Ca²⁺ is widely recognized as the primary and canonical secondary messenger in rice cold stress signaling pathways (Figure 1) (Ding et al., 2019). The cold-induced cytoplasmic Ca²⁺ influx (triggered by the COLD1-RGA1 complex) is mainly mediated by cyclic nucleotide-gated channels (CNGCs), including OsCNGC9, OsCNGC14, and OsCNGC16 (Cui et al., 2020; Wang et al., 2021). Additionally, the endoplasmic reticulum (ER)-localized Ca²⁺-binding protein calreticulin (OsCRT3) contributes to the cold-induced elevation of cytosolic Ca²⁺ concentrations (Table 1) (Guo et al., 2023).

Recent studies have identified 2’,3’-cyclic adenosine monophosphate (2’,3’-cAMP) as a crucial secondary messenger in rice cold signaling and other biotic stress signaling (Figure 1) (Yu et al., 2022; Luo et al., 2024). Under normal temperature conditions, COLD6 interacts with RGA1 at the plasma membrane. Upon exposure to cold stress, cold-induced osmotin-like protein 1 (OSM1) binds to COLD6, displacing RGA1 from the COLD6-RGA1 complex. This displacement triggers an increase in intracellular 2’,3’-cAMP levels, which in turn enhances chilling tolerance (Luo et al., 2024). Functional analyses have shown that COLD6 negatively regulates cold tolerance and depends on OSM1 for its function in cold stress responses. Both the accumulation of OSM1 and the interaction between COLD6 and OSM1 significantly elevate 2’,3’-cAMP concentrations, which further activate the expression of cold-responsive (COR) genes, such as members of the dehydration-responsive element-binding protein (OsDREB) family (Dubouzet et al., 2003; Ito et al., 2006; Luo et al., 2024). Collectively, OSM1 competes with RGA1 for binding to COLD6. Under cold stress, RGA1 is released from the COLD6-RGA1 complex and subsequently interacts with COLD1 to induce Ca²⁺ influx, while free COLD6 binds to OSM1 to trigger 2’,3’-cAMP accumulation (Table 1) (Ma et al., 2015; Luo et al., 2024). Notably, this coordinated interplay ensures the integration of Ca²⁺ and 2’,3’-cAMP signaling pathways in response to cold stress.

Reactive oxygen species (ROS) also function as secondary messengers in rice responses to cold stress, as well as to numerous other abiotic and biotic stresses (Waszczak et al., 2018; Sinenko et al., 2021; Mittler et al., 2022; Wu et al., 2023; Sood, 2025; Zheng et al., 2025). ROS encompass superoxide ion (O₂^•–), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), ozone, hydroxyl radical (OH^•), and organic and inorganic peroxides, which play pivotal role in initiating plant defense responses against various stresses (Mittler et al., 2022). However, excessive accumulation of ROS can induce substantial cellular damage by triggering the overoxidation of essential macromolecules, including lipids, amino acids and DNA. To ensure normal growth and stress resilience, ROS homeostasis is tightly regulated by a dynamic balance between ROS production and scavenging systems (Waszczak et al., 2018; Mittler et al., 2022). It is well established that cold stress induces ROS accumulation in rice (Li et al., 2022). Wang et al. demonstrated that OsPUS1, a pseudouridine synthase, is critical for maintaining ROS homeostasis in rice under cold stress (Wang et al., 2022); ospus1 mutants exhibit overproduction of O₂^•– from the mitochondrial complex 1 and display albino seedlings at low temperature (Wang et al., 2022; Zu et al., 2023). Further investigations revealed that SOP10, a mitochondrial pentatricopeptide repeat protein, contributes to overproduction of O₂^•–in ospus1 mutants, and knockout of SOP10 can rescue the albino phenotype of ospus1 under cold stress (Zu et al., 2023). Additional evidence from the same study indicated that mitochondrial superoxide is essential for rice cold stress responses, as overexpression of superoxide-scavenging enzymes can enhance rice cold tolerance (Zu et al., 2023). Although these studies have uncovered certain mechanism underlying ROS accumulation and scavenging in rice cold stress responses, more efforts are required to fully elucidate the entire regulatory network maintaining ROS homeostasis under cold stress.

