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Cold acclimation, the necessary prerequisite for promotion of freezing tolerance, is affected by both low temperature and enhanced far-red/red light (FR/R) ratio. The impact of FR supplementation to white light, created by artificial LED light sources, on the hormone levels, metabolism, and expression of the key hormone metabolism-related genes was determined in winter barley at moderate (15 °C) and low (5 °C) temperature. FR-enhanced freezing tolerance at 15 °C was associated with promotion of abscisic acid (ABA) levels, and accompanied by a moderate increase in indole-3-acetic acid (IAA) and cis -zeatin levels. The most prominent impact on the plants' freezing tolerance was found after FR pre-treatment at 15 °C (for 10 days) followed by cold treatment at FR supplementation (7 days). The response of ABA was diminished in comparison with white light treatment, probably due to the elevation of stress tolerance during FR pre-treatment. Jasmonic acid (JA) and salicylic acid (SA) were transiently reduced. When the plants were exposed directly to a combination of cold (5 °C) and FR supplementation, ABA increase was higher than in white light, and was associated with enhanced elevation of JA and, in the longer term (after 7 days), with IAA and cis -zeatin increase, which indicates a stronger stress response and better acclimation. Cold hardening was more efficient when FR light was applied in the early developmental stage of the barley plants (three-leaf stage, 18 days), rather than in later stages (28-days). The dynamics of the phytohormone changes are well supported by the expression profiles of the key hormone metabolism-related genes. This series of treatments serves as evidence for the close relationship between plant hormones, light quality, and low temperature at the beginning of cold acclimation. Besides the timing of the FR treatments, plant age also represents a key factor during light spectrum-dependent cold acclimation.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeum vulgare ( Hv ) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBF s, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance., Competing Interests: The authors declare no conflicts of interest.