Metabolism of fructans in temperate grasses dynamically fluctuates before and during winter and is involved in the overwintering activity of plants. We monitored three candidate factors that may be involved in seasonal fructan metabolism in timothy (Phleum pratense): transcription levels of two fructosyltransferase (PpFT1 and PpFT2) genes and one fructan exohydrolase (Pp6-FEH1) gene during fall and winter and under artificially cold conditions. Functional analysis using a recombinant enzyme for PpFT2, a novel fructosyltransferase cDNA, revealed that it encoded sucrose:fructan 6-fructosyltransferase, with enzymatic properties different from previously characterized PpFT1. PpFT1 transcripts decreased from September to December as the amount of fructans increased, whereas PpFT2 transcripts increased in timothy crowns. PpFT2 was transcriptionally more induced than PpFT1 in response to cold and sucrose in timothy seedlings. A rapid increase in Pp6-FEH1 transcripts and increased monosaccharide content were observed in timothy crowns when air temperature was continuously below 0. °C and plants were not covered by snow. Transcriptional induction of Pp6-FEH1 by exposure to -3. °C was also observed in seedlings. These findings suggest Pp6-FEH1 involvement in the second phase of hardening. PpFT1 and PpFT2 transcription levels decreased under snow cover, whereas Pp6-FEH1 transcription levels were constant, which corresponded with the fluctuation of fructosyltransferase and fructan exohydrolase activities. Inoculation with snow mold fungi (Typhula ishikariensis) increased Pp6-FEH1 transcription levels and accelerated hydrolysis of fructans. These results suggest that transcriptional regulation of genes coding fructan metabolizing enzymes is partially involved in the fluctuation of fructan metabolism during cold acclimation and overwintering. © 2014 Elsevier GmbH.