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SUMMARY: Alfalfa is one of the most widely cultivated forage crops worldwide. However, soil salinization restricts alfalfa growth and development and affects global productivity. The plant cell wall is the first barrier against various stresses. Therefore, elucidating the alterations in cell wall architecture is crucial for stress adaptation. This study aimed to clarify the impact of myo‐inositol oxygenase 2 (MsMIOX2) on cell wall pectin and hemicellulose biosynthesis under saline–alkali stress and identify the upstream transcription factors that govern MsMIOX2. MsMIOX2 activation induced cell wall pectin and hemicellulose accumulation under saline–alkali stress. The effects of MsMIOX2 in saline–alkali tolerance were investigated by characterizing its overexpression and RNA interference lines. MsMIOX2 overexpression positively regulated the antioxidant system and photosynthesis in alfalfa under saline–alkali stress. MsMIOX2 exhibited myo‐inositol oxygenase activity, which increased polysaccharide contents, facilitated pectin and hemicellulose biosynthesis, and extended the cell wall thickness. However, MsMIOX2 RNA interference decreased cell wall thickness and alleviated alfalfa saline–alkali stress tolerance. In addition, MsbZIP53 was identified as an upstream transcriptional MsMIOX2 regulator by yeast one‐hybrid, electrophoretic mobility shift assay, dual‐luciferase, and beta‐glucuronidase assays. MsbZIP53 overexpression increased MsMIOX2 expression, elevated MIOX activity, reinforced the antioxidant system and photosynthesis, and increased saline–alkali stress tolerance in alfalfa. In conclusion, this study presents a novel perspective for elucidating the molecular mechanisms of saline–alkali stress tolerance in alfalfa and emphasizes the potential use of MsMIOX2 in alfalfa breeding. [ABSTRACT FROM AUTHOR]

Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn2+-dependent enzyme that plays a significant character in the plant's response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa MsFtsH8 by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both MsFtsH8-overexpressing Arabidopsis and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H2O2 accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of MsFtsH8 enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that MsFtsH8 mitigates oxidative damage caused by drought by modulating the plant's antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering. [ABSTRACT FROM AUTHOR]

Alfalfa (Medicago sativa L.) grown in acidic soils is often affected by phosphorus (P) deficiency, which results in reduced mineral nutrient content and forage quality. In this context, the effects of phosphorus (P) fertiliser remain unclear. In this study, we analysed the effects of P application on mineral nutrient content and forage quality in aluminium (Al)-sensitive (Longzhong) and Al-tolerant (Trifecta) alfalfa cultivars cultivated in two acidic soil environments. Mineral nutrient content and quality were affected by genotype, soil type, and P treatment concentration (p < 0.001). In limestone soil, for Longzhong and Trifecta, the optimal potassium (K), calcium (Ca), and magnesium (Mg) contents as well as crude protein content (CP) and ether extract (EE) values were observed at 20 mg P kg−1, that of the P content was observed at 40 mg P kg−1, and the minimum neutral detergent fibre (NDF) acid detergent lignin (ADL) values were observed at 40 mg P kg−1. In yellow soil, the maximum K, Ca, Mg, and P contents in Longzhong and Trifecta were observed at 40 mg P kg−1, whereas the maximum CP, EE, and ADL values were observed at 20 mg P kg−1. Our study provides an empirically based framework for optimising alfalfa fertilisation programmes in acidic soils. [ABSTRACT FROM AUTHOR]

Alfalfa species Medicago sativa L. (MS) and Medicago falcata L. (MF), globally prominent perennial leguminous forages, hold substantial economic value. However, our comprehension of the molecular mechanisms governing their resistance to cold stress remains limited. To address this knowledge gap, we scrutinized and compared MS and MF cold-stress responses at the molecular level following 24 h and 120 h low-temperature exposure (4 °C). Our study revealed that MF had superior physiological resilience to cold stress compared with MS, and its morphology was healthier under cold stress, and its malondialdehyde content and superoxide dismutase activity increased, first, and then decreased, while the soluble sugar content continued to accumulate. Transcriptome analysis showed that after 120 h of exposure, there were different gene-expression patterns between MS and MF, including 1274 and 2983 genes that were continuously up-regulated, respectively, and a total of 923 genes were included, including star cold-resistant genes such as ICE1 and SIP1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed numerous inter-species differences in sustained cold-stress responses. Notably, MS-exclusive genes included a single transcription factor (TF) gene and several genes associated with a single DNA repair-related pathway, whereas MF-exclusive genes comprised nine TF genes and genes associated with 14 pathways. Both species exhibited high-level expression of genes encoding TFs belonging to AP2-EREBP, ARR-B, and bHLH TF families, indicating their potential roles in sustaining cold resistance in alfalfa-related species. These findings provide insights into the molecular mechanisms governing cold-stress responses in MS and MF, which could inform breeding programs aimed at enhancing cold-stress resistance in alfalfa cultivars. [ABSTRACT FROM AUTHOR]

To explore the feasibility of using cold plasma as a mutagenesis breeding technology for forage crops, in this study we used the Medicago sativa L. cultivar, Zhongcao No. 3, as the experimental material. The effects of plasma treatments on Medicago sativa L. were analyzed through the use of plasma and activated water. Treatments with plasma and activated water inhibited plant height but promoted root growth. By creating a closed environment, adding a dielectric barrier plate, and combining these two treatment methods, the greatest impact can be had on the growth of Medicago sativa L. seeds. After treatment, the plant heights were approximately half those of the control group, and the root lengths were approximately 1.6 times those of the control group. Through emission spectroscopy, it was found that active particles such as O, NO 2 , and N 2 * were present and could be considered to have produced plasma-activated water through contact with the water surface, thus affecting the survival and growth of the seeds. Whole-genome resequencing (WGRS) was performed on the wild-type and selected mutants after treatment, with an average sequencing depth of 115.93×, an average genome alignment rate of 91.72 %, and an average genome coverage rate of 91.85 %. Various types of mutations were detected and annotated. After filtering, 7,822,324 SNP (single nucleotide polymorphisms) sites, 2,161,917 indel sites, 200,544 SV sites, 238 CNV (copy number variation) sites. The SNPs, indels (insertions/deletions), and SVs (structural variations) were mainly heterozygous, with heterozygosity rates of 87.13 %, 92.16 %, and 83.49 %, respectively. The CNVs were dominated by low copy numbers, accounting for 53.77 %. These results indicate that plasma treatment has significant effects on plant growth and genome of Medicago sativa L. [Display omitted] • The experimental material is the earliest and largest and most important forage grass cultivated in the world - Medicago sativa L. • Mutagenesis breeding uses a combination of physical and chemical mutagenesis to carry out mutagenesis. • In previous literature and research, but the use of plasma in biological mutagenesis breeding is still a new technology, and there are few relevant reports. • After mutagenesis treatment, the plant traits changed significantly. [ABSTRACT FROM AUTHOR]

The growth and biological decline of alfalfa may be linked to the rhizosphere microbiome. However, plant–microbe interactions in the rhizosphere of alfalfa and associated microbial community variations with stand age remain elusive. This study explored the successional pattern of rhizosphere microbial communities across different aged alfalfa stands and its relationship with alfalfa decline. Rhizosphere soils were collected from 2- and 6-year-old alfalfa stands. Control soils were collected from interspaces between alfalfa plants in the same stands. Soil bacterial and fungal communities were characterized by 16S and ITS rRNA gene sequencing, respectively. Specific microbial taxa colonized the rhizosphere soils, but not the control soils. The rhizosphere-specific taxa mainly included potentially beneficial genera (e.g., Dechloromonas, Verrucomicrobium) in the young stand and harmful genera (e.g., Peziza, Campylocarpon) in the old stand. Alfalfa roots regulated soil microbial communities by selective promotion or inhibition of distinct taxa. The majority of time-enriched taxa were reported as harmful fungi, whose relative abundances were negatively correlated with plant traits. Time-depleted taxa were mostly known as beneficial bacteria, which had relative abundances positively correlated with plant traits. The relative abundances of functional bacterial genes associated with vancomycin biosynthesis, zeatin biosynthesis, and amino acid metabolism trended lower in rhizosphere soils from the old stand. An upward trend was observed for fungal pathogens and wood saprotrophs with increasing stand age. The results suggest that root activity drives the negative succession of rhizosphere microbial communities during alfalfa decline in old stands. [ABSTRACT FROM AUTHOR]

