Abstract: The experiment studied germination and early development of white clover (Trifolium repens cv. ‘Ladino’) seeds subject to drought stress induced by 18% polyethylene glycol (PEG) 6000 solution. Effects on starch metabolism, root activity, osmotic adjustment, antioxidant defense, and gene differential expression, of seed soaking with mannose solution (MAS), were analyzed during the germination period. Different MAS concentrations were investigated including 0 (Control), 1.0, 2.0, 5.0 and 10.0 mmol·L^-1. Seed soaking with low concentration (0.5, 1.0, 2.0 and 5.0 mmol·L^-1) of MAS significantly increased seed germination under drought stress, with the 2.0 mmol·L^-1 MAS concentration having the most pronounced beneficial effects. However, higher MAS concentration (10.0 mmol·L^-1) significantly reduced seed germination under drought stress. Further experiments found that seeds soaked with 2.0 mmol·L^-1 MAS displayed significant increases in root growth, root activity, and amylase activities than seeds soaking with 0 mmol·L^-1 MAS during germination under drought stress. The MAS pretreatment effectively alleviated the stress-induced inhibition of amylolysis, significantly increased the accumulation of free proline, and resulted in a more negative seed osmotic potential during germination. As compared to Control seedlings, the total antioxidant capacity, activities of superoxide dismutase (SOD) and peroxidase (POD), and transcriptional levels of MnSOD and POD significantly increased in the seedlings with 2.0 mmol·L^-1 MAS treatment under drought stress. MAS treatment also resulted in significantly lower levels of reactive oxygen species, electrolyte leakage, and malondialdehyde in cells indicating lower oxidative damage. However, 2.0 mmol·L^-1 of MAS soaking pretreatment did not increase the soluble sugar content during seed germination under drought stress, which indicated that the more negative osmotic potential induced by MAS was mainly related to the accumulation of free proline. In summary, the MAS-promoted production of soluble sugars through accelerated amylolysis is potentially useful to maintain seedling growth under stress. These results indicate that MAS significantly improves drought tolerance of white clover seeds during germination and early development. This response is closely associated with promotion of starch breakdown, enhancement of osmotic adjustment, and improvement of antioxidant defense during seed germination. Seed soaking with a low concentration of MAS may also be useful to promote seed germination of white clover in conditions where water is more freely available.

Key words: starch metabolism, gene differential expression, growth, antioxidant defense, osmotic adjustment