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Amending soil with biochar can reduce the toxic effects of heavy metals (HM) on plants and the soil. However, the effects of different concentrations of biochar on the properties and microbial activities in lead (Pb)-contaminated soils are unclear. In this study, two Pb concentrations were set (low, 1000 mg/kg; high, 5000 mg/kg), and five corn straw biochar (CSB) concentrations (0, 2.5, 5, 10 and 15%) were used to determine the response of the growth and rhizosphere of red clover (Trifolium pretense L.) (in terms of soil properties and bacteria) to CSB and Pb application. The results showed that 5% CSB better alleviated the toxicity of Pb on the shoot length of red clover, the biomass increased by 74.55 and 197.76% respectively and reduced the enrichment factor (BCF) and transport factor (TF) of red clover. Pb toxicity reduced soil nutrients, catalase (CAT), acid phosphatase (ACP) and urease activity, while the addition of CSB increased soil pH, soil organic matter (SOM) content and soil enzyme activity. 16S rDNA amplicon sequencing analysis showed that Pb toxicity reduced the diversity of rhizosphere bacteria in red clover and reduced the relative abundance of plant growth-promoting rhizobacteria such as Gemmatimonas, Devosia and Bryobacter. Spearman correlation analysis showed that the addition of alkaline CSB restored the relative abundance of rhizobacteria positively correlated with pH, such as Chitinophaga, Sphingomonas, Devosia and Pseudomonas, and thus restored the rhizosphere soil environment. This study demonstrates that 5% CSB can better alleviate the toxicity of Pb to red clover and soil. We also provide a theoretical basis for the subsequent use of beneficial bacteria to regulate the repair efficiency of red clover.

In recent years, drought stress caused by global warming has become a major constraint on agriculture. The thiamine thiazole synthase (THI1) is responsible for controlling thiamine production in plants displaying a response to various abiotic stresses. Nonetheless, most of the THI1 activities in plants remain largely unknown. In this study, we extracted MsTHI1 from alfalfa and demonstrated its beneficial impact on improving the resistance of plants to stress conditions. The highest levels of MsTHI1 expression were identified in alfalfa leaves, triggered by exposure to cold, drought, salt, or alkaline conditions. The upregulation of MsTHI1 in drought-stressed transgenic plants resulted in enhanced accumulation of vitamin B1 (VB1), chlorophyll a (Chl a), chlorophyll b (Chl b), soluble protein, higher soil and plant analyzer development (SPAD) value, and the activity of peroxidase (POD), maintained Fv/Fm, and decreased lipid peroxidation. Moreover, overexpression of MsTHI1 upregulated the transcription of THI4, TPK1, RbcX2, Cu/Zn-SOD, CPK13, and CPK32 and downregulated the transcription of TH1 and CPK17 in transgenic alfalfa under drought stress. These results suggested that MsTHI1 enhances drought tolerance by strengthening photosynthesis, regulating the antioxidant defense system, maintaining osmotic homeostasis, and mediating plant signal transduction.

