Transcriptional analysis reveals sodium nitroprusside affects alfalfa in response to PEG-induced osmotic stress at germination stage

Drought is one of the most common environmental factors that affect alfalfa germination and development. Nitric oxide (NO) could mediate stress tolerance in plants. The goal of this study was to determine exogenous NO donor–mediated drought adaption molecular mechanisms during the alfalfa germination stage. In this study, physiological and transcriptome analyses were performed on 7 days of the growth period seedlings by sodium nitroprusside (SNP) and polyethylene glycol (PEG) treatment. The results showed that SNP supplementation alleviated malondialdehyde accumulation, increased levels of proline and soluble sugars, and enhanced antioxidant enzyme activity under osmotic stress conditions. RNA-Seq experiments identified 5828 genes exhibiting differential expression in seedlings treated with PEG, SNP, or SNP+PEG relative to seedlings treated with distilled water. Of these DEGs, 3235 were upregulated, and 2593 were downregulated relative to the controls. Fifteen DEGs were amplified by qRT-PCR to verify the changes in expression determined by RNA-Seq, revealing that PIF3, glnA, PLCG1, and RP-S11e exhibited enhanced expression under the SNP+PEG treatment. SNP was found to modulate redox homeostasis-related genes such as GSTs, SOD2, GPX, and RBOH, and triggered calcium signaling transduction. It also induced some key genes relating to the abscisic acid, ethylene, and auxin signaling transduction in response to PEG stress. Conversely, genes associated with secondary metabolite biosynthesis and the metabolism of starch and sucrose during osmotic stress were downregulated by SNP. These results provide new insights into SNP-mediated drought adaption mechanisms at transcriptome-wide in alfalfa and reveal key drought tolerance pathways in this species.