Successful winter survival of the plants needs a programmed capacity to activate appropriate mechanisms in development of frost tolerance (FT). Cold hardening is a complicated operation facilitating the overwintering and is associated by extensive changes in metabolome. The purpose of the present investigation is to simplify the association between metabolites changes and FT in winter rye varieties (Puma and Anatolien) acclimated under field situation in NW Iran, regions with long winters. Plants were sampled on five dates (Oct. 31, Nov. 29, Dec. 17, Jan. 4, and Feb. 1) as different cold hardening periods. FT, as measured by LT50 (the temperature at 50% lethality) significantly expanded during the autumn and winter seasons. Cold adaptation persuaded accumulation of sucrose and sorbitol in both varieties; however, the accumulation was more profound in Puma. The content of proline, aspartic acid, glutamic acid, tyrosine, and glycine betaine linearly expanded in the Anatolien variety during the overwintering. However, content of glycine betaine, proline, and aspartic acid in Puma was considerably higher than Anatolien. Hydrogen peroxide reached to its greatest quantities at November and December in Anatolien and Puma, respectively. These levels significantly reduced up to the next months. Glutathione and ascorbic acid contents, as important antioxidants, noticeably expanded during overwintering. There was a significant positive association between FT, total protein content, free amino acid, soluble sugars, and glycine betaine. These data suggest that the development of FT during the cold adaptation and overwintering is partly resulted from metabolome changes and such modifications are strongly linked to the genetic potential.
