Low temperatures adversely affect plant quality and productivity and function as a determinant of geographical distribution and growth [1–3]. Plants achieve cold tolerance following gradual exposure to low but non-freezing temperatures, a phenomenon called cold acclimation [4–6]. Cold acclimation is accompanied by changes at the physiological, molecular and biochemical levels [7, 8].
Low temperatures initiate signaling pathways that control the expression of genes encoding determinants necessary for chilling tolerance . Until now, the ICE1-CBF (Inducer of CBF Expression - CRT/DRE Binding Factor)-cold-response pathway has been one of the dominant cold signaling mechanisms mediating cold tolerance in Arabidopsis[1, 2, 9, 10]. Cold-regulated genes (COR) encode functional hydrophilic proteins, controlling cell osmoregulation and stabilization under freezing stress [11, 12]. DRE/CRT cis-elements containing the core sequence CCGAC have been identified from these COR promoters [13, 14]. Transcription factors known as CBFs (CRT binding factors) or DREB1s (DRE binding factors) induce transcription of downstream COR genes via interaction with DRE/CRT elements [15–17]. The genes encoding CBF transcription factors are up-regulated by cold. The three CBF genes encoding DREB1B/CBF1, DREB1A/CBF3, and DREB1C/CBF2 in Arabidopsis play a role in the cold acclimation pathway . Numerous reports have demonstrated that CBF overexpression alleviated damage associated with freezing stress in Arabidopsis, rice and non-model plants [15, 18, 19].
Several factors involved in regulation of CBF/DREB1 expression have been identified genetically in Arabidopsis. Direct regulators of CBF/DREB1 expression include HOS1 (high expression of osmotically responsive genes) , ICE1 (inducer of CBF/DREB1 expression 1)  and MYB15. The ICE1 gene encodes a MYC-like bHLH transcription factor that binds directly to canonical MYC cis-elements (CANNTG) in the CBF3/DREB1A promoter . ICE2 encodes a homolog of ICE1, and primarily influences the expression of CBF1/DREB1B but not that of CBF3/DREB1A. Overexpression of two ICEs has been associated with enhanced chilling tolerance in Arabidopsis, rice, apples and tobacco [1, 2, 22, 23]. Interestingly, ICE1/SCREAM is also involved in stomatal differentiation, suggesting that ICE1 mediates transcriptional regulation of environmental adaptation and stomatal development in plants . In addition, protein interaction analysis reveals that ICE1 post-translational modification occurs during cold acclimation. Freezing tolerance is negatively regulated by HOS1-induced degradation of ICE1 and positively regulated by SIZ1-mediated sumoylation and stabilization of ICE1 [9, 10]. Recent data indicate that serine 403 of ICE1 plays a role in the regulation of transactivation and cold-induced degradation via the ubiquitin/26S proteasome pathway, which is probably mediated by HOS1 . Further investigation revealed that several jasmonate ZIM-domain (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors, decreasing the freezing stress response of Arabidopsis.
MicroRNAs (miRNAs), a class of small non-protein coding RNAs containing 20 to 24 nucleotides (nt), have been increasingly investigated as key regulators of gene expression [27, 28]. Recent evidence indicates that plant miRNAs play a role in biotic and abiotic stress responses [29–31]. Cold-responsive miRNAs in different species enable development of breeding strategies for cold tolerance [30, 32–34]. The miR398 is a repressor of Cu-Zn superoxide dismutase genes (At1g08830, CSD1; At2g28190, CSD2), which act as reactive oxygen species (ROS) scavengers . MiR398 is regulated in response to oxidative stress, salt, abscisic acid (ABA), sucrose treatment and different ambient temperatures, resulting in an immediate change in CSD levels [35–40]. Over expression of CSD protects plants from oxidative stress and enhances freezing stress tolerance in transgenic plants . A recent report suggested that miR398-CSD positively regulated heat tolerance .
The exact miR398-CSD pathway involved in the mechanism of freezing tolerance, however, is not completely understood.
In a previous investigation of chrysanthemum freezing tolerance, we isolated CdICE1 from Chrysanthemum dichrum. In our present study, we further explored CdICE1 functions under two different cold acclimation conditions, which revealed that CdICE1 mediates freezing tolerance via CBF and miR398 pathways.