Characterization of a novel green-revertible albino mutant in alfalfa (Medicago sativa L.).

High-temperature-sensitive leaf color mutants are ideal materials for studying photosynthetic pigment biosynthesis and corresponding response mechanisms under heat stress. Here, we provide the first report of albinism occurrence in alfalfa and characterize the high-temperature albino regreen (har) mutant of alfalfa, which presents albino leaves when exposed to temperatures ≥35 °C and is not specific to developmental stage. Genetic analysis demonstrated that the albino trait exhibits dominant inheritance. Agronomic trait evaluations revealed that the har mutants were slightly but negatively affected by albinism. However, under high temperature, albino leaves had a severe negative effect on the photosynthesis-related traits of har mutants. Cytological analysis revealed that the albino leaf cells contained disintegrated chloroplasts, suggesting a defect in chloroplast development. Moreover, this study involved a comprehensive investigation of the enzymes associated with the photosynthetic pigment biosynthetic pathway of the har mutant under high-temperature stress using RNA sequencing. Notably, high-temperature-induced differential leaf color traits in alfalfa result in distinct photosynthetic pigment biosynthetic pathways. Twelve key regulatory genes involved in the chlorophyll biosynthesis and degradation pathways, as well as four key regulatory genes involved in carotenoid biosynthesis pathways, were identified. Our study aims to provide a theoretical foundation for further research into the intrinsic mechanisms underlying albino leaves in alfalfa har mutants subjected to high-temperature stress and for the breeding of new germplasms with desirable pigmented leaves.