5,5′-Dithiobis(2-nitrobenzoicacid) Enhances the Freezing Resistance of Hydrated Lettuce Seeds under Programmed Cooling Treatment

Abstract The hydrolysis of globulins is supposedly involved in freezing tolerance of hydrated lettuce seeds. Considering that globulins are constituted by two subunits with S-S bonds, lettuce seeds was imbibed in the aqueous solution of 5,5′-Dithiobis (2-nitrobenzoicacid )(DTNB), the Ellman’s reagent which can react with free sulfhydryl side chain of cysteine to form an S-S bond between the protein and thionitrobenzoic acid (TNB) residue. According to this study, DTNB improved seed freezing tolerance, as shown by the greater viability of hydrated seeds under programmed cooling (-1°C min⁻ ¹-) treatment. Calorimetric study showed delayed ice formation in DTNB treatment upon programmed cooling (-22.0°C in DTNB-treated embryos vs -19.3°C in control embryos). Two-dimensional (Two-D) electrophoresis showed that DTNB promoted the de-polymerization of seed storage globulins in cooling treatment. The globulins and their hydrolyzed polypeptides were extracted and the ice formation mode of the polypeptides with different molecular weight was detected with differential scanning calorimeter (DSC), which indicated that the smaller peptides gave higher onset temperature of ice formation and higher enthalpy of heat release. The concentration of disulfides bond and sulfhydryl groups in globulins was detected which indicated that DTNB could promote the formation of sulfhydryl groups, and reduce the amount of disulfide bonds under programmed cooling treatment. Moreover, DTNB increased the hydroxyl radical scavenging rate, which indicated that DTNB promotes the anti-oxidation of hydrated seeds.