Metabolites in Cellular Leachate as Biomarkers for the Nature and Severity of Injury Following a Short vs. Prolonged Freezing Stress

Freeze injury is typically quantified by measuring electrolyte leakage from plant tissues exposed to a freeze-thaw cycle, whereby higher leakage indicates greater injury. The severity of stress is also assessed by the ability (or lack thereof) of the injured tissue to recover, wherein reduced leakage during the post-thaw period relative to right after thaw indicates recoverable injury. In this study, spinach leaves were injured to various degrees by freezing them for different durations at a fixed sub-zero temperature. We explored the cellular nature of freeze-thaw injury by comparing the profiles of cellular metabolites that were leaked from reversibly and/or irreversibly injured leaves. A total of 47 metabolites were, thus, detected in the leachate, including 8 sugars, 20 amino acids, GABA, uracil, adenine, palmitic acid, ferulic, coumaric acid, and other organic/inorganic acids involved in primary and secondary metabolism (e.g., citric acid, phosphoric acid, coumaric acid, etc.). Combined multivariate metabolome analysis, including principal component analysis, hierarchical clustering, and partial least square discriminant analysis, together identified 14 key metabolites mainly responsible for distinguishing the reversible vs. irreversible injury. These metabolites are also indicators for various lesions, including structural/functional perturbations to cellular components (cell wall, chloroplast, vacuole, plasma membrane, nucleic acids), disruption of metabolic homeostasis (antioxidant system, cytoplasmic pH, Kreb's cycle), protein degradation, and hypoxia.