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.

水分含量是影响田间种子存活和种子冷冻库成功的关键因素。研究水合种子的耐冻性可以为上述问题提供信息。我们先前的研究表明泛素介导的蛋白水解途径与水合生菜种子的耐冻性有关。在这项研究中，发现了以环指型泛素E3连接酶的阻遏物单苯乙内酯在慢冷处理中可显着降低生菜种子的耐冻性。差示扫描量热仪（DSC）证明，与对照处理相比，在缓慢冷却的条件下，小白菊内酯在水合种子的胚胎中导致更早和更多的冰晶形成。实时PCR分析表明，小白菊内酯改变了RING型E3连接酶COP1的转录节奏和冷诱导基因ICE1（CBF表达1的诱导物）。RNA-seq和实时PCR的结果表明，小白菊内酯会影响内质网相关降解（ERAD）通路的功能，其中缓慢冷却后Hsp90显着上调而Hsp20下调。总蛋白的二维电泳和可溶性蛋白的SDS-PAGE发现，在缓慢冷却的过程中，小白菊内酯抑制了种子贮藏球蛋白的蛋白水解。抑制蛋白水解的种子贮藏球蛋白和减少小分子分子伴侣，可以防止在单粉酚处理过的种子中错误折叠的蛋白质聚集，这可能是导致水合种子抗冻性降低的原因。