Alfalfa (Medicago sativa L.) is an economically important forage legume. As a result of the extensive and intensive cultivation of alfalfa planting and the gradual expansion of planting areas, pest occurrence has increased in frequency. Aphids are one of the main pests that threaten the growth and productivity of alfalfa. After an aphid outbreak, alfalfa yield and quality are commonly greatly reduced. At present, there are few studies on alfalfa plants infested with aphids, so it is imperative to study the regulatory mechanisms of aphid infestation tolerance in alfalfa. In this study, alfalfa plants from the variety "Wudi" were investigated, and pea aphids were selected for inoculation. The transcriptome and metabolome data were analyzed at three time points (0 d, 1 d, and 4 d), revealing 3458 differentially expressed genes and 358 differential metabolites. Trend analysis of DEGs and DAMs revealed that the former were significantly enriched in three distinct trends, whereas the latter were significantly enriched in only one. Moreover, 117 important hub genes associated with alfalfa response to aphid infestation were screened by WGCNA. By mapping DEGs and DAMs to KEGG pathways, it was found that the "phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "isoflavonoid biosynthesis" pathways play an important role in alfalfa responses to aphid infestation. These results further elucidate the regulatory mechanism of alfalfa in response to aphid infestation and provide valuable information for breeding new aphid-resistant plant varieties. [ABSTRACT FROM AUTHOR]

Abstract Background The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa. Results In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses. Conclusion This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance.

Abstract Sowing date and soil fertility are very important factors in the overwintering and production performance of alfalfa (Medicago sativa L.), yet there’s a knowledge gap in knowledge on how late-seeded alfalfa responds to phosphorus (P) fertilization. A field study was conducted in Inner Mongolia from 2020 to 2022 using a split-plot design. The main plots consisted of five sowing dates (31 July, 8, 16, and 24 August, and 1 September), while the subplots involved five P application rates (0, 40, 70, 100, and 130 kg P2O5 ha−1). Throughout the growing seasons, the overwintering rate, root traits, forage yield, and yield components were measured. The results revealed a consistent decrease in overwintering ability and productivity with the delayed sowing. This reduction in overwintering rate was mainly due to diminished root traits, while the decrease in forage yield was largely associated with a reduction in plants per square meter. However, P fertilizer application to late-seeded alfalfa demonstrated potential in enhancing the diameter of both the crown and taproot, thus strengthening the root system and improving the overwintering rate, the rate of increase ranges from 11.6 to 49%. This adjustment could also improve the shoots per square meter and mass per shoot, increasing by 9.4–31.3% and 15.0–27.1% respectively in 2 years, which can offset the decline in forage yield caused by late sowing and might even increase the forage yield. Regression and path analysis indicated that alfalfa forage yield is primarily affected by mass per shoot rather than shoots per square meter. This study recommended that the sowing of alfalfa in similar regions of Inner Mongolia should not be later than mid-August. Moreover, applying P fertilizer (P2O5) at 70.6–85.9 kg ha−1 can enhance the forage yield and persistence of late-seeded alfalfa. Therefore, appropriate late sowing combined with the application of P fertilizer can be used as an efficient cultivation strategy for alfalfa cultivation after a short-season crop harvest in arid and cold regions.

Background: The mitogen-activated protein kinase (MAPK) cascade is crucial cell signal transduction mechanism that plays an important role in plant growth and development, metabolism, and stress responses. The MAPK cascade includes three protein kinases, MAPK, MAPKK, and MAPKKK. The three protein kinases mediate signaling to downstream response molecules by sequential phosphorylation. The MAPK gene family has been identified and analyzed in many plants, however it has not been investigated in alfalfa. Results: In this study, Medicago sativa MAPK genes (referred to as MsMAPKs) were identified in the tetraploid alfalfa genome. Eighty MsMAPKs were divided into four groups, with eight in group A, 21 in group B, 21 in group C and 30 in group D. Analysis of the basic structures of the MsMAPKs revealed presence of a conserved TXY motif. Groups A, B and C contained a TEY motif, while group D contained a TDY motif. RNA-seq analysis revealed tissue-specificity of two MsMAPKs and tissue-wide expression of 35 MsMAPKs. Further analysis identified MsMAPK members responsive to drought, salt, and cold stress conditions. Two MsMAPKs (MsMAPK70 and MsMAPK75) responds to salt and cold stresses; two MsMAPKs (MsMAPK60 and MsMAPK73) responds to cold and drought stresses; four MsMAPKs (MsMAPK1, MsMAPK33, MsMAPK64 and MsMAPK71) responds to salt and drought stresses; and two MsMAPKs (MsMAPK5 and MsMAPK7) responded to all three stresses. Conclusion: This study comprehensively identified and analysed the alfalfa MAPK gene family. Candidate genes related to abiotic stresses were screened by analysing the RNA-seq data. The results provide key information for further analysis of alfalfa MAPK gene functions and improvement of stress tolerance. [ABSTRACT FROM AUTHOR]

SUMMARY: Cold and saline‐alkali stress are frequently encountered by plants, and they often occur simultaneously in saline‐alkali soils at mid to high latitudes, constraining forage crop distribution and production. However, the mechanisms by which forage crops respond to the combination of cold and saline‐alkali stress remain unknown. Alfalfa (Medicago sativa L.) is one of the most essential forage grasses in the world. In this study, we analyzed the complex response mechanisms of two alfalfa species (Zhaodong [ZD] and Blue Moon [BM]) to combined cold and saline‐alkali stress using multi‐omics. The results revealed that ZD had a greater ability to tolerate combined stress than BM. The tricarboxylic acid cycles of the two varieties responded positively to the combined stress, with ZD accumulating more sugars, amino acids, and jasmonic acid. The gene expression and flavonoid content of the flavonoid biosynthesis pathway were significantly different between the two varieties. Weighted gene co‐expression network analysis and co‐expression network analysis based on RNA‐Seq data suggested that the MsMYB12 gene may respond to combined stress by regulating the flavonoid biosynthesis pathway. MsMYB12 can directly bind to the promoter of MsFLS13 and promote its expression. Moreover, MsFLS13 overexpression can enhance flavonol accumulation and antioxidant capacity, which can improve combined stress tolerance. These findings provide new insights into improving alfalfa resistance to combined cold and saline‐alkali stress, showing that flavonoids are essential for plant resistance to combined stresses, and provide theoretical guidance for future breeding programs. [ABSTRACT FROM AUTHOR]

Sowing date and soil fertility are very important factors in the overwintering and production performance of alfalfa (Medicago sativa L.), yet there's a knowledge gap in knowledge on how late-seeded alfalfa responds to phosphorus (P) fertilization. A field study was conducted in Inner Mongolia from 2020 to 2022 using a split-plot design. The main plots consisted of five sowing dates (31 July, 8, 16, and 24 August, and 1 September), while the subplots involved five P application rates (0, 40, 70, 100, and 130 kg P2O5 ha−1). Throughout the growing seasons, the overwintering rate, root traits, forage yield, and yield components were measured. The results revealed a consistent decrease in overwintering ability and productivity with the delayed sowing. This reduction in overwintering rate was mainly due to diminished root traits, while the decrease in forage yield was largely associated with a reduction in plants per square meter. However, P fertilizer application to late-seeded alfalfa demonstrated potential in enhancing the diameter of both the crown and taproot, thus strengthening the root system and improving the overwintering rate, the rate of increase ranges from 11.6 to 49%. This adjustment could also improve the shoots per square meter and mass per shoot, increasing by 9.4–31.3% and 15.0–27.1% respectively in 2 years, which can offset the decline in forage yield caused by late sowing and might even increase the forage yield. Regression and path analysis indicated that alfalfa forage yield is primarily affected by mass per shoot rather than shoots per square meter. This study recommended that the sowing of alfalfa in similar regions of Inner Mongolia should not be later than mid-August. Moreover, applying P fertilizer (P2O5) at 70.6–85.9 kg ha−1 can enhance the forage yield and persistence of late-seeded alfalfa. Therefore, appropriate late sowing combined with the application of P fertilizer can be used as an efficient cultivation strategy for alfalfa cultivation after a short-season crop harvest in arid and cold regions. [ABSTRACT FROM AUTHOR]

