Integrated Agronomic Traits, Nutritional Quality, and Physiological Indices under Excessive Selenium: Anatomical Characters and miRNA-mRNA Expression Profiles Analyze Selenium-Tolerance in Alfalfa

Selenium (Se) is indispensable for the majority of organisms, yet it also possesses toxic properties, with a delicate balance between insufficiency and harmful levels. Some plants can be used for resistance of Se pollution. Here, to identify the Se tolerance and resistance potential of alfalfa, we comprehensively investigated the effects of excessive Na2SeO3 on the physio-biochemical, anatomical, mRNA, and miRNA expression profile alterations. Foliar spraying 500 mg/kg Se significantly reduced the biomass in alfalfa probably by inhibiting photosynthesis (20.2%). Excessive Se breaks the balance of antioxidant system, resulting in an increase of H2O2 and MDA content by 14.2 and 4.3%. Leaf ultra-structure damages subsequently revealed the phytotoxic effects of Se. And at 500 mg/kg, alfalfa synthesized more S-type lignin probably to increase the Se tolerance, but it resulted in lower nutritional quality. Through RNA-seq, we found 2112 Se-responsive differentially expressed genes, including 1322 up-regulated and 790 down-regulated mRNAs, many of which were related to plant hormone signal transduction, redox reaction, S-assimilation, and cell homeostasis. Moreover, we identified 296 known and 258 conserved miRNAs, and predicted 310 novel miRNAs, among which 77 differentially expressed miRNAs were induced by Se. miRNAs play a crucial role in forming Se tolerance in alfalfa under the precise regulation of redox, chlorophyll degradation, photosynthesis, cell wall synthesis, and other related target genes. Our results provided insights into the complex mechanism of the Se stress response in alfalfa.