Palmitoyl-protein thioesterase (PPT), involved in the fatty acid synthesis and the de-palmitoylation of protein, was induced under ecological cooling treatment in hydrated lettuce seeds. However, there was no significant difference in fatty acid levels between the control and the cooled samples. To further study the function of PPT, 2-bromopalmitic acid (2-Bp), an inhibitor of protein palmitoylation, was applied during the imbibition of hydrated lettuce seeds, which was followed by slow-cooling treatment (−3°C h−1). The application of 2-Bp (1 mM) significantly increased the survival rate of seeds from 6.70% (control imbibition) to 22.67% (2-Bp imbibition) after slow cooling to −20°C. Differential scanning calorimetry (DSC) analysis indicated that 2-Bp led to earlier onset of ice crystals in the endosperm than the control group. Two-dimensional electrophoresis (2D) confirmed that 2-Bp could promote the hydrolysis of seed globulins and the accumulation of globulin peptides with small molecular weights. High-efficiency hydrolysis of globulin induced by mercaptoethanol improved the freezing tolerance of hydrated lettuce seeds and led to the accumulation of small globulin peptides, which further proved the positive function of small globulin polypeptides in enhancing the freezing tolerance of hydrated lettuce seeds. DSC of small globulin peptides showed that the smaller the molecular weight, the earlier the appearance of ice crystals and the higher the enthalpy of heat release. For the smallest peptides, the 2-Bp-4 in 2-Bp group exhibited higher enthalpy in exothermic peak than the control group (c-4). In conclusion, the hydrolysis of seed globulins and accumulation of small-molecule globulin peptides could be the major reason for improving the freezing tolerance of hydrated seeds after de-palmitoylation treatment.

棕榈酰蛋白硫酯酶（PPT）参与脂肪酸合成和蛋白质去棕榈酰化，是在水合生菜种子的生态冷却处理下诱导产生的。然而，对照样品和冷却样品之间的脂肪酸水平没有显着差异。为了进一步研究PPT的功能，在水合生菜种子的吸胀过程中使用蛋白质棕榈酰化抑制剂2-溴棕榈酸（2-Bp），然后进行缓慢冷却处理（-3°C h -1）。缓慢冷却至-20°C后，施用2-Bp (1 mM)显着提高了种子的存活率，从6.70%（对照吸胀）增加到22.67%（2-Bp吸胀）。差示扫描量热法 (DSC) 分析表明，与对照组相比，2-Bp 导致胚乳中冰晶的出现更早。二维电泳(2D)证实2-Bp可以促进种子球蛋白的水解和小分子量球蛋白肽的积累。巯基乙醇诱导球蛋白高效水解，提高了水合生菜种子的耐冻性，并导致小球蛋白肽的积累，进一步证明了小球蛋白多肽在增强水合生菜种子耐冻性方面的积极作用。小球蛋白肽的DSC表明，分子量越小，冰晶出现越早，放热焓越高。对于最小的肽，2-Bp组中的2-Bp-4在放热峰中表现出比对照组(c-4)更高的焓。综上所述，种子球蛋白的水解和小分子球蛋白肽的积累可能是去棕榈酰化处理后水合种子耐冻性提高的主要原因。