Field trials were conducted (2022–2023) in a randomized complete block design to evaluate the combined use of a microbial biostimulant, P-K Stim, which contains phosphate and potassium solubilizing bacteria (109 Colony Forming Units mL−1), various fertilization rates, and herbicide use on weed growth and the productivity of alfalfa (Medicago sativa L.), oilseed rape (Brassica napus L.), and durum wheat (Triticum durum Desf.). The following treatment list was the same on all trial fields: (1) 100% basal fertilization (100N), (2) 20% basal fertilization and application of microbial biostimulant P-K Stim (20N + PK), (3) 100% basal fertilization and post-emergence herbicide treatment (100N + H), (4) 20% basal fertilization together with the application of P-K Stim and a post-emergence herbicide treatment (20N + PK + H), and (5) 50% basal fertilization together with the application of P-K Stim and a post-emergence herbicide treatment (50N + PK + H). The combined use of fertilization, biostimulants, and herbicides significantly affected crop yield, its components and weed biomass (p ≤ 0.05). The concentrations for potassium and phosphorus were higher in the 20N + PK + H and 50N + PK + H treatments for all crops compared to other treatments. Nutrient concentrations were remarkably high across all crops, closely approximating the values of the recommended nitrogen fertilization. Crop yield and its components were positively influenced by the 20N + PK + H and 50N + PK + H treatments. Weed biomass was significantly lower in these plots compared to other treatments. [ABSTRACT FROM AUTHOR]

Medicago sativa L. (alfalfa) is a herbaceous plant of great importance in agriculture. The chemical composition of the substrate affects the mineral supply as well as the growth, development and metabolism of the plants. Therefore, the aim of this experiment is to determine the influence of zinc, copper and platinum complexes ([ZnCl 2 (en)], [CuCl 2 (en)], cis -[PtCl 2 (NH 3) 2 ], en = ethylenediamine) on the germination and growth of alfalfa and on the content of phenolic compounds. Alfalfa seeds were sown in three separate groups and treated with the above complexes, the fourth group being the control. The growth of the vegetative part was observed after the tenth, twentieth and thirtieth day. The percentage of germination was recorded for each experimental group. At the end of the treatment, the aerial parts of each group were dried and prepared for extraction with methanol, after which the quantitative composition of phenolic compounds was analyzed by liquid chromatography.The results obtained show that [CuCl 2 (en)] has the best effect on the germination percentage, while the treatment with the platinum(II) complex - cis -[PtCl 2 (NH 3) 2 ] showed a significant effect on the growth of alfalfa. The results of chromatographic analysis showed that [ZnCl 2 (en)] and [CuCl 2 (en)] improved the synthesis of phenolic compounds of alfalfa, with a significant content of catechins in the treatment with the copper complex compared to the control group. Based on the obtained results, it was concluded that the complex compounds have a significant effect on the germination, growth and phenolic compounds content of alfalfa, with different effects for each of the studied processes. [Display omitted] • Medicago sativa L. (alfalfa) is plant with high nutritional value. • The influence of metal complexes on the germination and growth of alfalfa was investigated. • The effects of these complexes on the content of phenolic compounds was also evaluated. • The complexes had a positive effect on seed germination and cis -[PtCl2(NH3)2] had a significant effect on increasing the concentration of almost all phenolic compounds. [ABSTRACT FROM AUTHOR]

Silage preservation is critical for livestock's stable forage supply during cold seasons and fostering the recycling of agricultural byproducts. We assessed the effects of adding previously fermented juices (PFJ) and different proportions of Sorghum Sudanense (Piper) Stapf (Sudan grass) on the silage quality of Medicago sativa L. (alfalfa). A 50% alfalfa and 50% Sudan grass (M50) mixture exhibited superior performance in sensory evaluation and fermentation quality. The addition of PFJ (PFJI group) further enhanced quality, and increasing the proportion of alfalfa improved the nutritional quality. However, PFJI had no significant effect on the degradation rates of these nutritional indicators (p > 0.05). As alfalfa proportion increased, the disappearance rates of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber in silage increased. Principal component, correlation, and membership function analyses revealed that treatment with M50 without PFJ (PFJ0+M50) had the best effect, followed by treatment with 25% alfalfa and 75% Sudan grass (PFJ0+M25) and 100% alfalfa (PFJ0+M0). Therefore, the mixing ratio of alfalfa and Sudan grass should be maintained between 25% and 50% to optimize the nutritional and fermentation qualities of silage. These findings offer crucial guidance for alfalfa silage preparation, promoting enhanced livestock industry productivity and sustainable agricultural development. [ABSTRACT FROM AUTHOR]

The stem base of alfalfa is a critical part for its overwintering, regeneration, and yield. To better understand the specificity and importance of the stem base, we analyzed the structure, metabolic substances, and transcriptome of the stem base using anatomical techniques, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and RNA sequencing (RNA-seq), and compared it with stems and roots. The anatomical structure shows that the ratio of xylem to phloem changes at the base of the stem. A total of 801 compounds involved in 91 metabolic pathways were identified from the broadly targeted metabolome. Transcriptome analysis revealed 4974 differentially expressed genes (DEGs) at the stem base compared to the stem, and 5503 DEGs compared to the root. Comprehensive analyses of differentially accumulated compounds (DACs) and DEGs, in the stem base vs. stem, identified 10 valuable pathways, including plant hormone signal transduction, zeatin biosynthesis, α-Linolenic acid metabolism, histidine metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, pentose phosphate pathway, galactose metabolism, and fructose and mannose metabolism. The pathways of plant hormone signal transduction and carbon metabolism were also identified by comparing the stem base with the roots. Taken together, the stem base of alfalfa is the transition region between the stem and root in morphology; in terms of material metabolism, its growth, development, and function are regulated through hormones and sugars. [ABSTRACT FROM AUTHOR]

The forage quality of alfalfa (Medicago sativa) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa. The forage quality of alfalfa (Medicago sativa) stems is greater than the leaves. Starch and sucrose metabolism provide energy during alfalfa stem development. Different varieties of alfalfa have different sucrose, starch and related enzyme activities. Hollow stems will be formed during the rapid growth of alfalfa, which may be related to the redistribution of carbohydrates in the pith tissue. Our results may provide a new theoretical basis for understanding the molecular mechanism of sucrose and starch accumulation in alfalfa stems. [ABSTRACT FROM AUTHOR]

[Objective] In order to explore the effects of soil phosphorus (P) fertilizer and salt and their interaction on the plant growth, P nutrition and salt tolerance of alfalfa (Medicago sativa). [Methods] Alfalfa "Gannon Ⅶ" was planted by adding phosphate fertilizer (0, 40, 80, 160 mg/kg, in the form of KH2PO4) and NaCl (0, 0.4, 0.8, 1.6 g/kg) with different concentration gradients to the loess soil. [Results] The aboveground and underground biomass of alfalfa increased with the increase of P application level, but decreased with the increase of NaCl addition level. When P was added, P uptake by plants increased. When P was added at 160 mg/kg, P concentrations in roots, stems, and leaves all reached the maximum. However, the addition of salinity inhibited the absorption of P. When NaCl was added at 1.6 g/kg, P concentrations in roots, stems, and leaves all significantly decreased, when compared with that without NaCl. The content of tartrate in rhizosphere of alfalfa decreased with the increase of P compared with that without P, and the addition of exogenous salt significantly increased the content of tartrate in rhizosphere. The rhizosphere soil pH of most treatments was lower than that of non-rhizosphere pH, the decrease of soil pH under salt stress might increase the availability of soil P and P uptake by plants. A high-dose (160 mg/kg) of P significantly reduced the P-uptake efficiency of P-utilization efficiency of alfalfa. [Conclusion] In summary, there was a significant interaction between soil P fertilization and salinity, and the increase in salinity exacerbated plant P deficiency. Appropriate application of P-fertilizers can improve the salt tolerance of alfalfa and enhance its productivity in saline soils.

