Integrated Transcriptomic and Metabolomic Analyses Reveal the Auxin-Mediated Regulatory Network Governing Alfalfa Responses to Phosphorus Deficiency Stress

Auxin plays a positive role in plant responses to low-phosphorus stress. However, the molecular mechanisms underlying indole-3-acetic acid (IAA)-mediated responses to phosphorus deficiency in alfalfa remain poorly understood. Alfalfa (Medicago sativa L.) cultivar ‘Zhongmu No. 3’ was subjected to two treatments: normal phosphorus (NP, 1000 μM KH2PO4) and low phosphorus supplemented with 1 μM IAA (LP + IAA, 10 μM KH2PO4). Morphological traits, physiological parameters, transcriptomic profiles, and metabolite accumulation were analyzed at 48 h and 10 days following treatment. Compared with the NP group, plants in the LP + IAA group showed marked changes in growth-related traits. At 48 h, plant height increased by 34%, total root length was 1.24-fold that of the NP group, and lateral root number increased by 23.48%. After 10 days, root fresh weight increased by 28%, total root length was 1.25-fold that of the NP group, lateral root number increased by 36.58%, and root volume increased by 22.2%, whereas plant height, stem diameter, and shoot fresh weight remained comparable to those of the NP group. Root acid phosphatase activity was 114.8% higher than that of the NP group at 48 h. Transcriptome analysis identified 1274 and 2277 differentially expressed genes (DEGs) between the NP and LP + IAA groups at 48 h and 10 days, respectively. At 48 h, the up-regulated genes were amino phospholipid ATPase 9 and amino alcohol phosphotransferase 1, whereas phosphate transporter 1 and purple acid phosphatase 12 were up-regulated at 10 days. Metabolomic analysis identified 308 and 1296 differentially accumulated metabolites (DAMs) at 48 h and 10 days, respectively. Early responses were enriched in purine metabolism and involved (5′-phosphoribosyl)-5-formamido-4-imidazolecarboxamide (FAICAR), whereas prolonged treatment involved L-aspartic acid, adenine, and cAMP. Integrated analyses identified tryptophan metabolism, cysteine and methionine metabolism, glycerophospholipid metabolism, and plant hormone signal transduction as major regulatory pathways. CDP-choline accumulation and changes in lipid-remodeling genes further indicated enhanced membrane phospholipid remodeling. Overall, these results provide insight into the morphological, physiological, transcriptional, and metabolic responses of alfalfa to low phosphorus in the presence of exogenous IAA.