De-polymerisation and hydrolysis of seed storage protein is involved in the freezing tolerance of hydrated Brassica oleracea seeds

Hydrolysis of globulins might be involved in the freezing tolerance of hydrated lettuce seeds. The mechanism was further explored in hydrated brassica ( Brassica oleracea ) seeds, by application of 5,5'-dithiobis(2-nitro­benzoicacid) (DTNB), the Ellman’s reagent, which can bind to the free sulphydryl side chain of cysteine. DTNB increased the viability of hydrated brassica seeds after slow cooling (-3°C hour -1 ) to -17°C. Two- dimensio­nal electrophoresis and mass spectra analysis indicated the accumulation of small peptides derived from 12S globulins (cruciferin) in DTNB-treated seeds after slow cooling. The seed storage peptides with different molecular weight were cooled and the ice formation during cooling observed by cryomicroscopy, which proved that peptides of smaller molecular size produced smaller and rounder crystals. The smallest peptide in DTNB-treated seeds produced the smallest and roundest crystals during cooling. The levels of ASA, DHA and GSH were specifically up-regulated after DTNB treatment and following slow cooling, which indicates that DTNB enhanced the circulation of ASA and GSH to resist the oxidative stress. Consistent with this finding, RNA-seq and real time PCR indicated that glutathione reductase (GR) was up-regulated after DTNB treatment.