The aim of this study was to investigate the antioxidant and free-radical scavenging activity of extract and fractions from various parts of Elsholtzia ciliata. The inflorescences, leaves, stems and roots of E. ciliata were extracted separately and two phenolic component enrichment methods: ethyl acetate-water liquid-liquid extraction and macroporous resin adsorption-desorption, were adopted in this study. The antioxidant activities of water extracts and fractions of E. ciliata were examined using different assay model systems in vitro. The fraction root E (purified by HPD300 macroporous resin) exhibited the highest total phenolics content (497.2 ± 24.9 mg GAE/g), accompanied with the highest antioxidant activity against various antioxidant systems in vitro compared to other fractions. On the basis of the results obtained, E. ciliata extracts can be used potentially as a ready accessible and valuable bioactive source of natural antioxidants.

The mitochondrial calcium uniporters (MCUs) are a family of calcium unidirectional transporters important for cytoplasmic Ca²⁺ signals. Though the MCU proteins in several plant species have been investigated, genome-wide analysis of MCUs in alfalfa is lacking. Here, via genome-wide analysis, a total of 5, 20, and 6 MCU genes were identified in three different alfalfa cultivars, namely Medicago truncatula Jemalong A17, Medicago sativa XinJiangDaYe, and M. sativa Zhongmu No. 1, respectively. They were further phylogenetically classified into three subfamilies. Most MCU genes have only one intron, and gene duplication events of MCU genes were observed within each alfalfa accession and between different accessions. All alfalfa MCU proteins contained a highly conserved MCU domain and 10 conserved motifs, featuring two transmembrane domains and a DI/VME motif. According to the tissue expression data of M. sativa Zhongmu No. 1, MsMCU6.2 was the most abundant transcript with the highest expression in the leaf, and MsMCU5 and MsMCU1.2 showed higher expression levels in the stem than other tissues. We analyzed the expression profiles of five MCU genes (MsMCU1.1/1.2/5/6.1/6.2) under salt, drought, and cold stresses via qRT-PCR assays. All five MCU genes were induced by drought stress, except MsMCU5, whose expression was up-regulated by salt stress, while cold stress slightly altered MsMCU expression. Nine potential interacting proteins and three miRNAs targeting MtMCUs were predicted. These results provide detailed knowledge of alfalfa MCU genes and suggest their potential functions in abiotic stress response.

Introduction: Alfalfa ( Medicago sativa L. ) is the most important perennial forage crop cultivated globally. However, extreme environmental conditions, such as freezing stress, can significantly impact alfalfa’s growth and development. The potential mechanisms through which alfalfa responds to freezing stress remain largely unexplored. Methods: In this study, we analyzed the physiological indices, transcriptomes and proteomes of the cold-tolerant alfalfa cultivar “Dongnong NO.1” and the cold-sensitive cultivar “Bara 218TR” at -5°C. Results: The results indicated that the levels of antioxidant enzyme and osmoregulatory substances in “Dongnong NO.1” were significantly higher than in “Bara 218TR”. Additionally, the levels of malondialdehyde (MDA) and relative electrolyte leakage (REL) were found be lower in “Dongnong NO.1” than in “Bara 218TR”. Further transcriptomic analysis revealed that the differentially expressed genes (DEGs) found in both alfalfa cultivars were predominantly enriched in the AP2/ERF-ERF transcription factor family and in multiple signaling pathways. Weighted gene co-expression network analysis (WGCNA) revealed that the physiological processes associated with freezing stress tolerance in the two alfalfa cultivars are closely linked to DEGs that regulate protein synthesis, calcium signaling, the inhibition of iron toxicity, and the reduction of cell wall stiffness. Proteomics analysis indicates that differentially abundant proteins (DAPs) respond to frost damage by maintaining protein stability, antioxidant defense, and metabolic regulation. Integrated transcriptomic and proteomic analyses indicate that pathways related to carbohydrate metabolism, biotic stress defense, cell wall modification, and phenylpropanoid biosynthesis are key to alfalfa’s response to frost damage. Discussion: This study improves our understanding of the molecular mechanisms underlying alfalfa’s freezing resistance and provides insights for the further screening and in-depth investigation of candidate genes with potential functions against freezing stress.

Background: Alfalfa ( Medicago sativa L.) is globally important forage crop whose survival and productivity at higher latitudes depend on cold-stress tolerance. However, the molecular mechanisms of how alfalfa defends against freezing stress remain unclear. Results: In this study, the candidate gene MsCBF4 was cloned from the cold-tolerant alfalfa genotype “Dongnong NO.1”. MsCBF4 belongs to the APETALA2/Ethylene Response Factor (AP2/ERF) transcription factor family and functions as a transcriptional activator via a C-terminal activation domain. The encoded protein localized to the nucleus. MsCBF4 is induced by cold and freezing conditions and is expressed in roots, stems, leaves, flowers, and pods, with the highest expression levels in roots and leaves. Overexpression of MsCBF4 in Arabidopsis thaliana seedlings significantly enhanced root length, lateral root number, and fresh weight under cold stress. Lines overexpressing MsCBF4 demonstrated enhanced frost tolerance at both the seedling and mature stages, likely through improved physiological scavenging of reactive oxygen species (ROSs), thereby limiting membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance were upregulated in transgenic Arabidopsis . The MsCBF4 promoter exhibits transcriptional activity and can be directly activated by cold responses. Yeast one-hybrid (Y1H) and dual-luciferase reporter (Dual-LUC) assays showed that MsERF6 specifically binds to the MsCBF4 promoter and positively regulates its expression. Conclusion: MsCBF4 enhances frost tolerance in Arabidopsis by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.