Serpentine environment prevails over geographic distribution in shaping the genetic diversity of Medicago lupulina L.

Edaphic conditions of serpentine soils, naturally rich in heavy metals, act as a strong selection pressure that shapes specific metal-tolerant ecotypes. Medicago lupulina L. (black medick) is not only a widespread plant species that prefers calcareous and dry soil types but also grows at the borders of serpentine formations. It can also be found in waste and disturbed habitats. This is a species with reported phytoremediation potential, however, there is no published data regarding the impact of the environment on the genetic distribution of this species. The aim of our research was to explore how selection pressure of serpentine soils affects genetic diversity of M. lupulina and to test heavy-metal accumulation capacity of this species. Specimens of 11 M. lupulina populations were collected from serpentine outcrops located in Central and Eastern Bosnia as well as from non-serpentine sites. Soil and plant samples were analyzed for the total contents of heavy metals using air-acetylene flame atomic absorption spectroscopy. Genetic diversity was analyzed using AFLP (Amplified Fragment Length Polymorphism) markers. Serpentine soils showed high nickel, cobalt, chromium and iron concentrations. Nickel and manganese concentrations in soil samples and plant material showed statistically significant correlation. Although plants in two populations show the ability to extract Ni, M. lupulina does not show hyperaccumulating properties. Despite severe selective pressure, genetic diversity in serpentine populations is not reduced. Analyses of intrapopulation and interpopulation genetic diversity showed significant genetic differentiation among populations which is not related to their geographic distance. Population from non-metalliferous soil showed clear separation from all other populations. Diversity data suggest that serpentine populations maintain genetic diversity by undetected mechanisms and that edaphic factors rather than geography influence genetic structure analyzed M. lupulina populations.

Yield of white clover and orchard grass cultivated in mining passives adding black earth and compost as a substrate.

Mining environmental liabilities generate environmental pollution. The objective of the present study was to determine the yield of white clover (Trifolium repens) and orchard grass (Dactylis glomerata) cultivated in mining environmental passives adding black earth and compost as a substrate in the Buenaventura Julcani Huancavelica Company. The treatments were the combinations: 4: 3.1: 3.1: 3:1.1 kg of RP: RP, TN: RP, C: RP, TN, C respectively. They were distributed in four treatments with twelve experimental units for each species of leguminous and gramineous grass, we worked according to the completely randomized design (DCA) with a 2 x 4 factorial arrangement, the experimental unit being a treatment with twelve repetitions. The variables evaluated were: germination percentage (TG) and stem survival percentage (TST). For the statistical analysis, the SPSS software was used.

MrERF039 transcription factor plays an active role in the cold response of Medicago ruthenica as a sugar molecular switch.

Cold stress severely restricts plant development, causing significant agricultural losses. We found a critical transcription factor network in Medicago ruthenica was involved in plant adaptation to low-temperature. APETALA2/ethylene responsive factor (AP2/ERF) transcription factor MrERF039 was transcriptionally induced by cold stress in M. ruthenica. Overexpression of MrERF039 significantly increased the glucose and maltose content, thereby improving the tolerance of M. ruthenica. MrERF039 could bind to the DRE cis-acting element in the MrCAS15A promoter. Additionally, the methyl group of the 14th amino acid in MrERF039 was required for binding. Transcriptome analysis showed that MrERF039 acted as a sugar molecular switch, regulating numerous sugar transporters and sugar metabolism-related genes. In addition, we found that MrERF039 could directly regulate ß-amylase gene, UDP glycosyltransferase gene, and C2H2 zinc finger protein gene expression. In conclusion, these findings suggest that high expression of MrERF039 can significantly improve the cold tolerance of M. ruthenica root tissues during cold acclimation. Our results provide a new theoretical basis and candidate genes for breeding new legume forage varieties with high resistance.

Spermidine or spermine pretreatment regulates organic metabolites and ions homeostasis in favor of white clover seed germination against salt toxicity.

White clover is widely cultivated as a leguminous forage or ground cover plant worldwide. However, soil salinization decreases its yield and quality. Aims of the present experiment were to elucidate the impact of seed pretreatment with spermidine (Spd) or spermine (Spm) on amylolysis, Na+/K+ accumulation, and metabolic homeostasis during germination. Seed was soaked in distilled water (control), Spd or Spm solution and then germinated under optimal or salt stress conditions for 7 days. Results showed that germination vigor, germination percentage, or seed vigour index of seeds pretreatment with Spd increased by 7%, 11%, or 70% when compared with water-pretreated seeds under salt stress, respectively. Germination percentage or seed vigour index of seeds pretreatment with Spm increased by 17% or 78% than water-pretreated seeds under saline condition, respectively. In response to salt stress, accelerated amylolysis via activation of ß-amylase activity was induced by Spd or Spm pretreatment. Spd or Spm pretreatment also significantly enhanced accumulation of diverse amino acids, organic acids, sugars, and other metabolites (putrescine, myo-inositol, sorbitol, daidzein etc.) associated with enhanced osmotic adjustment, antioxidant capacity, and energy supply during germination under salt stress. In addition, Spd or Spm pretreatment not only significantly reduced salt-induced K+ loss and overaccumulation of Na+, but also improved the ratio of K+ to Na+, contributing to Na+ and K+ balance in seedlings. In response to salt stress, seeds pretreatment with Spd or Spm up-regulated transcription level of NHX2 related to enhancement in compartmentation of Na+ from cytoplasm to vacuole, thus reducing Na+ toxicity in cytoplasm. Spm priming also uniquely up-regulated transcription levels of SKOR, HKT1, and HAL2 associated with K+ and Na + homeostasis and decline in cytotoxicity under salt stress.

