metrics and adequate controls. No serious side effects from administration of homeopathic agents or vitamins were identified. CONCLUSIONS: The current literature does not support the use of AM, vitamin K oxide, R. tomentosum, or Melilotus extract for reducing ecchymosis after oculofacial surgery or pulsed dye laser surgery.

Genome-Wide Analysis of the Rab Gene Family in Melilotus albus Reveals Their Role in Salt Tolerance.

Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein-protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.

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-Wide Identification of the GRAS Family Genes in Melilotus albus and Expression Analysis under Various Tissues and Abiotic Stresses.

The GRAS gene family is a plant-specific family of transcription factors, which play an important role in many metabolic pathways, such as plant growth and development and stress response. However, there is no report on the comprehensive study of the GRAS gene family of Melilotus albus. Here, we identified 55 MaGRAS genes, which were classified into 8 subfamilies by phylogenetic analysis, and unevenly distributed on 8 chromosomes. The structural analysis indicated that 87% of MaGRAS genes have no intron, which is highly conservative in different species. MaGRAS proteins of the same subfamily have similar protein motifs, which are the source of functional differences of different genomes. Transcriptome and qRT-PCR data were combined to determine the expression of 12 MaGRAS genes in 6 tissues, including flower, seed, leaf, stem, root and nodule, which indicated the possible roles in plant growth and development. Five and seven MaGRAS genes were upregulated under ABA, drought, and salt stress treatments in the roots and shoots, respectively, indicating that they play vital roles in the response to ABA and abiotic stresses in M. albus. Furthermore, in yeast heterologous expression, MaGRAS12, MaGRAS34 and MaGRAS33 can enhance the drought or salt tolerance of yeast cells. Taken together, these results provide basic information for understanding the underlying molecular mechanisms of GRAS proteins and valuable information for further studies on the growth, development and stress responses of GRAS proteins in M. albus.

Coumarin-Induced Hepatotoxicity: A Narrative Review.

Coumarin is an effective treatment for primary lymphoedema, as well as lymphoedema related to breast cancer radiotherapy or surgery. However, its clinical use is limited in several countries due to the possible occurrence of hepatotoxicity, mainly in the form of mild to moderate transaminase elevation. It is worth noting that only a few cases of severe hepatotoxicity have been described in the literature, with no reported cases of liver failure. Data available on coumarin absorption, distribution, metabolism, and excretion have been reviewed, focusing on hepatotoxicity studies carried out in vitro and in vivo. Finally, safety and tolerability data from clinical trials have been thoroughly discussed. Based on these data, coumarin-induced hepatotoxicity is restricted to a small subset of patients, probably due to the activation in these individuals of alternative metabolic pathways involving specific CYP450s isoforms. The aim of this work is to stimulate research to clearly identify patients at risk of developing hepatotoxicity following coumarin treatment. Early identification of this subset of patients could open the possibility of more safely exploiting the therapeutical properties of coumarin, allowing patients suffering from lymphoedema to benefit from the anti-oedematous activity of the treatment.

Comparative Metabolomic Analysis of Four Fabaceae and Relationship to In Vitro Nematicidal Activity against Xiphinema index.

The grapevine fanleaf virus (GFLV), responsible for fanleaf degeneration, is spread in vineyards by the soil nematode Xiphinema index. Nematicide molecules were used to limit the spread of the disease until they were banned due to negative environmental impacts. Therefore, there is a growing interest in alternative methods, including plant-derived products with antagonistic effects to X. index. In this work, we evaluated the nematicidal potential of the aerial parts and roots of four Fabaceae: sainfoin (Onobrychis viciifolia), birdsfoot trefoil (Lotus corniculatus), sweet clover (Melilotus albus), and red clover (Trifolium pratense), as well as that of sainfoin-based commercial pellets. For all tested plants, either aerial or root parts, or both of them, exhibited a nematicidal effect on X. index in vitro, pellets being as effective as freshly harvested plants. Comparative metabolomic analyses did not reveal molecules or molecule families specifically associated with antagonistic properties toward X. index, suggesting that the nematicidal effect is the result of a combination of different molecules rather than associated with a single compound. Finally, scanning electron microscope observations did not reveal the visible impact of O. viciifolia extract on X. index cuticle, suggesting that alteration of the cuticle may not be the primary cause of their nematicidal effect.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

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.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Genome-wide development and application of miRNA-SSR markers in Melilotus genus.

