Plant cysteine oxidase (PCO) catalyzes the oxidation of cysteine residues in the N-degron pathway, thereby regulating the stability and activity of the seventh group of ethylene response factors (ERF-VII), which play a crucial role in reactive oxygen species (ROS)-mediated signal transduction. By regulating the degradation of ERF-VII, the PCO family genes control hormone signaling, which is highly valuable for plant growth and abiotic stress responses. However, systematic studies on PCO genes in Medicago sativa, a key forage legume, remain lacking. Herein, 35 MsPCO genes were identified from the alfalfa (Medicago sativa) genome, and their biological characteristics were comprehensively analyzed via bioinformatics approaches. The results showed that MsPCO genes are asymmetrically distributed across 18 chromosomes and clustered into 5 subgroups phylogenetically. Most MsPCO proteins are hydrophilic and localized in the cytoplasm. A total of 56 duplication events were detected, with most duplicated pairs undergoing purifying selection (Ka/Ks analysis). Collinearity analysis revealed close evolutionary relationships between Medicago sativa and Medicago truncatula, Arabidopsis thaliana or Glycine max. Promoter cis-acting elements in MsPCO genes are involved in light response, stress adaptation, hormone signaling, and growth regulation. Transcriptomic data indicated differential expression of MsPCO genes under abiotic stresses. MsPCO20 is dispersed throughout the cell membrane and nucleus, whereas MsPCO19 is localized to the nucleus, according to subcellular localization experiments. These findings provide candidate genes and a theoretical basis for further functional characterization of PCO genes in alfalfa.

The PIN-LIKES (PILS) gene family is crucial for regulating auxin homeostasis and stress adaptation in plants; nevertheless, a comprehensive study on this family in alfalfa (Medicago sativa) remains insufficient. This research found 46 MsPILS genes within the tetraploid alfalfa genome and categorized them into four subfamilies. The genes are irregularly allocated throughout 16 chromosomes, with tandem duplications acting as a primary catalyst for family expansion. Analysis indicated that all MsPILS proteins contain the conserved Mem_trans domain. The promoter study revealed that MsPILS genes had many cis-elements that respond to abiotic stressors and hormones. qRT-PCR research indicated that MsPILS genes exhibit variable expression across several tissues and respond to multiple abiotic stressors. Protein–protein interaction (PPI) research revealed PIN3, PIN5, and PIN6 as principal interacting partners of the MsPILS proteins. Subcellular localization studies indicated that MsPILS1c is in the nucleus, plasma membrane, and endoplasmic reticulum (ER). This research offers significant genetic resources and a theoretical framework for elucidating the activities of PILS genes and for molecular breeding aimed at improving stress tolerance in alfalfa.

RNA splicing of chloroplast-encoded genes contributes to plant responses to abiotic stress, yet its regulation remains poorly understood in forage legumes. Here, we investigated RNA splicing of the chloroplast ndhB gene in alfalfa (Medicago sativa L.) under abiotic stress and its association with photosynthetic and physiological responses under temperature stress. Using cultivar ‘Xinjiang Daye’, we characterized MsndhB structure and analyzed splicing efficiency across tissues, developmental stages, and four stress treatments (high temperature, low temperature, salt, and drought). Under high- and low-temperature stress, photosynthetic gas exchange, chlorophyll fluorescence, ATP, and malondialdehyde (MDA) were measured to evaluate associations with MsndhB splicing efficiency. The results showed that high temperature reduced splicing efficiency and caused pre-mRNA accumulation, whereas low temperature enhanced splicing; salt and drought affected transcript abundance but not splicing efficiency. Splicing efficiency was higher in green tissues and at the branching stage. Under temperature stress, splicing efficiency was correlated with photosynthetic and oxidative stress indicators, but these associations do not provide evidence of functional control. These findings suggest that MsndhB splicing is temperature-responsive and may be involved in chloroplast responses to temperature stress, providing a foundation for elucidating the role of post-transcriptional regulation of chloroplast genes in alfalfa responses to temperature stress.