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.