A metabolomics study of ascorbic acid-induced in situ freezing tolerance in spinach (Spinacia oleracea L.)
Freeze-thaw stress is one of the major environmental constraints that limit plant growth and reduces productivity and quality. Plants exhibit a variety of cellular dysfunction following freeze-thaw stress, including accumulation of reactive oxygen species (ROS). This means that enhancement of antioxidant capacity by exogenous application of antioxidants could potentially be one of major strategies for improving freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an antioxidant, has been shown to improve plant tolerance against abiotic stresses but its effect on FT has not been investigated. We evaluated the effect of AsA-feeding on FT of spinach (Spinacia oleracea L.) at whole-plant and excised leaf level, and conducted metabolite profiling of leaves before and after AsA-treatment to explore metabolic explanation for change in FT. AsA-application did not impede leaf-growth; instead slightly promoted it. Temperature-controlled freeze-thaw tests revealed AsA-fed plants were more freezing tolerant as indicated by: 1) less visual damage/mortality; 2) lower ion-leakage; and 3) less oxidative-injury, lower abundance of free radicals (O2 *- and H2O2). Comparative leaf metabolic profiling revealed clear separation of metabolic phenotypes for control vs. AsA-fed leaves. Specifically, AsA-fed leaves had greater abundance of antioxidants (AsA, glutathione, alpha-& gamma-tocopherol) and compatible solutes (proline, galactinol, myo-inositol). AsA-fed leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, catalase). These changes, together, may improve FT via alleviating freeze-induced oxidative stress as well as protecting membranes from freeze-desiccation. Additionally, improved FT by AsA-feeding may potentially include enhanced cell wall/lignin augmentation and bolstered secondary metabolism as indicated by diminished level of phenylalanine and increased abundance of branched amino acids, respectively.