Parthenolide reduces the freezing tolerance of hydrated lettuce seeds by inhibiting the proteolysis of seed storage globulins

Moisture content is a key factor affecting success of both field seed survival and seed cryobanks. Studying the freezing tolerance of hydrated seeds can provide information for above issues. Our previous study indicated that the ubiquitin-mediated proteolysis pathway was involved in the freezing tolerance of hydrated lettuce seeds. In this study, parthenolide, a repressor of ring-finger-type ubiquitin E3 ligase, was found to significantly reduce the freezing tolerance of lettuce seeds in slow cooling treatment. Differential scanning calorimeter (DSC) proved that parthenolide led to earlier and more ice crystal formation in the embryo of hydrated seeds in slow cooling, compared with control treatment. Real time PCR analysis showed that parthenolide changed the transcription rhythm of the RING-type E3 ligase COP1 and cold-induced gene ICE1 (Inducer of CBF Expression 1). Results of RNA-seq and real time PCR indicated that parthenolide affected the function of endoplasmic reticulum associated degradation (ERAD) pathway, in which Hsp90 was significantly up-regulated and Hsp20 down-regulated after slow cooling. Two-dimensional electrophoresis of total proteins and SDS-PAGE of soluble proteins found that parthenolide inhibited the proteolysis of seed storage globulins in slow cooling treatment. The inhibition of proteolysis for seed storage globulins and the reduction of small molecular chaperons that can prevented aggregation of misfolded proteins in parthenolide-treated seeds may be responsible for the reduction of freezing tolerance of the hydrated seeds. Parthenolide can disrupt the expression pattern of COP1 and cold responsive genes, reduce the level of small molecular chaperons and the proteolysis of seed globulins, which makes hydrated seeds freezing-sensitive.