In addition to regulate the cytosolic Ca²⁺ concentration (Guo et al., 2023), OsCRT3 is also involved in Ca²⁺ signal decoding process (Figure 1). Under normal temperatures, OsCRT3 weakly interacts with OsCIPK7 (a CBL-interacting protein kinase), and the kinase activity of OsCIPK7 is repressed by its auto-inhibitory domain (Weinl and Kudla, 2009). When cold stress occurs, OsCRT3 undergoes a conformational change that strengthens its interaction with OsCIPK7, boosting the kinase activity of OsCIPK7. Moreover, the elevated cytosolic Ca²⁺ is bound and perceived by calmodulin-like proteins (OsCBL7 and OsCBL8), which then physically interact with OsCIPK7 at the plasma membrane to further activate the kinase activity of OsCIPK7, thereby promoting cold tolerance in rice (Guo et al., 2023). The critical role of OsCIPK7 in cold tolerance is further supported by the genetic evidence that a point mutation which increases the kinase activity of OsCIPK7 results in enhanced cold tolerance in rice plants (Zhang et al., 2019a).

Beyond OsCBL7, OsCBL8, and OsCIPK7, calcium-dependent protein kinases (CDPKs) also play essential roles in decoding Ca²⁺ signals during rice cold stress responses (Figure 1). The fine-tuned regulation of OsCPK17 (a CDPK family member) is critical for rice cold tolerance. Both knockout and overexpression of OsCPK17 render rice more sensitive to cold stress, indicating that the accumulation of OsCPK17 must be maintained within a narrow optimal range (Almadanim et al., 2017). Activated OsCPK17 phosphorylates target proteins including aquaporins (OsPIP1;2, OsPIP2;6) and sucrose-phosphate synthase (OsSPS4), which are hypothesized to contribute to cold tolerance by regulating water homeostasis and sugar metabolism, respectively (Almadanim et al., 2017). Another CDPK, OsCPK24, is induced by cold stress and acts as a positive regulator of cold tolerance (Liu et al., 2018b). Mechanistically, OsCPK24 phosphorylates OsGrx10, a glutathione-dependent thioltransferase, and inhibits the thioltransferase activity of OsGrx10. This inhibition maintains higher intracellular reduced glutathione levels, which enhances redox homeostasis and protects cells from cold-induced oxidative damage (Liu et al., 2018b). OsCDPK7, a third key CDPK, is also induced by cold stress and functions as a positive regulator of cold tolerance, though its specific downstream targets and detailed mechanism of action remain to be fully elucidated (Saijo et al., 2000).

As a central cold sensor in rice, the transcriptional regulation of COLD1 has been extensively studied (Figure 2). Histone deacetylases (HDACs) have emerged as critical regulators of COLD1 expression. Under cold stress, cold-induced histone deacetylase HDA705 reduces the acetylation of OsECT3, a plant m⁶A reader protein, at lysine 471 (K471) (Ma et al., 2025). This deacetylation enhances the m⁶A-binding activity of OsECT3, which then binds to and stabilizes the transcripts of COLD1 and other cold-response-related genes, thereby enhancing cold tolerance (Ma et al., 2025). Another histone deacetylase, OsHDA716, regulates cold stress responses by mediating the deacetylation of OsbZIP46. OsbZIP46 is a transcription factor that controls the expression of COLD1 and OsDREB1A (Kamble, 2024; Sun et al., 2024). Under normal temperatures, OsbZIP46 deacetylated by OsHDA716 is prone to proteasomal degradation; simultaneously, OsHDA716 represses the expression of OsbZIP46 target genes through histone deacetylation of their promoters. Under cold stress, cold signals induce the degradation of OsHDA716, which prevents the deacetylation of OsbZIP46 at K263, K281, and K294. The accumulated acetylated OsbZIP46 then activates the transcription of COLD1 and OsDREB1A, leading to enhanced cold tolerance (Sun et al., 2024). Additionally, OsDREB2B regulates COLD1 expression by forming a complex with OsSRO1c, a protein with intrinsic liquid-liquid phase separation properties, in the nucleus (Hu et al., 2024). The phase separation of the OsSRO1c-OsDREB2B complex dynamically responds to low temperatures and enhances the transcription of COLD1 and other COR genes (Hu et al., 2024). Given the central role of COLD1 in rice cold stress responses, it is anticipated that more transcription factors and regulatory mechanisms governing COLD1 expression will be discovered in future studies.

