Dual-phase cold acclimation in barley under field conditions: expression dynamics of ICE-CBF-COR and vernalization pathways in Nure and Tremois

Barley (Hordeum vulgare L.) thrives across diverse environments but yield in temperate regions like Italy’s Po Valley depends on adapting to seasonal cold. Survival relies on cold acclimation for freezing tolerance and vernalization to align flowering (Feng et al., 2025). While studies under controlled conditions helped to understand the base of ICE-CBF-COR and vernalization pathways, field-based work is needed to capture transcriptional dynamics under natural temperature and photoperiod fluctuations(Vítámvás et al., 2019). Cold acclimation in barley is driven by the ICE–CBF–COR pathway: ICE1 activates HvCBFs at FR-H2, which induce COR effectors genes that stabilize membranes and mitigate oxidative/dehydration stress (Caccialupi et al., 2023). In parallel, vernalization (VRN) and photoperiod regulators (PPD) genes integrate seasonal cues to coordinate stress tolerance with reproductive timing (Deng et al., 2015). To investigate the dynamics of these pathways under agronomic conditions, a time-series gene expression experiment was performed in the Po Valley, comparing the winter cultivar Nure with the spring cultivar Tremois. Plants were sampled weekly at sunset throughout the whole vegetative phase, and expression was quantified for ICE1, multiple HvCBF genes at FR-H2, the effector genes HvCOR14b and HvDHN5, and selected genes of the vernalization pathway (VRN-H1, 2, 3 and PPD-H1 and 2). Our results confirmed that HvCBF4 is a putative candidate to enhance freezing tolerance, that showed consistently stronger and more sustained induction in Nure than Tremois. Expression of effector genes was higher in Nure at the beginning of the experiment, followed by secondary peaks during later stages, reflecting a two-phase acclimation response. In contrast, no constitutively high expression of effector genes was observed in Tremois showing transient induction events that coincided with minimum field temperatures falling below 0 °C. Taken together, these findings support a dual-phase model of acclimation in barley: an early phase, where ICE-CBF-COR signaling primes stress-protective responses, and a later phase, where sustained cold maintains or reactivates effector genes expression to reinforce freezing tolerance. These results provide novel field-based evidence of how barley integrates stress signaling with seasonal adaptation, offering insights for breeding strategies aimed at improving winter hardiness under climate variability.