Microorganisms in the soil that have been contaminated with heavy metals for a long-time exhibited resistance to heavy metals. In recent years, the use of resistant microbes and heavy metal-tolerant plants to repair heavy metal-contaminated soil has become an increasingly hot subject. In this study, heavy metal-tolerant bacteria ZG7 was isolated from the mining soils and injected into Bidens pilosa L. and Medicago sativa L.. The results revealed that: (1) Compared to the non-inoculated group, inoculation with ZG7 notably increased the dry weight and chlorophyll content in plants under heavy metal stress, as well as promoted the content of several types of antioxidant chemicals; (2) Combined remediation improved soil enzyme activities, with soil catalase, sucrase, and phosphatase activities remarkably increased by 136.06%, 124.17% and 180.14% in the ZG7- Bidens pilosa L.; And soil catalase, urease, and sucrase activities promoted by 68.07%, 130.08% and 239.79% in the ZG7- Medicago sativa L.; (3) The content of Pb and Zn in the ZG7- Medicago sativa L. combination soil were considerably lower (9.28% and 6.2%, respectively) than no strain group, but there was no substantial change in soil heavy metal content when combined with the Bidens pilosa L. remediation when compared to no strain group. In summary, the combination of strain ZG7 and plants had a good effect on the remediation of heavy metal contaminated soils, and ZG7- Medicago sativa L. had the best effect, which is a reliable technique for effective control of heavy metal pollution. This study provided theoretical support for microbial-phytoremediation joint remediation. [ABSTRACT FROM AUTHOR]

The perennial legume alfalfa (Medicago sativa L.) is of high value in providing cheap and high-nutritive forages. Due to a lack of tillage during the production period, the soil in which alfalfa grows prunes to become compacted through highly mechanized agriculture. Compaction deteriorates the soil's structure and fertility, leading to compromised alfalfa development and productivity. However, the way alfalfa responses to different levels of soil compaction and the underlying molecular mechanism are still unclear. In this study, we systematically evaluated the effects of gradient compacted soil on the growth of different cultivars of alfalfa, especially the root system architecture, phytohormones and internal gene expression profile alterations. The results showed that alfalfa growth was facilitated by moderate soil compaction, but drastically inhibited when compaction was intensified. The inhibition effect was universal across different cultivars, but with different severity. Transcriptomic and physiological studies revealed that the expression of a set of genes regulating the biosynthesis of lignin and flavonoids was significantly repressed in compaction treated alfalfa roots, and this might have resulted in a modified secondary cell wall and xylem vessel formation. Phytohormones, like ABA, are supposed to play pivotal roles in the regulation of the overall responses. These findings provide directions for the improvement of field soil management in alfalfa production and the molecular breeding of alfalfa germplasm with better soil compaction resilience. [ABSTRACT FROM AUTHOR]

Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn2+-dependent enzyme that plays a significant character in the plant’s response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa MsFtsH8 by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both MsFtsH8-overexpressing Arabidopsis and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H2O2 accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of MsFtsH8 enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that MsFtsH8 mitigates oxidative damage caused by drought by modulating the plant’s antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering.

Alfalfa (Medicago sativa L.) grown in acidic soils is often affected by phosphorus (P) deficiency, which results in reduced mineral nutrient content and forage quality. In this context, the effects of phosphorus (P) fertiliser remain unclear. In this study, we analysed the effects of P application on mineral nutrient content and forage quality in aluminium (Al)-sensitive (Longzhong) and Al-tolerant (Trifecta) alfalfa cultivars cultivated in two acidic soil environments. Mineral nutrient content and quality were affected by genotype, soil type, and P treatment concentration (p < 0.001). In limestone soil, for Longzhong and Trifecta, the optimal potassium (K), calcium (Ca), and magnesium (Mg) contents as well as crude protein content (CP) and ether extract (EE) values were observed at 20 mg P kg−1, that of the P content was observed at 40 mg P kg−1, and the minimum neutral detergent fibre (NDF) acid detergent lignin (ADL) values were observed at 40 mg P kg−1. In yellow soil, the maximum K, Ca, Mg, and P contents in Longzhong and Trifecta were observed at 40 mg P kg−1, whereas the maximum CP, EE, and ADL values were observed at 20 mg P kg−1. Our study provides an empirically based framework for optimising alfalfa fertilisation programmes in acidic soils.

Alfalfa species Medicago sativa L. (MS) and Medicago falcata L. (MF), globally prominent perennial leguminous forages, hold substantial economic value. However, our comprehension of the molecular mechanisms governing their resistance to cold stress remains limited. To address this knowledge gap, we scrutinized and compared MS and MF cold-stress responses at the molecular level following 24 h and 120 h low-temperature exposure (4 °C). Our study revealed that MF had superior physiological resilience to cold stress compared with MS, and its morphology was healthier under cold stress, and its malondialdehyde content and superoxide dismutase activity increased, first, and then decreased, while the soluble sugar content continued to accumulate. Transcriptome analysis showed that after 120 h of exposure, there were different gene-expression patterns between MS and MF, including 1274 and 2983 genes that were continuously up-regulated, respectively, and a total of 923 genes were included, including star cold-resistant genes such as ICE1 and SIP1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed numerous inter-species differences in sustained cold-stress responses. Notably, MS-exclusive genes included a single transcription factor (TF) gene and several genes associated with a single DNA repair-related pathway, whereas MF-exclusive genes comprised nine TF genes and genes associated with 14 pathways. Both species exhibited high-level expression of genes encoding TFs belonging to AP2-EREBP, ARR-B, and bHLH TF families, indicating their potential roles in sustaining cold resistance in alfalfa-related species. These findings provide insights into the molecular mechanisms governing cold-stress responses in MS and MF, which could inform breeding programs aimed at enhancing cold-stress resistance in alfalfa cultivars.

The growth and biological decline of alfalfa may be linked to the rhizosphere microbiome. However, plant–microbe interactions in the rhizosphere of alfalfa and associated microbial community variations with stand age remain elusive. This study explored the successional pattern of rhizosphere microbial communities across different aged alfalfa stands and its relationship with alfalfa decline. Rhizosphere soils were collected from 2- and 6-year-old alfalfa stands. Control soils were collected from interspaces between alfalfa plants in the same stands. Soil bacterial and fungal communities were characterized by 16S and ITS rRNA gene sequencing, respectively. Specific microbial taxa colonized the rhizosphere soils, but not the control soils. The rhizosphere-specific taxa mainly included potentially beneficial genera (e.g., Dechloromonas, Verrucomicrobium) in the young stand and harmful genera (e.g., Peziza, Campylocarpon) in the old stand. Alfalfa roots regulated soil microbial communities by selective promotion or inhibition of distinct taxa. The majority of time-enriched taxa were reported as harmful fungi, whose relative abundances were negatively correlated with plant traits. Time-depleted taxa were mostly known as beneficial bacteria, which had relative abundances positively correlated with plant traits. The relative abundances of functional bacterial genes associated with vancomycin biosynthesis, zeatin biosynthesis, and amino acid metabolism trended lower in rhizosphere soils from the old stand. An upward trend was observed for fungal pathogens and wood saprotrophs with increasing stand age. The results suggest that root activity drives the negative succession of rhizosphere microbial communities during alfalfa decline in old stands.