OsbHLH002/OsICE1 (INDUCER OF CBF EXPRESSION 1) is a master transcription factor in the rice cold stress signaling pathway (Figure 3). Unlike its homolog in Arabidopsis (AtICE1), OsbHLH002/OsICE1 expression is minimally affected by cold stress (Chinnusamy et al., 2003; Nakamura et al., 2011), its function is primarily regulated at the post-transcriptional and post-translational levels. It was reported that the expression of OsbHLH002/OsICE1 is negatively regulated by the OsEIN2-OsEIL1/2 module, which is a key component of the ethylene signaling pathway (Zhai et al., 2024). Specifically, OsEIL1 and OsEIL2, two master regulators of ethylene signaling, form a heterodimer and directly bind to the promoter of OsbHLH002/OsICE1, thereby synergistically suppressing its transcription (Zhai et al., 2024).

Post-translational modifications (PTMs) play a critical role in regulating the accumulation and activity of OsbHLH002/OsICE1, and thus mediate cold stress signaling (Figure 3). The E3 ubiquitin ligase OsHOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1) mediates the ubiquitination of OsbHLH002/OsICE1, marking it for degradation by the 26S proteasome (Lourenco et al., 2013). Under cold stress, OsMAPK3 and OsMAPK6 interact with and phosphorylate OsbHLH002/OsICE1, which prevents its ubiquitination by OsHOS1 and promotes its accumulation (Zhang et al., 2017; Liu et al., 2023a). The accumulated phosphorylated OsbHLH002/OsICE1 directly binds to the promoter of OsTPP1 (trehalose-6-phosphate phosphatase 1) and activates its expression, leading to increased trehalose biosynthesis and cold tolerance (Ge et al., 2008; Zhang et al., 2017). OsPP2C27, a type 2C protein phosphatase, functions as a negative regulator of the OsMAPK3/6-OsbHLH002/OsICE1-OsTPP1 pathway (Xia et al., 2021). It directly dephosphorylates both OsMAPK3/6 and OsbHLH002/OsICE1, thereby attenuating the activity of this pathway (Xia et al., 2021). This negative regulation is essential for preventing the sustained activation of cold stress responses under cold conditions, which could otherwise compromise normal plant growth and development. Together, OsbHLH002/OsICE1 orchestrates multiple signaling pathways through various post-translation modifications, including ubiquitination, phosphorylation, and dephosphorylation, in response to cold stress in rice.

In addition to OsTPP1, OsDREBs are key direct targets of OsbHLH002/OsICE1, forming the core OsbHLH002/OsICE1-OsDREBs-COR regulatory axis in the rice cold stress signaling pathway (Figure 3) (Zhang et al., 2019b). Other transcription factors also contribute to the regulation of OsDREBs. OsERF52, an AP2/ERF family member, and IPA1, a SPL family member, directly bind to the promoters of OsDREBs and regulate their expression (Jia et al., 2022; Xu et al., 2024). Furthermore, OsSAPK9, a sucrose non-fermenting 1-related protein kinase 2, phosphorylates OsERF52 at serine 261 (S261), which enhances the protein stability of OsERF52 and strengthens its interaction with IPA1 and OsbHLH002/OsICE1. These interactions synergistically activate the transcription of OsDREBs, thereby initiating cold stress responses (Xu et al., 2024). Similar to OsERF52, IPA1 is phosphorylated by a kinase, OsSAPK6, and the phosphorylation increases its protein stability. Genetic studies have confirmed that serine 213 (S213) is a critical phosphorylation site mediating the function of IPA1 in rice cold stress responses (Jia et al., 2022). Additionally, OsMAPK6 phosphorylates IPA1, further modulating its activity and influencing cold stress tolerance in rice (Liu et al., 2023a).

