RNA Splicing of the MsndhB Gene and Photosynthetic Characteristics in Alfalfa (Medicago sativa) Under High- and Low-Temperature Stress

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.