Genetic diversity of plants is the brace of biodiversity and diversity within species, between species, and of ecosystems. SSR markers are the most preferable molecular marker tool that has been successfully used to study the genetic diversity of plant species. Development of miRNA-SSR markers has been deed in animals but is still limited in plants. In this study, 365 precursors miRNA were extracted from Melilotus albus (Ma) genome and used to design Ma miRNA-SSR primers. 137 Ma primer pairs (79 from known and 58 from novel pre-miRNAs) were obtained. 66 pairs of Ma miRNA-SSR primers were selected with polymorphisms and expected fragment size. The polymorphisms of primers were evaluated in 60 individuals of 15 Ma accessions. A total of 66 primer pairs showed high polymorphism, with average polymorphic information content of 0.49 among 15 Ma accessions and 0.63 among 18 Melilotus species, indicating that these primers have high polymorphisms. The number of alleles produced per primer ranged from 2 to 6 with an average of 3.6 alleles per locus in Ma accessions, and 2 to 10 numbers of alleles with a mean of 5.24 alleles per locus in Melilotus spp. For further studies, the genetic relationship was examined and the cluster analysis showed that 15 Ma accessions were grouped in three groups, on the other hand, 18 Melilotus species clustered into two groups. The analysis of molecular variance (AMOVA) revealed that 64.82% of the variation was found within the species and 35.18% between the species. The population structure analysis showed similar results with PCA analysis in that 18 species were grouped in two groups. In addition, 16,450 miRNA target genes were identified and used for GO and KEGG analysis. This is the first study to develop miRNA-SSR molecular markers in Melilotus spp., which has a great potential for marker-assisted, genetic improvement, genotyping applications, QTL analysis, and molecular-assisted selection studies for plant breeders and other researchers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01086-z.

Identifying similar transcripts in a related organism from de Bruijn graphs of RNA-Seq data, with applications to the study of salt and waterlogging tolerance in Melilotus.

BACKGROUND: A popular strategy to study alternative splicing in non-model organisms starts from sequencing the entire transcriptome, then assembling the reads by using de novo transcriptome assembly algorithms to obtain predicted transcripts. A similarity search algorithm is then applied to a related organism to infer possible function of these predicted transcripts. While some of these predictions may be inaccurate and transcripts with low coverage are often missed, we observe that it is possible to obtain a more complete set of transcripts to facilitate possible functional assignments by starting the search from the intermediate de Bruijn graph that contains all branching possibilities. RESULTS: We develop an algorithm to extract similar transcripts in a related organism by starting the search from the de Bruijn graph that represents the transcriptome instead of from predicted transcripts. We show that our algorithm is able to recover more similar transcripts than existing algorithms, with large improvements in obtaining longer transcripts and a finer resolution of isoforms. We apply our algorithm to study salt and waterlogging tolerance in two Melilotus species by constructing new RNA-Seq libraries. CONCLUSIONS: We have developed an algorithm to identify paths in the de Bruijn graph that correspond to similar transcripts in a related organism directly. Our strategy bypasses the transcript prediction step in RNA-Seq data and makes use of support from evolutionary information.

Isolation of Tricin as a Xanthine Oxidase Inhibitor from Sweet White Clover (Melilotus albus) and Its Distribution in Selected Gramineae Species.