Among the COR genes directly targeted by OsDREBs, OsCNGC9, a cyclic nucleotide-gated channel, is a well-studied target of OsDREB1A. OsDREB1A directly activates the expression of OsCNGC9 (Figure 3) (Wang et al., 2021). Recent studies have shown that OsCNGC9 is phosphorylated by two kinases, OsSAPK8 and OsCTK1 (Wang et al., 2021; Wu et al., 2024). Specifically, the phosphorylation of OsCNGC9 at serine 645 (S645) by OsSAPK8 significantly enhances its channel activity, promoting cytosolic Ca²⁺ influx under cold stress and amplifying the cold signal (Wang et al., 2021). Beyond OsCNGC9, OsCTK1 phosphorylates other substrates involved in cold tolerance, including the acidic ribosomal protein OsP3B and the dual-specific mitogen-activated protein kinase phosphatase OsMKP1 (Wu et al., 2024). Notably, OsMKP1 negatively regulates the kinase activity of OsMAPK3/6 that can phosphorylate OsbHLH002/OsICE1 and IPA1 (Zhang et al., 2017; Liu et al., 2023a), forming a negative feedback loop that fine-tunes the MAPK signaling cascade during cold stress responses.

Cold stress-induced biosynthesis and accumulation of osmolytes play a crucial role in osmotic adjustment, counteracting the osmotic pressure imposed by cold stress (Li et al., 2022). Among these osmolytes, trehalose is an important outcome of OsbHLH002/OsICE1-OsDREBs-COR core module, and enhances rice cold tolerance (Figure 3). Its biosynthesis is tightly regulated by two critical enzymes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) (Lunn et al., 2014; Eh et al., 2025). The genes encoding these enzymes, OsTPS2 (TPS) and OsTPP1 (TPP), are subject to complex transcriptional regulation in response to cold stress (Lunn et al., 2014; Eh et al., 2025). OsTPP1 is not only regulated by OsbHLH002/OsICE1 but also by OsMYB30 (Yu et al., 2024). OsTPS2 is regulated by ABF1, a bZIP family transcription factor, which is induced by cold stress (Shu et al., 2023). OsSAPK10 phosphorylates ABF1, which strengthens its DNA-binding ability to the cis-acting elements in the OsTPS2 promoter, thereby enhancing OsTPS2 transcription and trehalose biosynthesis (Shu et al., 2023). Chromatin accessibility also plays a crucial role in regulating the expression of OsTPS2 and OsTPP1 under cold stress. The imitation switch (ISWI)-type chromatin remodeling factors OsCHR11 and OsCHR17 physically associate with the promoters of OsTPS2 and OsTPP1, increasing chromatin accessibility and ensuring the full induction of these genes under cold stress conditions (Li et al., 2024a).

It was validated that GA biosynthesis and signaling pathway are implicated in rice cold response. GA exhibits unique and indispensable regulatory functions, primarily centered on alleviating cold-induced growth inhibition and coordinating the balance between growth and stress tolerance. OsWRKY53 is induced by cold stress. It directly suppresses the expression of GA biosynthetic genes, such as GA20ox1, GA20ox3, and GA3ox1, leading to reduced GA biosynthesis and lower intracellular GA levels in anthers (Fang et al., 2017; Tang et al., 2022). The decrease in GA content promotes the accumulation of DELLA protein SLR1 (SLENDER RICE 1), a negative regulator of GA signaling. SLR1 interacts with and suppresses the transcriptional activity of UDT1 (UNDEVELOPED TAPETUM 1) and TDR (TAPETUM DEGENERATION RETARDATION), which is essential for tapetum development and pollen maturation (Tang et al., 2022). This interaction results in pollen infertility under cold stress, a major cause of yield loss in rice. SLR1 also interacts with OsGRF6 (GROWTH-REGULATING FACTOR 6). OsGRF6 is a transcription factor that promotes GA biosynthesis by repressing the expression of OsGA2ox1, a GA catabolic gene, to respond to cold stress in rice (Li et al., 2021). Under normal temperatures, OsGRF6 represses OsGA2ox1, thereby maintaining active GA levels to support plant growth. Under cold stress, SLR1 directly promotes OsGA2ox1 expression. Meanwhile, SLR1 interacts with OsGRF6, relieving the repression of OsGA2ox1. This leads to increased GA catabolism, reduced active GA levels, and enhanced cold tolerance in order to balance growth and stress responses (Li et al., 2021). Other transcription factors also regulate GA metabolism in response to cold stress. CTB5, a HD-Zip family member, interacts with OsHox12, another HD-Zip family member, to activate the expression of the GA biosynthetic gene OsGA3ox1 and inhibit the expression of OsGA2ox6, a GA catabolic gene (Guo et al., 2025). This regulation promotes GA accumulation in anthers, facilitating tapetum development and pollen fertility under cold stress (Guo et al., 2025). Additionally, CTB5 directly binds to the promoter of PYL9, an ABA receptor-encoding gene, enhancing cold tolerance at the seedling stage by reducing ROS accumulation. This regulation mechanism highlights the crosstalk between GA and ABA signaling in cold responses (Guo et al., 2025). Evolutionary studies have revealed that the favorable CTB5^KM allele, a natural variant of CTB5, was selected during the cold acclimation of japonica rice adapted to plateau habitats in Yunnan Province, China, underscoring the role of natural variation in shaping cold tolerance in rice (Guo et al., 2025).

