A perennial leguminous forage, Medicago ruthenica has outstanding tolerance to abiotic stresses. The genome of Medicago ruthenica is large and has a complex genetic background, making it challenging to accurately determine genetic information. However, the chloroplast genome is widely used for researching issues related to evolution, genetic diversity, and other studies. To better understand its chloroplast characteristics and adaptive evolution, chloroplast genomes of 61 Medicago ruthenica were assembled (including 16 cultivated Medicago ruthenica germplasm and 45 wild Medicago ruthenica germplasm). These were used to construct the pan-chloroplast genome of Medicago ruthenica, and the chloroplast genomes of cultivated and wild Medicago ruthenica were compared and analyzed. Phylogenetic and haplotype analyses revealed two main clades of 61 Medicago ruthenica germplasm chloroplast genomes, distributed in eastern and western regions. Meanwhile, based on chloroplast variation information, 61 Medicago ruthenica germplasm can be divided into three genetic groups. Unlike the phylogenetic tree constructed from the chloroplast genome, a new intermediate group has been identified, mainly consisting of samples from the eastern region of Inner Mongolia, Shanxi Province, and Hebei Province. Transcriptomic analysis showed that 29 genes were upregulated and three genes were downregulated. The analysis of these genes mainly focuses on enhancing plant resilience and adapting adversity by stabilizing the photosystem structure and promoting protein synthesis. Additionally, in the analysis of adaptive evolution, the accD, clpP and ycf1 genes showed higher average Ka/Ks ratios and exhibited significant nucleotide diversity, indicating that these genes are strongly positively selected. The editing efficiency of the ycf1 and clpP genes significantly increases under abiotic stress, which may positively contribute to plant adaptation to the environment. In conclusion, the construction and comparative analysis of the complete chloroplast genomes of 61 Medicago ruthenica germplasm from different regions not only revealed new insights into the genetic variation and phylogenetic relationships of Medicago ruthenica germplasm, but also highlighted the importance of chloroplast transcriptome analysis in elucidating the model of chloroplast responses to abiotic stress. These provide valuable information for further research on the adaptive evolution of Medicago ruthenica.

E3 ubiquitin ligases known as plant U-box (PUB) proteins regulate a variety of aspects of plant growth, development, and stress response. However, the functions and characteristics of the PUB gene family in alfalfa remain unclear. This work involved a genome-wide examination of the alfalfa U-box E3 ubiquitin ligase gene. In total, 210 members were identified and divided into five categories according to their homology with the members of the U-box gene family in Arabidopsis thaliana. The phylogenetic analysis, conserved motifs, chromosomal localization, promoters, and regulatory networks of this gene were investigated. Chromosomal localization and covariance analyses indicated that the MsPUB genes expanded MsPUB gene family members through gene duplication events during evolution. MsPUB genes may be involved in the light response, phytohormone response, growth, and development of several biological activities, according to cis-acting element analysis of promoters. In addition, transcriptome analysis and expression analysis by qRT-PCR indicated that most MsPUB genes were significantly upregulated under cold stress, drought stress, and salt stress treatments. Among them, MsPUBS106 and MsPUBS185 were significantly and positively correlated with cold resistance in alfalfa. MsPUBS110, MsPUBS067, MsPUBS111 and MsPUB155 were comprehensively involved in drought stress, low temperature, and salt stress resistance. All things considered, these discoveries offer fresh perspectives on the composition, development, and roles of the PUB gene family in alfalfa. They also provide theoretical guidance for further investigations into the mechanisms regulating the development, evolution, and stress tolerance of MsPUB.

Plants are frequently exposed to various abiotic stresses, among which low-temperature stress markedly impairs growth and physiological functions. WRKY transcription factors are key regulators in plant responses to abiotic stress. In this study, a novel WRKY transcription factor gene, TrWRKY79, was cloned from white clover. Functional characterization revealed that the full-length TrWRKY79 protein possesses typical features of transcription factors, including transcriptional activation activity located at its C-terminal domain. Heterologous expression of TrWRKY79 in Arabidopsis thaliana significantly enhanced cold tolerance under low-temperature stress. Physiological assays showed that the transgenic lines exhibited higher chlorophyll content and elevated activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) compared to wild-type plants. Furthermore, Protenix was employed to predict the potential target genes of TrWRKY transcription factors, and their expression profiles were analyzed to help elucidate the regulatory network underlying cold tolerance. qRT-PCR analysis confirmed that several cold-responsive genes, such as COR47 and ABI5, were significantly upregulated in the transgenic lines. Collectively, these findings indicate that TrWRKY79 plays a positive regulatory role in enhancing cold tolerance, providing valuable insights into the molecular mechanisms of cold resistance in white clover and offering promising candidate genes for improving stress resilience in forage crops.

Medicago sativa L. (alfalfa) is a major forage crop due to its high yield and stress resilience. However, its growth and productivity are often compromised by abiotic stresses, including cold, drought, and salinity. The plant defensin (PDF) gene family plays a crucial role in resistance to abiotic stress. In this study, a total of 11 MsPDF gene family members were identified in the alfalfa genome and classified into three groups. Phylogenetic and conserved motif analyses revealed that the MsPDF genes are highly conserved. Promoter analysis, gene regulatory network analysis (GRN), and gene ontology (GO)-enrichment analyses were used to infer the potential functions of MsPDF genes. The results showed that the gene actively responds to abiotic stress, participates in phytohormonal responses, and regulates plant growth and development through gene interactions. Transcriptome and qRT-PCR analyses showed that most of the MsPDF genes were significantly up-regulated under cold, drought, and salinity stresses. Among them, the MsPDF03 exhibited superior performance under cold stress. The MsPDF04, MsPDF08, and MsPDF09 genes were able to respond positively to drought and salt stresses. Finally, the monomeric, dimeric, and tetrameric structures of the proteins encoded by the MsPDF genes were predicted using AlphaFold 2 software. This study lays the foundation for the identification and evolutionary relationship analysis of the MsPDF gene family, and provides a new reference for subsequent research on abiotic stress resistance.

Trifolium repens L. (white clover) is a widely distributed perennial legume, which is regarded as one of the most important forages for its high protein content and excellent palatability. Low temperature limits the distribution and productivity of white clover, thereby reducing its economic returns. WRKY transcription factors are key regulators in stress defense and are involved in multiple abiotic stress responses in plants. In this study, a cold inducible gene named TrWRKY41 was cloned from white clover. The TrWRKY41 protein is predominantly localized in the nucleus and functions as a hydrophilic, acidic protein. Under cold stress, the overexpression plants had significantly higher chlorophyll (CHL) and proline (Pro) contents, significantly increased activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), and malondialdehyde (MDA) content significantly decreased. Compared to wild-type Arabidopsis thaliana, TrWRKY41-overexpressing plants exhibited better cold tolerance. In addition, target genes downstream of the TrWRKY41 transcription factor were predicted utilizing BLAST alignment and AlphaFold2 (version 0.2.0) software, the expression of six genes, including AtCOR47, AtCOR6.6, and AtABI5, was significantly up-regulated under cold stress. It suggests that TrWRKY41 may enhance cold tolerance in Arabidopsis by activating the ICE-CBF-COR cascade. This study provides candidate genes for research on enhancing the cold tolerance of white clover.