Xanthine oxidase, an enzyme present in significant levels in the intestine and liver, metabolizes hypoxanthine to xanthine and xanthine to uric acid in the purine catabolic pathway. An inhibitory compound acting against xanthine oxidase was isolated from sweet white clover (Melilotus albus) by bioassay and high-performance liquid chromatography guided separation. It was identified as tricin by spectroscopic analysis. Tricin possessed a potent xanthine oxidase inhibitory activity with an IC50 value of 4.13 µM. Further inhibition kinetics data indicated it to be a mixed-type inhibitor and Ki and KI values were determined to be 0.47 µM and 4.41 µM. To find a rich source of tricin, the distribution of tricin in seven different tissues from four Gramineae species was investigated by high-performance liquid chromatography analysis. The highest amount (1925.05 mg/kg dry materials) was found in the straw of wheat, which is considered as a potentially valuable source of natural tricin.

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.

Chemical Constituents and Antioxidant, Anti-Inflammatory and Anti-Tumor Activities of Melilotus officinalis (Linn.) Pall.

Two new p-hydroxybenzoic acid glycosides, namely p-hydroxybenzoic acid-4-O-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 1) and 4-O-α-l-rhamnopyran-osyl-(1 → 6)-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 2), and seven known compounds, compound 3, 6, 7 (acid components), compound 8, 9 (flavonoids), compound 4 (a coumarin) and compound 5 (an alkaloid), were isolated from the 70% ethanol aqueous extract of the aerial parts of Melilotus officinalis (Linn.) Pall. The structures of all compounds were elucidated by use of extensive spectroscopic methods Infrared Spectroscopy (IR), High resolution electrospray ionization mass spectrometry (HR-ESI-MS), and ¹H and 13C-NMR). Sugar residues obtained after acid hydrolysis were identified by high-performance liquid chromatography (HPLC). The antioxidant activity of all the compounds was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The anti-inflammatory effects of the compounds were also evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. All compounds were shown to inhibit LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, in LPS-stimulated RAW 264.7 cells. The inhibitory effect of all the compounds on MCF-7 cells was determined by Cell Counting Kit-8 (CCK-8) method. The results showed that compounds 1, 2, 7, 8, 9 exhibited better antioxidant activity compared to the other compounds. compounds 1-9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, of which compound 7 was the most effective against inflammatory factors. compounds 1 and 2 have better antitumor activity compared to other compounds. Further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.

[Proteomic Profile of the Bacterium Sinorhizobium meliloti Depends on Its Life Form and Host Plant Species].

The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccha-rides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

Polish Yellow Sweet Clover (Melilotus officinalis L.) Honey, Chromatographic Fingerprints, and Chemical Markers.

A case study of Polish Melilotus officinalis honey was presented for the first time. Gas chromatography-mass spectrometry (GC-MS) (after steam distillation, Soxhlet extraction, ultrasonic solvent extraction, and solid phase extraction (SPE)) and targeted high performance liquid chromatography with a photodiode array detector (HPLC-PAD) were applied to determine the characteristic components of honey. While ubiquitous in most honeys, carbohydrates, terpene derivatives, and phenylacetic acid dominated in the Soxhlet extracts (25.54%) and in the application of SPE (13.04%). In addition, lumichrome (1.85%) was found, and may be considered as a marker of this honey. Due to the presence of these compounds, Polish yellow sweet clover honey is similar to French lavender honeys. The major compounds determined in the methanolic extract were (+)-catechine (39.7%) and gallic acid (up to 30%), which can be regarded as specific chemical markers of the botanical origin of melilot honey. With respect to total phenolic and flavonoid contents, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were determined spectrophotometrically. The honey exhibited a moderate antioxidant activity, typical for light honeys, which correlates well with its phenolic and flavonoid composition.

Genome assembly of Melilotus officinalis provides a new reference genome for functional genomics.

