Palmitoylation mediates the proteolysis of seed storage proteins during the cooling process in hydrated lettuce seeds (Lactuca sativa)

Abstract

AbstractPalmitoyl-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.