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Polyamines (PAs) have been demonstrated to be involved in plant in response to abiotic stresses including chilling stress. The present study was designed to investigate the effects of seed priming with 5 mM of spermidine (Spd) and 8.5 mM of 5-Aminolevulinic acid (ALA) on seed polyamines metabolism associated with the improvement of chilling tolerance in two rice cultivars, Zhu Liang You 06 (ZY) and Qian You No.1 (QY). Germination percentage, seedling growth and seedling vigor index was decreased under chilling stress, but this physiological parameters was improved by Spd and ALA priming in both studied cultivars as compared with unprimed seeds. As well, total phenolics, flavonoids and glycine-betaine were improved by priming treatment. Contrarily, significant decrease of α-amylase activity, soluble sugars and soluble protein contents of both cultivars was observed in chilling stressed plants as compared with normal growth condition (25 °C). However, priming with Spd and ALA significantly increased α-amylase activity, soluble sugars and soluble protein contents with more prominent increase in QY cultivar. Results showed that chilling stress significantly improved superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and glutathione peroxidase (GPX), and further enhancement was observed by Spd and ALA-primed seeds. Spd and putrescine (Put) were decreased under chilling stress, while a reverse tendency was observed in case of spermine (Spm) content. The enzymes involved in the PAs biosynthesis, arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) was improved by priming treatment. The relative expressions of genes encoding enzymes involved in PAs biosynthesis increased by Spd and ALA priming. Additionally, priming treatment improved leaf cell and grain structure as compared with the unprimed seeds.