Physiological Mechanisms Only Tell Half Story: Multiple Biological Processes are involved in Regulating Freezing Tolerance of Imbibed Lactuca sativa Seeds

The physiological mechanisms by which imbibed seeds survive freezing temperatures in their natural environment have been categorized as freezing avoidance by supercooling and freezing tolerance by extracellular freeze-desiccation, but the biochemical and molecular mechanisms conferring seed freezing tolerance is unexplored. In this study, using imbibed Lactuca sativa seeds we show that fast cooled seeds (60 °C h-1) suffered significantly higher membrane damage at temperature between -20 °C and -10 °C than slow cooled (3 °Ch-1) seeds (P < 0.05), presumably explaining viability loss during fast cooling when temperature approaches -20 °C. Total soluble sugars increase in low temperature environment, but did not differ significantly between two cooling rates (P > 0.05). However, both SOD activity and accumulation of free proline were induced significantly after slow cooling to -20 °C compared with fast cooling. RNA-seq demonstrated that multiple pathways were differentially regulated between slow and fast cooling. Real-time verification of some differentially expressed genes (DEGs) revealed that fast cooling caused mRNA level changes of plant hormone and ubiquitionation pathways at higher sub-zero temperature, whilst slow cooling caused mRNA level change of those pathways at lower sub-zero ttemperatures. Thus, we conclude that imbibed seed tolerate low temperature not only by physiological mechanisms but also by biochemical and molecular changes.