Salicylic acid-induced freezing tolerance in spinach (Spinacia oleracea L.) leaves explored through metabolite profiling

Freezing tolerance and metabolome changes were investigated for spinach (Spinacia oleracea L. 'Reflect') seedlings treated with 0.5 mM salicylic acid by sub-fertigation. Experiments included seedlings treated with salicylic acid at both the ambient (non-acclimation; NA) and cold acclimation (CA) temperatures, i.e. NASA and CASA. Except for slight reduction in the leaf size, salicylic acid-fed plants exhibited, in general, similar growth performance as non-treated controls based on the dry weight/fresh weight ratio, percent water content, and dry weight/leaf area. Temperature-controlled in situ freeze-thaw of seedlings revealed NASA plants were more freeze-tolerant (visual estimates and ion-leakage test) and had higher salicylic acid content than NA control. Metabolite profiling revealed NASA had higher trehalose, ascorbic acid, γ-tocopherol, proline, and leucine, whereas lower mannose and aconitic acid than NA tissues. Excised leaf freeze-thaw tests revealed CASA leaves to be the most freeze-tolerant of the four conditions followed, respectively, by CA, NASA, and NA. Principal component analysis distinctly separated metabolic phenotypes for NA, NASA, CA, and CASA, indicating salicylic acid differentially affected metabolism at warm vs. cold. CASA leaves had higher compatible solutes (osmolytes), antioxidants, and salicylic acid than CA control. Our data suggests that altered accumulation of trehalose, ascorbic acid, and aconitic acid was a salicylic acid-specific response. Additionally, 7 metabolites (5-oxoproline, fructose, glucose, maltose, proline, sucrose, and tartaric acid) were quantitatively associated with the freezing tolerance levels across four conditions.