Legume alfalfa (Medicago sativa L.) is extensively planted to reduce chemical fertilizer input to the soil and remedy damaged fields. The soil mechanism of these effects is potentially related to the variations in alfalfa-mediated interactions of the soil microbial community. To understand the impact of planting alfalfa on the soil microbial community in degraded black soil cultivated land, a 4-year experiment was conducted in degraded black soil cultivated land. We assessed soil parameters and characterized the functional and compositional diversity of the microbial community by amplicon sequencing that targeted the 16S rDNA gene of bacteria and ITS of fungi in four systems under corn cultivation at the Harbin corn demonstration base (Heilongjiang, China): multiyear corn planting (more than 30 years, MC1); 2 years of alfalfa-corn rotation (OC); 3 years of alfalfa planting (TA); and 4 years of alfalfa planting (FA). It was found out that alfalfa led to changes in the alpha diversity of soil bacteria rather than in fungi in the degraded arable land. The abundance of the bacterial groups Gemmatimonadetes, Actinobacteria, Planctomycetes, and Chloroflexi was increased in OC, while Proteobacteria and Acidobacteria and the fungal group Glomeromycota were increased in TA and FA. OC, TA, and FA significantly increased the pH level but reduced soil electrical conductivity, but they had no impact on soil available nitrogen and soil available potassium at the 0–15 cm soil depth. However, with the years of alfalfa planting, soil available nitrogen and soil available potassium were reduced at the 15–30 cm soil depth. OC, TA, and FA significantly reduced the soil available phosphorus and soil total phosphorus at the 15–30 cm soil depth. There was no significant impact made on soil total nitrogen. FA significantly reduced the soil organic matter at the 15–30 cm soil depth. Planting alfalfa in degraded black soil cultivated land can reduce the salt content of the soil, and the nutrient content of soil planted with alfalfa without fertilization was equivalent to that of degraded corn cultivated land with annual fertilization. Besides, alfalfa recruited and increased contained taxa with the capacity to improve soil nutrient utilization and inhibit the harmful influences of pathogens for subsequent crops. Meanwhile, the planting of alfalfa can modify soil conditions by promoting the proliferation of specific beneficial microbiota groups.

Alkaline stress severely limits plant growth and yield worldwide. NF-YC transcription factors (TFs) respond to abiotic stress by activating gene expression. However, the biological function of NF-YC TFs in alfalfa (Medicago sativa L.) is not clear. In our study, an NF-YC2 gene was identified and transgenic plants were obtained by constructing overexpression vector and cotyledon node transformation system in alfalfa. The open reading frame of MsNF-YC2 is 879 bp with 32.4 kDa molecular mass. MsNF-YC2 showed tissue expression specificity and was induced by a variety of abiotic stresses including drought, salt, and alkali stress in alfalfa. Under alkali stress treatment, transgenic plants exhibited higher levels of antioxidant enzyme activities and proline (Pro), correlating with a lower levels of hydrogen peroxide (H2O2), superoxide anion (O2–) compared with wild-type (WT) plants. Transcriptomic results showed that overexpression of MsNF-YC2 regulated the expression of phytohormone signal transduction and photosynthesis-related genes under normal and alkaline stress treatments. These results suggest that the MsNF-YC2 gene plays crucial role enhance alkali adaptation abilities in alfalfa.

Low temperature (LT) is an important threat to the normal growth of plants. In this study, based on the full-length transcriptome sequencing results, the cold resistance genes were cloned from Caucasian clover with strong cold resistance. We cloned the CDS of

Alkaline stress severely limits plant growth and yield worldwide. NF-YC transcription factors (TFs) respond to abiotic stress by activating gene expression. However, the biological function of NF-YC TFs in alfalfa (

Phytoremediation is a promising remediation strategy for degraded soil restoration. Root exudates are the main carrier substances for information communication and energy transfer between plant roots and soil, which play non-negligible roles in the restoration process. This work investigated the adaptation of Leymus chinensis root exudates to different degraded levels of soil and the mechanism of rhizosphere restoration in a 3-year degraded soil field study. We found that the soil quality at each degradation level significantly increased, with the soil organic matter (SOM) content slightly increasing by 1.82%, moderately increasing by 3.27%, and severely increasing by 3.59%, and there were significant increases in the contents of available nutrients such as available phosphorus (AP), ammonia nitrogen (AN), and nitrate nitrogen (NN). The physiological activities indicated that root tissue cells also mobilize oxidative stress to respond to the soil environment pressure. A total of 473 main components were obtained from root exudates by gas chromatography–time-of-flight mass spectrometry (GC–TOFMS), including acids, alcohols, carbohydrates, and other major primary metabolites. OPLS-DA revealed that soil degradation exerted an important influence on the metabolic characteristics of root exudates, and the numbers of both up- and downregulated metabolic characteristic peaks increased with the increase in the degree of degradation. Forty-three metabolites underwent clear changes, including some defense-related metabolites and osmotic adjustment substances that were significantly changed. These changes mainly mobilized a series of lipid metabolism pathways to maintain the fluidity of membrane function and help plants adapt to unfavorable soil environmental conditions. The PPP energy metabolism pathway was mobilized in response to slight degradation, and TCA energy pathways responded to the environmental pressure of severe soil degradation.

