Molecular mechanisms underlying leaf enlargement in spaceflight-mutated 'Deqin'alfalfa.

backgroundAs a tetraploid plant, alfalfa (Medicago sativa L.) exhibits complex genetic mechanisms that pose significant challenges for the integration and stabilization of superior traits. In this study, space mutagenesis–a highly efficient and stochastic gene mutation induction technique–was employed to treat ‘Deqin’ alfalfa seeds. Through ground-based selection, a mutant line with enlarged leaves was identified. However, the molecular mechanisms underlying leaf enlargement remain unexplored.

resultsIntegrated transcriptomic, targeted phytohormone metabolomic, and microstructural analyses were used to reveal the mechanistic basis of leaf enlargement in the mutant. The results showed that cell size increased significantly, the palisade and spongy mesophyll tissues thickened notably, and photosynthesis was markedly enhanced. Transcriptome analysis identified a total of 28,060 expressed genes in the leaves, among which 2,009 differentially expressed genes were upregulated and 2,082 were downregulated. Enrichment analysis revealed that pathways such as ribosome, biosynthesis of amino acids, photosynthesis, plant-pathogen interaction, starch and sucrose metabolism, and phenylpropanoid biosynthesis were significantly involved in the leaf enlargement process. Targeted plant hormone metabolomics analysis showed that the levels of abscisic acid, jasmonic acid (JA), 3-indolebutyric acid, 3-indolepropionic acid, and 1-aminocyclopropane-1-carboxylic acid increased by 200.76%, 98.35%, 97.70%, 69.72%, and 27.89%, respectively, compared with those in the control. In contrast, salicylic acid and JA-isoleucine levels decreased by 62.21% and 14.01%, respectively. Combined transcriptomic and metabolomic analysis indicated that metabolic pathways, biosynthesis of secondary metabolites, and hormone signal transduction were the most significantly enriched pathways. Furthermore, a protein-protein interaction network analysis identified MsG0380013794.01, MsG0580027937.01, and MsG0780041014.01 as key genes involved in these pathways.

conclusionsThis study revealed the core regulatory roles of energy metabolism, secondary metabolite biosynthesis, and plant hormone signaling networks in the process of leaf enlargement in the mutant, and identified target genes. These findings lay a solid theoretical foundation for the breeding of high-yield, high-quality alfalfa varieties and provide valuable material resources.