Alfalfa (Medicago sativa L.) is an economically important forage legume. As a result of the extensive and intensive cultivation of alfalfa planting and the gradual expansion of planting areas, pest occurrence has increased in frequency. Aphids are one of the main pests that threaten the growth and productivity of alfalfa. After an aphid outbreak, alfalfa yield and quality are commonly greatly reduced. At present, there are few studies on alfalfa plants infested with aphids, so it is imperative to study the regulatory mechanisms of aphid infestation tolerance in alfalfa. In this study, alfalfa plants from the variety “Wudi” were investigated, and pea aphids were selected for inoculation. The transcriptome and metabolome data were analyzed at three time points (0 d, 1 d, and 4 d), revealing 3458 differentially expressed genes and 358 differential metabolites. Trend analysis of DEGs and DAMs revealed that the former were significantly enriched in three distinct trends, whereas the latter were significantly enriched in only one. Moreover, 117 important hub genes associated with alfalfa response to aphid infestation were screened by WGCNA. By mapping DEGs and DAMs to KEGG pathways, it was found that the “phenylpropanoid biosynthesis”, “flavonoid biosynthesis”, and “isoflavonoid biosynthesis” pathways play an important role in alfalfa responses to aphid infestation. These results further elucidate the regulatory mechanism of alfalfa in response to aphid infestation and provide valuable information for breeding new aphid-resistant plant varieties.

Proper water and fertilizer management strategies are essential for alfalfa cultivation in arid areas. However, at present, the optimal amounts of subsurface irrigation and nitrogen (N) supply for alfalfa (Medicago sativa L.) cultivation are still unclear. Therefore, a field experiment was conducted in 2022 in Yinchuan, Ningxia, China, to explore the effects of different subsurface irrigation levels (W1, 50% of ETC (crop evapotranspiration); W2, 75% of ETC; W3, 100% of ETC) and N application rates (N0, 0 kg/ha; N1, 75 kg/ha; N2, 150 kg/ha; N3, 225 kg/ha; N4, 300 kg/ha) on alfalfa yield, crop water productivity (CWP), N use efficiency (NUE), quality, and economic benefits. Besides, the least squares method and multiple regression analysis were used to explore the optimal water and N combination for alfalfa cultivation under subsurface irrigation. The results showed that the alfalfa yield, crude ash content, and partial factor productivity from applied N (PFPN) were the highest under W2 level, but there was no difference in PFPN compared with that under W3 level. The branch number (BN), leaf area index (LAI), yield, CWP, irrigation water productivity (IWP), crude protein content (CPC), and economic benefits increased and then decreased with the increase of N application rate, reaching a maximum at the N2 or N3 level, while the NUE and PFPN decreased with the increase of N application rate. Considering the yield, CWP, NUE, quality, and economic benefits, W2N2 treatment was the optimal for alfalfa cultivation under subsurface irrigation. Besides, when the irrigation volume and N application rate were 69.8 ~ 88.7% of ETC and 145 ~ 190 kg/ha, respectively (confidence interval: 85%), the yield, CPC, and economic benefits reached more than 85% of the maximum. This study will provide technique reference for the water and N management in alfalfa cultivation in Northwest China. [ABSTRACT FROM AUTHOR]

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Increasing pressure over water resources in the western U.S. is currently forcing alfalfa (Medicago sativa L.) producers to adopt water-saving irrigation techniques. Crop yield forecasting tools can be used to develop smart irrigation scheduling methods that can be used to estimate the future effects of a given irrigation amount applied during a current irrigation event on yield. In this work, a linear model and a random forest model were used to estimate the yield of irrigated alfalfa crops in northern Nevada. It was found that water (rain + irrigation), the occurrence of extreme temperatures, and wind have a greater effect on crop yield. Other variables that accounted for the photoperiod and the dormant period were also included in the model and are also important. The linear model had the best performance with an R2 of 0.854. On the other hand, the R2 value for the random forest was 0.793. The linear model showed a good response to water variability; therefore, it is a good model to consider for use as an irrigation decision support system. However, unlike the linear model, the random forest model can capture non-linear relationships occurring between the crop, water, and the atmosphere, and its results may be enhanced by including more data for its training. [ABSTRACT FROM AUTHOR]

New types of extruded snack pellets of wheat-corn blend base and fresh lucerne sprouts were developed. The aim of the study was to examine the effect of fresh lucerne sprouts addition on the water consumption, processing efficiency and the specific mechanical energy during production of wheat-maize snack pellets. Additionally, the total phenolic content and antiradical activity, as well as the water absorption and water solubility indices in samples processed under variable processing conditions were tested. The extrusion-cooking of blends consisted of 10, 20 and 30% of lucerne sprouts was carried out using a single screw extruder at screw speeds of 60 and 100 rpm, and at moisture contents of 32, 34 and 36%. Replacement of wheat-corn flour blends by fresh lucerne sprouts at various levels (10, 20 and 30%) enabled to sufficiently reduce technological water which is needed in extrusioncooking process of snacks pellets. The limitation of water was from 89 to 100% if fresh lucerne sprouts were used, depending on the recipe and dough moisture level tested. Total phenolic content and antioxidant activity increased significantly due to lucerne sprouts addition. Furthermore, higher water absorption and water solubility index were noted if increased initial moisture content was applied during the processing of snack pellets. It can be concluded that fresh lucerne sprouts can be valuable additives, enabling to save the technological water in production process and to obtain nutritionally valuable supplemented wheat-corn-based snack pellets. [ABSTRACT FROM AUTHOR]

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In this work, the utilization of phosphogypsum (PG), a waste coming from the manufacture of phosphate fertilizers, as fertilizer for alfalfa (Medicago sativa L.) crops was investigated using pot experiments. The objective of this study was to evaluate the effects of both phosphogypsum and red mud (RM) in two soils representative of the pasture production area in Southern Spain. The morpho-physiological parameters of biomass, plant height, number of stems and number of leaves, as well as the chemical parameters of soil content, were measured. High doses of PG inhibited seed germination in some treatments. In addition, the treatment substrate (2550 g soil + 50 g kg−1 PG + 100 g kg−1 RM) also affected seed germination, possibly due to the large amount of RM. The application of PG and RM to the soil increased the availability of important nutrients for alfalfa, such as phosphorus (P), calcium (Ca2+) and magnesium (Mg2+). The results demonstrate that the treatment with PG significantly improved the uptake of P in alfalfa.

Increasing pressure over water resources in the western U.S. is currently forcing alfalfa (Medicago sativa L.) producers to adopt water-saving irrigation techniques. Crop yield forecasting tools can be used to develop smart irrigation scheduling methods that can be used to estimate the future effects of a given irrigation amount applied during a current irrigation event on yield. In this work, a linear model and a random forest model were used to estimate the yield of irrigated alfalfa crops in northern Nevada. It was found that water (rain + irrigation), the occurrence of extreme temperatures, and wind have a greater effect on crop yield. Other variables that accounted for the photoperiod and the dormant period were also included in the model and are also important. The linear model had the best performance with an R2 of 0.854. On the other hand, the R2 value for the random forest was 0.793. The linear model showed a good response to water variability; therefore, it is a good model to consider for use as an irrigation decision support system. However, unlike the linear model, the random forest model can capture non-linear relationships occurring between the crop, water, and the atmosphere, and its results may be enhanced by including more data for its training.

Abstract Background Alfalfa (Medicago sativa L.) is a perennial leguminous forage that can improve the properties of saline soil. In addition, the supplementation with fertilizer to promote soil bacterial activity is critical to improve the productivity of coastal saline soils. However, the association between fertilizer application, bacterial community characteristics, and alfalfa yield in saline soil remains unclear. Aim To understand the interaction of different fertilizer and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil. Results A 4-year field study was conducted to examine the interaction of different fertilizer treatments and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil in China. Three organic fertilizer treatments (fulvic acid potassium, fulvic acid potassium + wood vinegar, and fulvic acid potassium + Bacillus), one biochar treatment (bio-charcoal), one inorganic fertilizer treatment (diammonium phosphate), and a control (no fertilizer) were included. The effects of the six treatments and 15 cutting times on alfalfa yield and soil bacterial community diversity were assessed. The productivity in fulvic acid potassium + Bacillus and fulvic acid potassium treatments was 68.37 and 67.90 t·hm−2 in 4 years, which was significantly higher than that in the bio-charcoal treatment and control. Hydrothermal conditions and timing of alfalfa harvest had significant effects on the soil bacterial community. Proteobacteria, Acidobacteria, and Actinobacteria were the principal bacterial phyla detected in the soil samples, collectively accounting for approximately 60% of the total bacterial abundance. The relative abundance of Bacteroidetes, Firmicutes, and Nitrospirae was significantly correlated with alfalfa yield, and the abundance of these phyla was also affected by the maximum temperature and precipitation. Fertilizer supplementation in coastal saline soil can effectively increase the yield of alfalfa. Among the fertilizers tested, fulvic acid potassium had the strongest effect, whereas bio-charcoal fertilizer had little effect on alfalfa yield. Graphical Abstract