Genomic and Metabolic Characterization of Plant Growth-Promoting Rhizobacteria Isolated from Nodules of Clovers Grown in Non-Farmed Soil.

The rhizosphere microbiota, which includes plant growth-promoting rhizobacteria (PGPR), is essential for nutrient acquisition, protection against pathogens, and abiotic stress tolerance in plants. However, agricultural practices affect the composition and functions of microbiota, reducing their beneficial effects on plant growth and health. Among PGPR, rhizobia form mutually beneficial symbiosis with legumes. In this study, we characterized 16 clover nodule isolates from non-farmed soil to explore their plant growth-promoting (PGP) potential, hypothesizing that these bacteria may possess unique, unaltered PGP traits, compared to those affected by common agricultural practices. Biolog profiling revealed their versatile metabolic capabilities, enabling them to utilize a wide range of carbon and energy sources. All isolates were effective phosphate solubilizers, and individual strains exhibited 1-aminocyclopropane-1-carboxylate deaminase and metal ion chelation activities. Metabolically active strains showed improved performance in symbiotic interactions with plants. Comparative genomics revealed that the genomes of five nodule isolates contained a significantly enriched fraction of unique genes associated with quorum sensing and aromatic compound degradation. As the potential of PGPR in agriculture grows, we emphasize the importance of the molecular and metabolic characterization of PGP traits as a fundamental step towards their subsequent application in the field as an alternative to chemical fertilizers and supplements.

Structural Reorganization in Two Alfalfa Mitochondrial Genome Assemblies and Mitochondrial Evolution in Medicago Species.

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution.

Research on the mechanism of plant root protection for soil slope stability.

In order to investigate the impact of herbaceous root development on soil slope stability in expansive soil areas, the research was conducted in the soil slope experimental area of Yaoshi Town, Shangzhou District, Shangluo City. Three types of herbaceous plants, namely Lolium perenne, Medicago, and Cynodon dactylon, were planted to examine their influence on slope stability. The results indicated that Lolium perenne had significantly higher root length density and root surface area density compared to Cynodon dactylon and Medicago. However, the root weight density of Cynodon dactylon was found to be highest. The roots of Lolium perenne, Cynodon dactylon, and Medicago were predominantly observed in diameter ranges of 0 < L ≤ 1.0 mm, 0 < L ≤ 2.5 mm, and 2.5 < L ≤ 3.0 mm, respectively. The roots of herbaceous plants have the ability to enhance water retention in soil, resist hydraulic erosion of slope soil, and reduce soil shrinkage and swelling. During the initial phase of herbaceous planting, there is an accelerated process of organic carbon mineralization in the soil. The roots of herbaceous plants play a crucial role in soil consolidation and slope protection. They achieve this by dispersing large clastic particles, binding small particles together, altering soil porosity, enhancing soil water retention, and reducing soil water infiltration. It was found that Lolium perenne and Medicago, which have well-developed roots, exhibited superior slope protection effects. These findings contribute to the theoretical understanding for the implementation of green ecological protection technology on soil slopes.

Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

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.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Epiphytic microbiota in Swedish grass-clover herbage and the effect of silage additives on fermentation profiles and bacterial community compositions of the resulting silages.

AIMS: To investigate the epiphytic microbiota in grass-clover herbage harvested at different sites and occasions and to explore the effect of different silage additives on the resulting silage microbiota. METHODS AND RESULTS: Herbage was harvested from grass-clover leys at geographically distributed sites in a long-term field experiment in Sweden, in early and late season of two consecutive years. Different silages were made from the herbage using: (1) no additive, (2) acid-treatment, and (3) inoculation by starter culture. Herbages were analysed for botanical and chemical composition, and the resulting silages for products of fermentation. Bacterial DNA was extracted from herbage and silage samples, followed by sequencing using Illumina 16S rRNA amplicon sequencing. Herbage microbiota showed no clear correlation to site or harvesting time. Silage additives had a major effect on the ensiling process; inoculation resulted in well fermented silages comprising a homogenous microbiota dominated by the genera Lactobacillus and Pediococcus. A minor effect of harvest time was also observed, with generally a more diverse microbiota in second-harvest silages. Untreated silages showed a higher relative abundance (RA) from non-lactic acid bacteria compared to acid-treated silages. In most silages, only a few bacterial amplicon sequence variants contributed to most of the RA. CONCLUSIONS: The epiphytic microbiota in grass-clover herbage were found to be random and not dependent on site. From a microbial point of view, the most predictable and preferable silage outcome was obtained by inoculation with a starter culture. Acid-treatment with formic- and propionic acid surprisingly resulted in a less preferable silage. Silage making without additives cannot be recommended based on our results.

