Ethylene-mediated regulation of root responses to deficient-phosphate stress based on transcriptomic and metabolomic analyses in alfalfa

Phosphorus is an essential nutrient for plant growth and development. However, the deficient available phosphorus in soil has become a critical factor limiting the improvement of forage yield and quality. Although ethylene is known to participate in plant responses to phosphorus starvation stress, its regulatory roles in alfalfa ( Medicago sativa ) remain poorly understood. In this study, the alfalfa roots were used to investigate the morphology and physiological traits, gene expression, and metabolite profiles after seven days of treatments under normal phosphorus (NP), deficient phosphate (DP) and DP supplemented with 1, 10, and 100 µM of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). The results showed that ACC significantly inhibited alfalfa root growth under DP conditions, reducing total root length, while concurrently stimulating root hair formation. ACC promoted the accumulation of starch, sucrose, and organic acids, and altered the levels of hormones such as abscisic acid (ABA), gibberellin (GA), and salicylic acid (SA). Transcriptome analysis identified 761, 2142, 2488, and 2607 differentially expressed genes (DEGs) in TDP, TACC1, TACC10 and TACC100 groups compared with TNP, respectively. ACC induced the expression of genes such as the glycerol-3-phosphate transporter , purple acid phosphatase , phosphate transporters . A total of 926 significantly differentially accumulated metabolites (DAMs), with a total of 266, 378, 504, and 570 identified in the groups of DP, ACC1, ACC10 and ACC100, respectively, versus NP conditions. ACC induced the accumulation of metabolites such as sucrose in the carbohydrate category, while it suppressed the accumulation of fatty acyls, sphingolipids, and the hormones ABA and SA. Integrated transcriptome and metabolome analysis revealed that DP and ACC co-regulated metabolic pathways including starch and sucrose metabolism, glutathione metabolism, flavonoid biosynthesis, fatty acid degradation, and hormone signal transduction. Ethylene also specifically induced glycolysis, the citrate cycle, tryptophan metabolism, and inositol phosphate metabolism in response to DP stress. These findings will provide a theoretical framework for improving phosphorus utilization efficiency in alfalfa.