Abstract The WUSCHEL-related homeobox (WOX) family members are plant-specific transcriptional factors, which function in meristem maintenance, embryogenesis, lateral organ development, as well as abiotic stress tolerance. In this study, 14 MsWOX transcription factors were identified and comprehensively analyzed in the cultivated alfalfa cv. Zhongmu No.1. Overall, 14 putative MsWOX members containing conserved structural regions were clustered into three clades according to phylogenetic analysis. Specific expression patterns of MsWOXs in different tissues at different levels indicated that the MsWOX genes play various roles in alfalfa. MsWUS, MsWOX3, MsWOX9, and MsWOX13-1 from the three subclades were localized in the nucleus, among which, MsWUS and MsWOX13-1 exhibited strong self-activations in yeast. In addition, various cis-acting elements related to hormone responses, plant growth, and stress responses were identified in the 3.0 kb promoter regions of MsWOXs. Expression detection of separated shoots and roots under hormones including auxin, cytokinin, GA, and ABA, as well as drought and cold stresses, showed that MsWOX genes respond to different hormones and abiotic stress treatments. Furthermore, transcript abundance of MsWOX3, and MsWOX13-2 were significantly increased after rhizobia inoculation. This study presented comprehensive data on MsWOX transcription factors and provided valuable insights into further studies of their roles in developmental processes and abiotic stress responses in alfalfa.

Silage preservation is critical for livestock’s stable forage supply during cold seasons and fostering the recycling of agricultural byproducts. We assessed the effects of adding previously fermented juices (PFJ) and different proportions of Sorghum Sudanense (Piper) Stapf (Sudan grass) on the silage quality of Medicago sativa L. (alfalfa). A 50% alfalfa and 50% Sudan grass (M50) mixture exhibited superior performance in sensory evaluation and fermentation quality. The addition of PFJ (PFJI group) further enhanced quality, and increasing the proportion of alfalfa improved the nutritional quality. However, PFJI had no significant effect on the degradation rates of these nutritional indicators (p > 0.05). As alfalfa proportion increased, the disappearance rates of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber in silage increased. Principal component, correlation, and membership function analyses revealed that treatment with M50 without PFJ (PFJ0+M50) had the best effect, followed by treatment with 25% alfalfa and 75% Sudan grass (PFJ0+M25) and 100% alfalfa (PFJ0+M0). Therefore, the mixing ratio of alfalfa and Sudan grass should be maintained between 25% and 50% to optimize the nutritional and fermentation qualities of silage. These findings offer crucial guidance for alfalfa silage preparation, promoting enhanced livestock industry productivity and sustainable agricultural development.

Field trials were conducted (2022–2023) in a randomized complete block design to evaluate the combined use of a microbial biostimulant, P-K Stim, which contains phosphate and potassium solubilizing bacteria (109 Colony Forming Units mL−1), various fertilization rates, and herbicide use on weed growth and the productivity of alfalfa (Medicago sativa L.), oilseed rape (Brassica napus L.), and durum wheat (Triticum durum Desf.). The following treatment list was the same on all trial fields: (1) 100% basal fertilization (100N), (2) 20% basal fertilization and application of microbial biostimulant P-K Stim (20N + PK), (3) 100% basal fertilization and post-emergence herbicide treatment (100N + H), (4) 20% basal fertilization together with the application of P-K Stim and a post-emergence herbicide treatment (20N + PK + H), and (5) 50% basal fertilization together with the application of P-K Stim and a post-emergence herbicide treatment (50N + PK + H). The combined use of fertilization, biostimulants, and herbicides significantly affected crop yield, its components and weed biomass (p ≤ 0.05). The concentrations for potassium and phosphorus were higher in the 20N + PK + H and 50N + PK + H treatments for all crops compared to other treatments. Nutrient concentrations were remarkably high across all crops, closely approximating the values of the recommended nitrogen fertilization. Crop yield and its components were positively influenced by the 20N + PK + H and 50N + PK + H treatments. Weed biomass was significantly lower in these plots compared to other treatments.

The stem base of alfalfa is a critical part for its overwintering, regeneration, and yield. To better understand the specificity and importance of the stem base, we analyzed the structure, metabolic substances, and transcriptome of the stem base using anatomical techniques, ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and RNA sequencing (RNA-seq), and compared it with stems and roots. The anatomical structure shows that the ratio of xylem to phloem changes at the base of the stem. A total of 801 compounds involved in 91 metabolic pathways were identified from the broadly targeted metabolome. Transcriptome analysis revealed 4974 differentially expressed genes (DEGs) at the stem base compared to the stem, and 5503 DEGs compared to the root. Comprehensive analyses of differentially accumulated compounds (DACs) and DEGs, in the stem base vs. stem, identified 10 valuable pathways, including plant hormone signal transduction, zeatin biosynthesis, α-Linolenic acid metabolism, histidine metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, pentose phosphate pathway, galactose metabolism, and fructose and mannose metabolism. The pathways of plant hormone signal transduction and carbon metabolism were also identified by comparing the stem base with the roots. Taken together, the stem base of alfalfa is the transition region between the stem and root in morphology; in terms of material metabolism, its growth, development, and function are regulated through hormones and sugars.

In recent decades, agriculture in Greece has undergone significant changes due to the intensification of land use. This intensification, contrary to the sustainable land management, has an impact on the healthiness of the environmental agroecosystem. This study aimed to investigate if these cultivation systems influence some of the main edaphic properties and plant species diversity and which are the main anthropogenic interventions which may have a decisive role in the changes of soil physicochemical properties and the reduction of plant species. To assess the environmental implications among different types of land-use, the most sensitive and reliable edaphic indicators were measured and plant species diversity was recorded. Three land-use types such as maize crops, alfalafa and abandoned land were selected and ten fields with an area of approximately 1 ha in each land use type were taken. The results indicate that among the edaphic parameters pH, sand, electrical conductivity (EC), soil organic matter (SOM), NO3- as well as Ca+2 and Cu+2 soil concentration presented statistically significant differences among thirty cultivated fields. Among three different land uses, the edaphic variables EC, SOM, BD, Cal & Na concentrations presented significant differences. A higher soil organic matter content was observed in abandoned land while bulk density and electric conductivity value as well as Na+ concentrations were lower compared to other land uses. In total 122 taxa belonging to 30 families were recorded, while the families with the greatest species diversity were Poaceae, Asteraceae and Fabaceae in all land uses. Therophytes was the dominant life-form, followed by the Hemicryptophytes and Geophytes. The findings of the present study imply that different agronomic practices influence soil quality parameters, which are likely to affect species diversity and the environmental implications among different land uses.