Control of arbuscule development by a transcriptional negative feedback loop in Medicago.

Most terrestrial plants establish a symbiosis with arbuscular mycorrhizal fungi (AMF), which provide them with lipids and sugars in exchange for phosphorus and nitrogen. Nutrient exchange must be dynamically controlled to maintain a mutually beneficial relationship between the two symbiotic partners. The WRI5a and its homologues play a conserved role in lipid supply to AMF. Here, we demonstrate that the AP2/ERF transcription factor MtERM1 binds directly to AW-box and AW-box-like cis-elements in the promoters of MtSTR2 and MtSTR, which are required for host lipid efflux and arbuscule development. The EAR domain-containing transcription factor MtERF12 is also directly activated by MtERM1/MtWRI5a to negatively regulate arbuscule development, and the TOPLESS co-repressor is further recruited by MtERF12 through EAR motif to oppose MtERM1/MtWRI5a function, thereby suppressing arbuscule development. We therefore reveal an ERM1/WRI5a-ERF12-TOPLESS negative feedback loop that enables plants to flexibly control nutrient exchange and ensure a mutually beneficial symbiosis.

Haplotype-Resolved, Chromosome-Level Assembly of White Clover (Trifolium repens L., Fabaceae).

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively.

Alleviation of gadolinium stress on Medicago by elevated atmospheric CO2 is mediated by changes in carbohydrates, Anthocyanin, and proline metabolism.

Rare earth elements (REE) like Gadolinium (Gd), are increasingly used in industry and agriculture and this is concomitant with the increasingly leaking of Gd into the environment. Under a certain threshold concentration, REE can promote plant growth, however, beyond this concentration, they exert negative effects on plant growth. Moreover, the effect of Gd on plants growth and metabolism under a futuristic climate with increasingly atmospheric CO2 has not yet been studied. To this end, we investigated the effect of soil contamination with Gd (150 mg/kg soil) on the growth, carbohydrates, proline, and anthocyanin metabolism of Medicago plants grown under ambient (aCO2, 410 ppm) or elevated CO2 (eCO2, 720 ppm) concentration. Gd negatively affected the growth and photosynthesis of plants and imposed oxidative stress i.e., increased H2O2 and lipid peroxidation (MDA) level. As defense lines, the level and metabolism of osmoprotectants (soluble sugars and proline) and antioxidants (phenolics, anthocyanins, and tocopherols) were increased under Gd treatment. High CO2 positively affected the growth and metabolism of Medicago plants. Moreover, eCO2 mitigated the negative impacts of Gd on Medicago growth. It further induced the levels of osmoprotectants and antioxidants. In line with increased proline and anthocyanins, their metabolic enzymes (e.g. OAT, P5CS, PAL, and CS) were also increased. This study advances our understanding of how Gd adversely affects Medicago plant growth and metabolism. It also sheds light on the biochemical mechanisms underlying the Gd stress-reducing impact of eCO2.

Genome-wide analysis of the WRKY genes and their important roles during cold stress in white clover.

Background: White clover (Trifolium repens L) is a high-quality forage grass with a high protein content, but it is vulnerable to cold stress, which can negatively affect its growth and development. WRKY transcription factor is a family of plant transcription factors found mainly in higher plants and plays an important role in plant growth, development, and stress response. Although WRKY transcription factors have been studied extensively in other plants, it has been less studied in white clover. Methods and Results: In the present research, we have performed a genome-wide analysis of the WRKY gene family of white clover, in total, there were 145 members of WRKY transcription factors identified in white clover. The characterization of the TrWRKY genes was detailed, including conserved motif analysis, phylogenetic analysis, and gene duplication analysis, which have provided a better understanding of the structure and evolution of the TrWRKY genes in white clover. Meanwhile, the genetic regulation network (GRN) containing TrWRKY genes was reconstructed, and Gene Ontology (GO) annotation analysis of these function genes showed they contributed to regulation of transcription process, response to wounding, and phosphorylay signal transduction system, all of which were important processes in response to abiotic stress. To determine the TrWRKY genes function under cold stress, the RNA-seq dataset was analyzed; most of TrWRKY genes were highly upregulated in response to cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiment, implying they are involved in various gene regulation pathways in response to cold stress. Conclusion: The results of this study provide insights that will be useful for further functional analyses of TrWRKY genes in response to biotic or abiotic stresses in white clover. These findings are likely to be useful for further research on the functions of TrWRKY genes and their role in response to cold stress, which is important to understand the molecular mechanism of cold tolerance in white clover and improve its cold tolerance.

Optimization of Protocols for the Induction of Callus and Plant Regeneration in White Clover (Trifolium repens L.).

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L-1 AgNO3 and 25 mg L-1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L-1 6-BA, 1 mg L-1 KT and 0.5 mg L-1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.