Enhanced Cold Hardiness in Pinus koraiensis Seedlings: Physiological Responses to Exogenous Organic Acids

Pinus koraiensis, a key species for afforestation in Northeast China, faces severe cold stress at the seedling stage. Non-salicylic acid (non-SA) low-molecular-weight organic acids (LMWOAs) have been reported to regulate plant stress resistance while their regulatory mechanisms on cold resistance of this conifer species remain largely unclear. This study aimed to investigate the regulatory effects and optimal application concentrations of non-SA LMWOAs (oxalic acid OA, citric acid CA, malic acid MA) on the cold hardiness of P. koraiensis seedlings, and explicitly propose the "nutrient activation-physiological regulation" synergistic mechanism as a testable scientific hypothesis. In this study, seedlings were sprayed with relevant concentrations of LMWOAs before being subjected to 4°C stress. The effects on membrane stability, antioxidant enzymes, osmoregulatory and chlorophyll were then measured. The results showed that 5.0 mmol·L^-1 of LMWOAs was the optimal concentration, with citric acid being the most effective, followed by malic acid and then oxalic acid. Specifically, 5.0 mmol·L^-1 citric acid reduced malondialdehyde (MDA) content by 62.6% (p<0.05) and relative electrolyte leakage to 12.5% (p<0.01) compared to the low-temperature control group. Exogenous organic acids enhance the cold hardiness of P. koraiensis seedlings through three pathways: chelating intracellular metal ions to stabilize cell membrane structures, activating antioxidant enzyme systems (SOD, POD, CAT, APX) to scavenge reactive oxygen species, and promoting the accumulation of osmotic regulatory substances (proline, soluble proteins). Meanwhile, exogenous LMWOAs effectively alleviated chlorophyll degradation under low-temperature stress. This study reveals the potential "nutrient activation-physiological regulation" synergistic mechanism of non-SA LMWOAs regulating cold hardiness of P. koraiensis seedlings, and provides a reliable theoretical basis and technical support for cold-tolerant seedling cultivation of P. koraiensis in northern cold regions.