Environmental pollution with heavy metals has become a problem of major interest due to the harmful effects of metal ions that constantly evolve and generate serious threats to both the environment and human health through the food chain. Recognizing the imperative need for toxicological assessments, this study revolves around elucidating the effects of Pb(II) and Cu(II) ions on three plant species; namely, Medicago sativa L., Triticum aestivum L., and Zea mays L. These particular species were selected due to their suitability for controlled laboratory cultivation, their potential resistance to heavy metal exposure, and their potential contributions to phytoremediation strategies. The comprehensive phytotoxicity assessments conducted covered a spectrum of critical parameters, encompassing germination inhibition, seedling development, and broader considerations regarding ecosystem health. The key metrics under scrutiny included the germination rate, the relative growth of root and stem lengths, the growth inhibition index, and the tolerance index. These accurately designed experiments involved subjecting the seeds of these plants to an array of concentrations of PbCl2 and CuCl2 solutions, enabling an exhaustive evaluation of the phytotoxic potential of these metal ions and their intricate repercussions on these plant species. Overall, this study provides valuable insights into the diverse and dynamic responses of different plant species to Pb(II) and Cu(II) metal ions, shedding light on their adaptability and resilience in metal-contaminated environments. These findings have important implications for understanding plant–metal interactions and devising phytoremediation strategies in contaminated ecosystems. [ABSTRACT FROM AUTHOR]

Drought and climate change have decreased water availability for agriculture, especially in the desert of southwestern USA. Efficiency enhancements in irrigation management aimed at conserving water are key to adjust to limits in water supply, improve profitability and sustainability of alfalfa production in arid and semiarid areas. This study intended to conduct a field-scale analysis to develop yield and ET estimation tools for the effective use of irrigation water in a desert alfalfa production system. Extensive data collection and trials were carried out over three years in nine fully irrigated commercial alfalfa fields in the low deserts of California. The seasonal crop water consumption measured using the residual of energy balance method varied from 1381 mm to 1596 mm across the experimental sites and crop seasons. Variable seasonal dry mater (DM) yields ranged from 23.01 Mg ha−1 to 29.90 Mg ha−1. The results indicated that the first five cuttings each year were the most productive cuttings with a mean DM value ranged between 3.29 (cut 1) and 4.21 (cut 4) Mg ha−1 but declined in later cuttings. An average annual water productivity (WP) value of 17.0 kg ha−1 mm−1 was determined across the sites varying from 15.5 to 18.9 kg ha−1 mm−1. The findings suggested that one may lose up to 1.44 Mg ha−1 alfalfa yields with moderate summer deficit irrigation strategies, using 40% less water applied than full irrigation practices over the summer period of July–September. A more severe summer water deficit, with no irrigation event over the summer period of July–September may result in a potential water savings of 0.234–0.246 (ha·m) ha−1 and 19–21% seasonal yield losses in the desert environment. This study describes the seasonal yield pattern, the crop water use-production function, and the crop coefficient values for various harvest cycles over the crop season. These tools may assist farmers to quantify water savings and estimate yield losses for more accurate and effective irrigation management strategies to meet water conservation objectives and for the resiliency of alfalfa production in the desert region. [ABSTRACT FROM AUTHOR]

Background: Alfalfa (Medicago sativa L.) is a perennial leguminous forage that can improve the properties of saline soil. In addition, the supplementation with fertilizer to promote soil bacterial activity is critical to improve the productivity of coastal saline soils. However, the association between fertilizer application, bacterial community characteristics, and alfalfa yield in saline soil remains unclear. Aim: To understand the interaction of different fertilizer and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil. Results: A 4-year field study was conducted to examine the interaction of different fertilizer treatments and environmental factors on soil bacterial diversity and alfalfa yield in coastal saline soil in China. Three organic fertilizer treatments (fulvic acid potassium, fulvic acid potassium + wood vinegar, and fulvic acid potassium + Bacillus), one biochar treatment (bio-charcoal), one inorganic fertilizer treatment (diammonium phosphate), and a control (no fertilizer) were included. The effects of the six treatments and 15 cutting times on alfalfa yield and soil bacterial community diversity were assessed. The productivity in fulvic acid potassium + Bacillus and fulvic acid potassium treatments was 68.37 and 67.90 t·hm−2 in 4 years, which was significantly higher than that in the bio-charcoal treatment and control. Hydrothermal conditions and timing of alfalfa harvest had significant effects on the soil bacterial community. Proteobacteria, Acidobacteria, and Actinobacteria were the principal bacterial phyla detected in the soil samples, collectively accounting for approximately 60% of the total bacterial abundance. The relative abundance of Bacteroidetes, Firmicutes, and Nitrospirae was significantly correlated with alfalfa yield, and the abundance of these phyla was also affected by the maximum temperature and precipitation. Fertilizer supplementation in coastal saline soil can effectively increase the yield of alfalfa. Among the fertilizers tested, fulvic acid potassium had the strongest effect, whereas bio-charcoal fertilizer had little effect on alfalfa yield. [ABSTRACT FROM AUTHOR]

The WUSCHEL-related homeobox (WOX) family members are plant-specific transcriptional factors, which function in meristem maintenance, embryogenesis, lateral organ development, as well as abiotic stress tolerance. In this study, 14 MsWOX transcription factors were identified and comprehensively analyzed in the cultivated alfalfa cv. Zhongmu No.1. Overall, 14 putative MsWOX members containing conserved structural regions were clustered into three clades according to phylogenetic analysis. Specific expression patterns of MsWOXs in different tissues at different levels indicated that the MsWOX genes play various roles in alfalfa. MsWUS, MsWOX3, MsWOX9, and MsWOX13-1 from the three subclades were localized in the nucleus, among which, MsWUS and MsWOX13-1 exhibited strong self-activations in yeast. In addition, various cis-acting elements related to hormone responses, plant growth, and stress responses were identified in the 3.0 kb promoter regions of MsWOXs. Expression detection of separated shoots and roots under hormones including auxin, cytokinin, GA, and ABA, as well as drought and cold stresses, showed that MsWOX genes respond to different hormones and abiotic stress treatments. Furthermore, transcript abundance of MsWOX3, and MsWOX13-2 were significantly increased after rhizobia inoculation. This study presented comprehensive data on MsWOX transcription factors and provided valuable insights into further studies of their roles in developmental processes and abiotic stress responses in alfalfa. [ABSTRACT FROM AUTHOR]

Abstract Background Alfalfa (Medicago sativa L.) as an important legume plant can quickly produce adventitious roots (ARs) to form new plants by cutting. But the regulatory mechanism of AR formation in alfalfa remains unclear. Results To better understand the rooting process of alfalfa cuttings, plant materials from four stages, including initial separation stage (C stage), induction stage (Y stage), AR primordium formation stage (P stage) and AR maturation stage (S stage) were collected and used for RNA-Seq. Meanwhile, three candidate genes (SAUR, VAN3 and EGLC) were selected to explore their roles in AR formation. The numbers of differentially expressed genes (DEGs) of Y-vs-C (9,724) and P-vs-Y groups (6,836) were larger than that of S-vs-P group (150), indicating highly active in the early AR formation during the complicated development process. Pathways related to cell wall and sugar metabolism, root development, cell cycle, stem cell, and protease were identified, indicating that these genes were involved in AR production. A large number of hormone-related genes associated with the formation of alfalfa ARs have also been identified, in which auxin, ABA and brassinosteroids are thought to play key regulatory roles. Comparing with TF database, it was found that AP2/ERF-ERF, bHLH, WRKY, NAC, MYB, C2H2, bZIP, GRAS played a major regulatory role in the production of ARs of alfalfa. Furthermore, three identified genes showed significant promotion effect on AR formation. Conclusions Stimulation of stem basal cells in alfalfa by cutting induced AR production through the regulation of various hormones, transcription factors and kinases. This study provides new insights of AR formation in alfalfa and enriches gene resources in crop planting and cultivation.

The perennial legume alfalfa (Medicago sativa L.) is of high value in providing cheap and high-nutritive forages. Due to a lack of tillage during the production period, the soil in which alfalfa grows prunes to become compacted through highly mechanized agriculture. Compaction deteriorates the soil’s structure and fertility, leading to compromised alfalfa development and productivity. However, the way alfalfa responses to different levels of soil compaction and the underlying molecular mechanism are still unclear. In this study, we systematically evaluated the effects of gradient compacted soil on the growth of different cultivars of alfalfa, especially the root system architecture, phytohormones and internal gene expression profile alterations. The results showed that alfalfa growth was facilitated by moderate soil compaction, but drastically inhibited when compaction was intensified. The inhibition effect was universal across different cultivars, but with different severity. Transcriptomic and physiological studies revealed that the expression of a set of genes regulating the biosynthesis of lignin and flavonoids was significantly repressed in compaction treated alfalfa roots, and this might have resulted in a modified secondary cell wall and xylem vessel formation. Phytohormones, like ABA, are supposed to play pivotal roles in the regulation of the overall responses. These findings provide directions for the improvement of field soil management in alfalfa production and the molecular breeding of alfalfa germplasm with better soil compaction resilience.