Alfalfa is widely grown worldwide as a perennial high-quality legume forage and as a good ecological landcover. The cysteine synthase (CSase) gene family is actively involved in plant growth and development and abiotic stress resistance but has not been systematically investigated in alfalfa. We identified 39 MsCSase genes on 4 chromosomes of the alfalfa genome. Phylogenetic analysis demonstrated that these genes were clustered into six subfamilies, and members of the same subfamily had similar physicochemical properties and sequence structures. Overexpression of the CSase gene in alfalfa increased alkali tolerance. Compared with control plants, the overexpression lines presented higher proline, soluble sugars, and cysteine and reduced glutathione contents and superoxide dismutase and peroxidase activities as well as lower hydrogen peroxide and superoxide anion contents after alkali stress. The relative expression of γ-glutamyl cysteine synthetase gene (a downstream gene of CSase) in the overexpression lines was much higher than that in the control line. The CSase gene enhanced alkalinity tolerance by regulating osmoregulatory substances and improving antioxidant capacity. These results provide a reference for studying the CSase gene family in alfalfa and expanding the alkali tolerance gene resources of forage plants.

Amphicarpaea edgeworthii, an annual twining herb, is a widely distributed species and an attractive model for studying complex flowering types and evolutionary mechanisms of species. Herein, we have generated a high-quality assembly of A. edgeworthii by using a combination of PacBio, 10× Genomics libraries, and Hi-C mapping technologies. The final 11 chromosome-level scaffolds covered 90.61% of the estimated genome (343.78Mb), which is a chromosome-scale assembled genome of an amphicarpic plant. Subsequently, we characterized the genetic diversity and population structure of A. edgeworthii species by resequencing individuals collected from their natural area of distribution. Using transcriptome profiling, we observed that specific phenotypes are regulated by a complex network of light, hormones, and MADS-box gene families. These data are beneficial for the discovery of genes that control major agronomic traits and spur genetic improvement of and functional genetic studies in legumes, as well as supply comparative genetic resources for other amphicarpic plants.

Pinewood nematode (PWN), the causal agent of pine wilt disease (PWD), causes massive global losses of Pinus species each year. Bacteria and fungi existing in symbiosis with PWN are closely linked with the pathogenesis of PWD, but the relationship between PWN pathogenicity and the associated microbiota is still ambiguous. This study explored the relationship between microbes and the pathogenicity of PWN by establishing a PWN-associated microbe library, and used this library to generate five artificial PWN–microbe symbiont (APMS) assemblies with gnotobiotic PWNs. The fungal and bacterial communities of different APMSs (the microbiome) were explored by next-generation sequencing. Furthermore, different APMSs were used to inoculate the same Masson pine (Pinus massoniana) cultivar, and multi-omics (metabolome, phenomics, and transcriptome) data were obtained to represent the pathogenicity of different APMSs at 14 days post-inoculation (dpi). Significant positive correlations were observed between microbiome and transcriptome or metabolome data, but microbiome data were negatively correlated with the reactive oxygen species (ROS) level in the host. Five response genes, four fungal genera, four bacterial genera, and nineteen induced metabolites were positively correlated with the ROS level, while seven induced metabolites were negatively correlated. To further explore the function of PWN-associated microbes, single genera of functional microbes (Mb1–Mb8) were reloaded onto gnotobiotic PWNs and used to inoculate pine tree seedlings. Three of the genera (Cladophialophora, Ochroconis, and Flavobacterium) decreased the ROS level of the host pine trees, while only one genus (Penicillium) significantly increased the ROS level of the host pine tree seedlings. These results demonstrate a clear relationship between associated microbes and the pathogenicity of PWN, and expand the knowledge on the interaction between PWD-induced forest decline and the PWN-associated microbiome.

