Abstract: Gentiana macrophylla is a widely used ingredient in traditional Chinese medicine for more than 2 000 years.Now, it is under third-class protection in China and on the list of National Key Protected Wild Herbs.G.macrophylla is a seed-propagated plant with extremely low germination rate.Short seed vigor, lower seed yield and after-ripening severely hinder its seedling establishment and significantly raise nursery cost.Various priming methods have shown that ferrous priming is better than other liquid priming, having higher germination performance under both optimal and adverse environments than non-primed seeds.The objectives of this study was to investigate the physiological mechanism of FeSO₄ priming increasing seed germination performance of G.macrophylla to lay the theoretical basis for FeSO₄ priming in abiotic stress response and application of FeSO₄ priming technology in other plants seeds.Compare with non-priming of seeds, we investigated the effects of 0.6% FeSO₄ priming on seed membrane permeability, stored material metabolism, energy metabolism, hormone homeostasis and enzyme activity.In this study, we noted that FeSO₄ reduced seed membrane permeability and improved seed vigor by decreasing electrical conductivity by 6.61% at 12 h, 11.67% at 24 h.The mobilization of stored materials such as sugars, proteins and fatty acids were activated, sucrose content decreased by 9.57%, and soluble protein content increased by 49.63%.With regard fatty acids, content of saturated fatty acids increased by 4.93% for myristic acid and 9.03% for methyl lignocerate.Then the content of unsaturated fatty acids decreased by 6.73% for behenic acid, 8.18% for oleic acid, 8.40% for linoleic acid and 6.70% for inolenic acid.In addition, FeSO₄ priming altered hormone homeostasis of seeds between ABA and GA, of which ABA content decreased by 64.78% and GA content increased by nearly 22 times.In terms of energy metabolism, FeSO₄ priming remarkably improved energy level, increased ATP content by 2.16 times and increased cytochrome C oxidase activity by 67.91%.Moreover, FeSO₄ priming acted as an initial stress-exposure, both enzyme-catabolized and non-enzyme-catabolized systems response to environmental stress were activated.This was specifically so for the activities of SOD, CAT, APX of enzyme-catabolized elements, which increased by 2.85 times, 1.79 times and 19.6%, respectively.The contents of non-enzyme-catabolized elements were increased by 74.08% for glutathione and 10.89% for ascorbic acid.In summary, our study indicated that regulation of FeSO₄ priming was a complex process that promoted stored material mobilization, ATP biosynthesis, key enzyme activation, phytohormone homeostasis between ABA and GA; and simultaneously regulated both enzymatic-catabolized and non-enzymatic-catabolized effecters in biotic and an-biotic stress.On the one hand, priming induced more advanced development in seeds towards complete germination than unprimed ones.On the other hand, priming was considered as a pre-germination stress-exposure that left seeds with "stress-memory" and improved anti-adversity ability.These changes were not eliminated even when seeds dried to original moisture content.This was the main reason why priming enhanced seed vigor and rapid, uniform seed germination performance.