In this study, artificial neural networks (ANNs), Multivariate Adaptive Regression Splines (MARS) algorithm and multiple regression analysis (MLR) were used for plant stem weight prediction. Stem length, stem diameter, number of lateral branch, branch length, leaf number, stipule length and distance between stipules have been selected as input variables in these mentioned methods. A total of 150 plants were examined. Fifty plants from each of Gea, Bilensoy and Basbag alfalfa cultivars were analyzed separately. Our alfalfa varieties in this study are Gea, Bilensoy and Başbağ. In the ANN method, 70% of the data were allocated for training 20% for validation and 10% for testing. ANN training data were used in MARS algorithm and MLR. To measure which models can predict better, the coefficient of determination (R2) and mean square error (MSE) were compared each other. Correlation coefficients (r) of ANN, MARS and MLR in Stem Weight estimation were 0.801, 0.999 and 0.753 for Gea clover variety, respectively; 0.864, 0.997 and 0.711 for Bilensoy variety, respectively, and 0.781, 0.998 and 0561 for the Basbag variety, respectively. In the same models, R2 was 0.642, 0.998 and 0.567 for the Gea variety, respectively, 0.746, 0.994 and 0.505 for the Bilensoy variety, respectively, and 0.610, 0.997 and 0.315 for the Basbag variety, respectively. MSE values were 0.023, 0.008 and 2.498 for the Gea variety, respectively, 0.113, 0.014 and 1.409 for the Bilensoy variety, respectively, and 0.151, 0.017 and 4.641 for the Basbag variety, respectively. According to these criteria, the MARS algorithm provides a more realistic prediction than ANNs and MLR. The order of used algorithms in obtaining better prediction results in stem weight estimation in alfalfa plants was MARS> ANN> MLR. [ABSTRACT FROM AUTHOR]

To protect the body against environmental threats, cosmetics can play important antiinflammatory and anti-aging roles. Many researchers have developed cosmetic ingredients using natural plant extracts or the active compounds of such extracts. In this study, we assessed the ability of extracts from the seeds of alfalfa (Medicago sativa L.) to affect skin cells. In a keratinocyte-derived cell line (HaCaT cells), alfalfa polysaccharide extract (APE) treatment triggered cornified envelope formation and increased the gene expression levels of filaggrin and involucrin, suggesting that APE increased epidermal cell differentiation. In addition, APE dramatically increased the mRNA and protein levels of claudin1, suggesting that it enhances tight junction formation. Finally, APE exerted antiinflammatory effects in keratinocytes by decreasing the LPSinduced production of proinflammatory cytokines. In primary normal human dermal fibroblasts (NHDF), APE increased procollagen synthesis and decreased elastase activity and metalloproteinase-1 (MMP) secretion. APE also showed superoxide dismutase (SOD)-like activity in a cell-free system, resulting in antioxidant effects. Finally, we found that the polysaccharides from alfalfa seeds used in our experiments consist mainly of galactose and mannose. Based on our findings, we conclude that APE may have potent skin-protective and skin-improving effects and could be developed as an important ingredient in cosmetics and medicines. [ABSTRACT FROM AUTHOR]

Polysaccharides have multiple pharmacological activities without toxicity or side effects. In the present study, the structure, antioxidant and lipid-lowering activities of alfalfa (Medicago sativa L.) polysaccharides (APS) were reported. Three alfalfa root polysaccharides (RAPS), RAPS-1, RAPS-2, and RAPS-3 were isolated, purified, and characterized. The structural analyses demonstrated that the homogeneous RAPS had molecular weights of 22.60, 26.95, and 38.25 kDa, respectively. Furthermore, RAPS-1 was composed of rhamnose, arabinose, xylose, glucuronic acid, glucose, and galactose with molar ratios of 10.25%, 12.53%, 6.58%, 4.56%, 15.74%, and 36.30%, RAPS-2 comprised rhamnose, arabinose, xylose, glucose, and galactose in the molar ratios of 24.07%, 14.48%, 1.24%, 15.05%, and 39.37%, while RAPS-3 was composed of rhamnose, arabinose, xylose, glucuronic acid, mannose, glucose, and galactose in the molar ratios of 31.61%, 11.78%, 1.93%, 5.49%, 3.18%, 14.56%, and 24.63%. The SEM analysis suggested that with the increase of the molecular weight of polysaccharides, its structure is more compact and the stability is stronger. In vitro , RAPS showed strong scavenging ability against DPPH and ABTS free radicals. In addition, RAPS had potent antioxidant activity and reduced the accumulation of lipids in oleic acid-induced NAFLD in HepG2 cells, with RAPS-1 being the most effective. Furthermore, RAPS-1 decreased MDA production, improved the activities of SOD and the total antioxidant capacity of cells, demonstrating that RAPS could effectively restrict lipid accumulation in HepG2 cells induced by oleic acid by restoring the antioxidant system. Western blot analysis indicated that the RAPS-1 can activate the Nrf2/HO-1 signaling pathway to relieve oxidative stress induced by oleic acid, thus playing a role in the treatment of NAFLD. [Display omitted] • Three new acidic polysaccharides were isolated from the roots of Medicago sativa L. • The polysaccharides exhibited considerable antioxidant and lipid-lowering activities. • The polysaccharides with antioxidant effect by activating Nrf2/HO-1 signaling pathway. [ABSTRACT FROM AUTHOR]

The study of winter stress tolerance in perennial legumes needs to consider the complete symbiotic system including both plants and bacteria since these two partners are differentially affected by stress conditions. Here, we compared the regrowth after a freezing stress of four different associations of two alfalfa populations differing in freezing tolerance (A-TF0 and A-TF7) inoculated with two Sinorhizobium (Ensifer) meliloti strains (B399 and NRG34) of contrasted adaptation to cold. To understand the contribution of each partner to a better regrowth performance of an association after freezing, we identified molecular traits having major roles in cold acclimation, freezing tolerance, and those involved in the crosstalk between alfalfa and its symbiotic partner. Regrowth after exposure to a freezing stress was 35% larger in the A-TF7 × NRG34 than in the A-TF0 × B399 association. The metabolomic study of roots, crowns and, more specifically, nodules, revealed profound changes in these organs, switching from a sink to support cold acclimation to a source of reserves enabling regrowth after deacclimation. Marked increases in concentrations of stachyose and raffinose, two sugars of the raffinose-family oligosaccharides (RFO), and in the expression level of a gene of the RFO synthetic pathway were observed in response to cold acclimation supporting the importance of a protective role for RFO in alfalfa. Both cold-adapted partners of the symbiotic association contributed to increases in arginine concentration in nodules in response to cold acclimation and deacclimation underscoring the importance of N storage and remobilization for a successful overwintering in alfalfa. [ABSTRACT FROM AUTHOR]

Alfalfa (Medicago sativa L.) is an important forage for the development of herbivorous animal husbandry, which is widely planted in the cold climate areas of northern China, where there is low overwintering and forage yield in production, and fertilization is a vital production strategy. A field study was conducted to determine the response of alfalfa's winter survival rate, yield, and yield components to different gradients of phosphate (P) fertilizer (0, 45, 90, and 135 kg P2O5 ha−1) in two types of low-phosphorus alkaline soils. The results show that the winter survival rates and forage yield increased at first and then decreased with the increase of P application; the greater winter survival rates were achieved at 45~90 kg ha−1 of P fertilizer applied, while the greater forage yield were achieved at 90 kg ha−1 in the two sites, and the suitable P fertilizer application rates for greater winter survival were less than those for greater forage yield; plant height and shoot mass made a greater contribution to forage yield than other yield components. To pursue greater forage yield, the suitable P fertilization rates for aeolian sandy soil and silt loam soil are 108.1 and 78.3 kg ha−1, respectively, based on the regression equations. Therefore, in cold winter and soil P deficiency areas, applying P fertilizer can be used as an effective strategy to improve alfalfa's forage yield and persistence, and the most appropriate amount of fertilizer should be determined according to climate and soil conditions. [ABSTRACT FROM AUTHOR]