Alfalfa is widely grown worldwide as a perennial high-quality legume forage and as a good ecological landcover. The cysteine synthase (CSase) gene family is actively involved in plant growth and development and abiotic stress resistance but has not been systematically investigated in alfalfa. We identified 39 MsCSase genes on 4 chromosomes of the alfalfa genome. Phylogenetic analysis demonstrated that these genes were clustered into six subfamilies, and members of the same subfamily had similar physicochemical properties and sequence structures. Overexpression of the CSase gene in alfalfa increased alkali tolerance. Compared with control plants, the overexpression lines presented higher proline, soluble sugars, and cysteine and reduced glutathione contents and superoxide dismutase and peroxidase activities as well as lower hydrogen peroxide and superoxide anion contents after alkali stress. The relative expression of γ-glutamyl cysteine synthetase gene (a downstream gene of CSase) in the overexpression lines was much higher than that in the control line. The CSase gene enhanced alkalinity tolerance by regulating osmoregulatory substances and improving antioxidant capacity. These results provide a reference for studying the CSase gene family in alfalfa and expanding the alkali tolerance gene resources of forage plants.

Carex meyeriana lowland meadow is an important component of natural grasslands in Hulun Buir. However, in Hulun Buir, fewer studies have been conducted on C. meyeriana lowland meadows than on other grassland types. To determine the most appropriate utilization mode for C. meyeriana lowland meadows, an experiment was conducted in Zhalantun city, Hulun Buir. Unused, moderately grazed, heavily grazed and mowed meadow sites were selected as the research objects. The analysis of experimental data from 4 consecutive years showed that relative to the other utilization modes, mowing and moderate grazing significantly increased C. meyeriana biomass. Compared with non-utilization, the other three utilization modes resulted in a higher plant diversity, and the moderately grazed meadow had the highest plant community stability. Moreover, principal component analysis (PCA) showed that among the meadow sites, the mowed meadow had the most stable plant community and soil physicochemical properties. Structural equation modeling (SEM) showed that grazing pressure was less than 0.25 hm2/sheep unit and that plant biomass in C. meyeriana lowland meadow increases with increasing grazing intensity, temperature and precipitation.

Alkaline salts (e.g., NaHCO3 and Na2CO3) causes more severe morphological and physiological damage to plants than neutral salts (e.g., NaCl and Na2SO4) due to differences in pH. The mechanism by which plants respond to alkali stress is not fully understood, especially in plants having symbotic relationships such as alfalfa (Medicago sativa L.). Therefore, a study was designed to evaluate the metabolic response of the root-nodule symbiosis in alfalfa under alkali stress using comparative metabolomics. Rhizobium-nodulized (RI group) and non-nodulized (NI group) alfalfa roots were treated with 200 mmol/L NaHCO3 and, roots samples were analyzed for malondialdehydyde (MDA), proline, glutathione (GSH), superoxide dismutase (SOD), and peroxidase (POD) content. Additionally, metabolite profiling was conducted using gas chromatography combined with time-of-flight mass spectrometry (GC/TOF-MS). Phenotypically, the RI alfalfa exhibited a greater resistance to alkali stress than the NI plants examined. Physiological analysis and metabolic profiling revealed that RI plants accumulated more antioxidants (SOD, POD, GSH), osmolytes (sugar, glycols, proline), organic acids (succinic acid, fumaric acid, and alpha-ketoglutaric acid), and metabolites that are involved in nitrogen fixation. Our pairwise metabolomics comparisons revealed that RI alfalfa plants exhibited a distinct metabolic profile associated with alkali putative tolerance relative to NI alfalfa plants. Data provide new information about the relationship between non-nodulized, rhizobium-nodulized alfalfa and alkali resistance.