Unlike gibberellic acid (GA) which focuses on growth recovery, ABA primarily functions as a key regulator of cold stress defense, orchestrating a series of physiological and molecular responses to enhance rice tolerance to low temperatures. Direct evidence indicating the important role of ABA in rice cold response is null mutants of ABA biosynthetic genes OsNCED4 and OsNCED5 display reduced cold tolerance (Xiang et al., 2024, 2025). OsNAC5 is a cold-induced NAC family transcription factor. It modulates rice cold tolerance by fine-tuning the ABA signaling pathway (Takasaki et al., 2010; Li et al., 2024b). OsNAC5 directly activates the expression of OsABI5 (ABA INSENSITIVE 5), which is a key transcription factor in ABA signaling pathway. Meanwhile, it physically interacts with OsABI5 to enhance its protein stability (Li et al., 2024b). In vivo and in vitro assays have shown that OsABI5 directly regulates the expression of three classes of genes. First, it targets OsPP2C30 that encode a type 2C protein phosphatase and negatively regulates ABA signaling (Kim et al., 2011). Second, it regulates OsPRX70, which encodes a ROS-scavenging peroxidase that reduces cold-induced oxidative damage. Third, it controls COR genes including OsDREB1A and OsMYB20, integrating ABA signaling with the core cold response pathway (Li et al., 2024b).

The bZIP family transcription factors also play an important role in rice cold tolerance by mediating ABA biosynthesis. bZIP73^jap, a japonica-specific variant of bZIP73, and bZIP71 form a heterodimer that regulates cold tolerance through four distinct signaling pathways. First, the bZIP73^jap:bZIP71 heterodimer inhibits the expression of OsNCED3 and OsNCED5, reducing ABA levels and preventing excessive growth inhibition under cold stress (Liu et al., 2018a; Li et al., 2025). Second, the bZIP73^jap:bZIP71 heterodimer targets to the promoters of four peroxidase (POX) precursor genes (LOC_Os01g22249, LOC_Os03g02920, LOC_Os03g32050, and LOC_Os04g59210) and activates their expression, thereby reducing the ROS accumulation. Third, it directly targets the promoters of sugar transport-related genes (OsMST7, OsMST8, and OsINV4), enhancing soluble sugar transport and partitioning. Soluble sugars act as compatible solutes and cryoprotectants, protecting cells from cold-induced damage (Bolouri-Moghaddam et al., 2010). Fourth, the bZIP73^jap:bZIP71 heterodimer also activates the expression of qLTG3-1^Nip, a natural variant of qLTG3–1 from Nipponbare (Fujino et al., 2008). qLTG3-1^Nip interacts with bZIP73^jap and enhances the transcriptional activity of bZIP73^jap, forming a positive feedback loop that amplifies cold tolerance (Liu et al., 2019). Additionally, OsWRKY71^ZH11, a variant of OsWRKY71 from Zhonghua 11, directly suppresses the expression of OsABA8ox1, an ABA catabolic gene, under cold stress, increasing ABA levels and enhancing cold tolerance, which further highlights the role of ABA metabolism in rice cold responses (Li et al., 2025).

In recent years, many findings have indicated that many other phytohormones, such as auxin, jasmonate, salicylic acid, ethylene, and brassinosteroid are also involved in rice cold response (Mao et al., 2019; Jiang et al, 2020; Cheng et al., 2024; Cui et al., 2025; Xu et al., 2025). However, the molecular mechanisms remain to be explored.