The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

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Climate change (rainfall events and global warming) affects the survival of alfalfa (Medicago sativa L.) in winter. Appropriate water management can quickly reduce the mortality of alfalfa during winter. To determine how changes in water affect the cold resistance of alfalfa, we explored the root system traits under different rainfall events and the effects on cold resistance in three alfalfa cultivars. These were exposed to three simulated rainfall events (SRE) × two phases in a randomized complete block design with six replications. The three cultivars were WL168, WL353 and WL440, and the three SRE were irrigation once every second day (D2), every four days (D4) and every eight days (D8). There were two phases: before cold acclimation and after cold acclimation. Our results demonstrated that a period of exposure to low temperature was required for alfalfa to achieve maximum cold resistance. The root system tended toward herringbone branching under D8, compared with D2 and D4, and demonstrated greater root biomass, crown diameter, root volume, average link length and topological index. Nevertheless, D8 had less lateral root length, root surface area, specific root length, root forks and fractal dimensions. Greater root biomass and topological index were beneficial to cold resistance in alfalfa, while more lateral roots and root forks inhibited its ability to survive winter. Alfalfa roots had higher proline, soluble sugar and starch content in D8 than in D2 and D4. In contrast, there was lower malondialdehyde in D8, indicating that alfalfa had better cold resistance following a longer irrigation interval before winter. After examining root biomass, root system traits and physiological indexes we concluded that WL168 exhibited stronger cold resistance. Our results contribute to greater understanding of root and cold stress, consequently providing references for selection of cultivars and field water management to improve cold resistance of alfalfa in the context of changes in rainfall patterns.

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