BACKGROUND: Sweet yellow clover (Melilotus officinalis) is a diploid plant (2n = 16) that is native to Europe. It is an excellent legume forage. It can both fix nitrogen and serve as a medicine. A genome assembly of Melilotus officinalis that was collected from Best corporation in Beijing is available based on Nanopore sequencing. The genome of Melilotus officinalis was sequenced, assembled, and annotated. RESULTS: The latest PacBio third generation HiFi assembly and sequencing strategies were used to produce a Melilotus officinalis genome assembly size of 1,066 Mbp, contig N50 = 5 Mbp, scaffold N50 = 130 Mbp, and complete benchmarking universal single-copy orthologs (BUSCOs) = 96.4%. This annotation produced 47,873 high-confidence gene models, which will substantially aid in our research on molecular breeding. A collinear analysis showed that Melilotus officinalis and Medicago truncatula shared conserved synteny. The expansion and contraction of gene families showed that Melilotus officinalis expanded by 565 gene families and shrank by 56 gene families. The contacted gene families were associated with response to stimulus, nucleotide binding, and small molecule binding. Thus, it is related to a family of genes associated with peptidase activity, which could lead to better stress tolerance in plants. CONCLUSIONS: In this study, the latest PacBio technology was used to assemble and sequence the genome of the Melilotus officinalis and annotate its protein-coding genes. These results will expand the genomic resources available for Melilotus officinalis and should assist in subsequent research on sweet yellow clover plants.

Non-destructive distinction of single seed for Medicago sativa and Melilotus officinalis by capillary electrophoresis with laser-induced fluorescence detection.

Flavonoids are a class of natural polyphenolic compounds with great health benefits, and the development of methods for their analysis is of continuing interest. In this work, apigenin, kaempferol and formononetin were selected as the typical representatives of flavone, flavonol and isoflavone, three subclasses of flavonoids. Fluorescence studies revealed that tetraborate complexation could significantly sensitize the weak intrinsic fluorescence of flavonoids in solution, with a maximum of 137-fold for kaempferol. Subsequently, an integrated strategy of derivatization and separation was proposed for the universal analysis of flavonoids by capillary electrophoresis (CE) with 405 nm laser-induced fluorescence (LIF) detection. Using a running buffer consisting of 20 mM sodium tetraborate, 10 mM SDS and 10% methanol (pH 8.5), the dynamic derivatization was realized in the capillary, and the baseline separation was achieved within 10 min, with the detection limits of 0.92-35.46 nM (S/N=3) for the total of 9 flavonoids. The developed CE-LIF method was employed to the quantitative analysis of some flavonoids in Medicago sativa (alfalfa) plants and granulated alfalfa with the recoveries of 80.55-94.25%. Combined with the principal component analysis, the developed method was successfully applied to the non-destructive distinction of single seed for alfalfa and Melilotus officinalis (sweet clover), two forage grass seeds with very similar apparent morphology. Furthermore, this method was used to continuously monitor the substance metabolism during the soaking process at the level of single seed.

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

The trihelix gene family is a plant-specific family of transcription factors that play an important role in many metabolic pathways, including plant growth and development and stress responses. Drought stress is a major factor limiting the distribution and yield of Melilotus albus. However, the distribution of this gene family in M. albus and its biological functions in response to drought stress have not been reported. To investigate the responses of functional genes to drought stress in M. albus, in this study, a total of 34 MaGTs were identified and characterized, of which 32 MaGT proteins were predicted to be nuclear-localized. Based on conserved motif and phylogenetic analyses, the MaGTs could be divided into five subgroups (GT-1, GT-2, SH4, GT-γ, SIP1). Seven potential candidate genes for drought tolerance were screened and identified via qRT-PCR based on a transcriptome data analysis of drought stress in M. albus. The results indicated that MaGT17 was not only significantly upregulated in the roots after 24 h of drought stress, but also showed a significant induction in the shoots. This finding further confirms that MaGT17 is capable of responding to drought stress in M. albus. Taken together, these results will offer essential insights for understanding the underlying molecular mechanisms of the trihelix proteins and useful data for further research on the growth, development and stress responses of trihelix proteins in M. albus.