The key stage for the formation of freezing-resistance in cold-tolerant plants and the main timing of death of non cold-tolerant plants is during the freeze-thaw cycle induced by rapidly fluctuating temperatures in early winter. However, it is not clear that how cells in the leaves of cold-tolerant plants survive during freeze-thaw cycles in winter. The objective of this study was to investigate the changes in the activities of antioxidant enzymes and content of osmolytes, lipid peroxidantion and membrane permeability in leaves of white clover grown in different micro-environment in order to know the relationship between changes in the antioxidant enzymes activities and the contents of osmolytes with freeze-tolerance during the freeze-thaw cycles in winter. Three separate white clover plantings were established in 2006 in full sun(roadside, enough sunlight), in partial shade (woodland) and in full shade(behind the building, shade until about 4 o'clock in the afternoon). In the fall of 2008, weeds and smaller and older leaves of white clover were pulled out to provide uniform leaves for sampling and the areas were fenced to avoid penetration by animals. Sampling of leaves of The white clover was took before and after snowfall. The results showed that in thaw-freeze stage when average daily temperature rapidly decreased from 9℃ to -4.5℃, the leaves of white clover were frozen, membrane permeability initially declined, then rapidly increased up to 2 fold, the contents of malonaldehyde (MDA) and proline and the activities of catalase (CAT EC 1.11.1.6), peroxidase (POD EC1.11.1.7), and superoxide dismutase (SOD EC 1.15.1.1) increased in the frozen leaves. Thus there was a negative relationship between antioxidant enzymes activity and the contents of osmolytes during declining temperatures in thaw-freeze stage. In early freeze-thaw stage, when average daily temperature increased from -5℃ up to 0℃, the leaves were in condition of freeze-thaw, the membrane permeability, malonaldehyde content and catalase activity increased, while the activities of superoxide dismutase and peroxidase decreased. When the temperature further increased to 5℃, the leaves were totally thaw, the membrane permeability, catalase activity, and malonaldehyde content declined while the activities of superoxide dismutase and peroxidase increased. There were no significant differences between the three separate populations of white clover.
In the thaw-freeze stage, antioxidant enzymes rapidly increase to scavenge active oxygen species activated by formatted-ice in the leaves in order to protect membrane integrity. Likewise, in the freeze-thaw stage, lower lipid peroxidation and higher membrane elasticity were highly correlated with antioxidant enzyme activity. It suggested that white clover has higher capacity to activate antioxidant enzyme system to control the lipid peroxidation and increase osmolytes in response to freezing temperature. Antioxidant enzymes activity and osmolytes contents enhance fastly prevent lipid peroxidation and protect cell membrane integrality, this plays a key role in surviving winter freezing-thaw cycles.