Being sessile organisms, plants have evolved relative elaborate mechanism to sense and cope with the ever-changing temperature. Cold signals are sensed by receptors through the cell membranes, which then participate in the regulation of intracellular signaling networks or cell-cell communication (Ahuja et al., 2010; Norman et al., 2011; Jogaiah et al., 2013). Much efforts have been made to discover the cold sensors ( Figure 2 ). The CHILLING TOLERANCE DIVERGENCE 1 (COLD1) is one of the currently identified cold sensors in rice, which could mediate the chilling tolerance of rice by regulating calcium channels and OsCBF1 gene expression (Ma et al., 2015). It deserved to further investigate whether the function of COLD1 gene is conservative in tea or other plant species. In addition to COLD1, the receptor-like kinases (RLKs) and histidine kinases (HKs) were also able to sense environmental signals. During cold acclimation of tea plants, almost all CsRLK and CsHK genes were particularly found to be up-regulated, suggesting their crucial roles for cold tolerance in tea plants (Wang et al., 2013). RLK members constitute the largest gene family of plant membrane signaling proteins, while HKs are the most abundant and diverse membrane receptors. Both of them are potential cold sensors in plants (Osakabe et al., 2013). They usually regulate the expression of many cold-inducible genes through abscisic acid signaling pathway and/or calcium/calmodulin signaling or other manners under cold stimuli (Murata and Los, 2006; Yang et al., 2010; Xu et al., 2020).

Besides, previous studies demonstrated that the light receptors were also closely related to cold response of bacteria and plants. They reported that cold-stress signaling pathways showed to be closely associated with the light perception and circadian clock, but it remains unknown how plants sense and transmit stress signals to regulate gene expression (Chen et al., 2004; Gould et al., 2013; Estravis-Barcala et al., 2020; Kidokoro et al., 2021; Kidokoro et al., 2022). An activation of the phytochromes under long day conditions triggers PIF7 (phytochrome interacting factor 7) to interact with the circadian oscillator TOC1, subsequently bind to a G-box sequence (CAGTG) in the CBF promoter, and thereby downregulates CBF expression (Wisniewski et al., 2014). The blue light receptor phytotropin perceives cold signals in liverwort Marchantia polymorpha at its photoactivated state (Fujii et al., 2017). In Arabidopsis, phytochrome B (phyB) photoreceptor integrates light and ambient temperature by reversible photoconversion between active Pfr (far-red) state and the inactive Pr (red) light-absorbing state (Legris et al., 2016). The physical interaction of phyB and CBF1 abrogated the interaction of phyB and PIF3/4 to promote the photomorphogenesis at 22/17°C in the light (Lu et al., 2020) (Dong et al., 2020) Jung et al., 2016; Legris et al., 2016). In contrast, the cold-induced CBFs stabilized the phyB thermosensor to enhance plant cold tolerance at 4°C (Jiang et al., 2020). In plants, phytochrome signaling pathway related gene AtFHY3/FAR1 (FAR-RED ELONGATED HYPOCOTYL 3/FAR-RED-IMPAIREDRESPONSE 1) not only modulates the phyA activity by directly activating the expression of FHY1/FHL but also positively regulates cold response through regulating JA (jasmonic acid) signaling pathway (Liu et al., 2019; Dai et al., 2022). Unfortunately, little research has been done on the interaction of low temperature and light signals in tea plants, with the exception of the expression analysis of CsFHY3/CsFAR1. Almost all of the CsFHY3/CsFAR1 family members were down-regulated under cold stress of tea plants, suggesting their negative roles for regulating cold tolerance in tea plants (Liu et al., 2021).

An increased expression in a set of genes related to red-light perception (GRAVITROPIC IN THE LIGHT), blue and UV-light perception (CRYPTOCHROME 1, EARLY LIGHT-INDUCABLE PROTEIN 1, and UVB-RESISTANCE 8), chloroplasts relocation (CHLOROPLAST UNUSUAL POSITIONING 1), and regulation of chlorophyll biosynthesis (Cs213 putative cold-inducible protein), stomatal movement (PHOSPHOGLUCOMUTASE), PSII associated light-harvesting complex II catabolic process (FILAMENTATION TEMPERATURE-SENSITIVE H) were also observed in tea plant under the long term cold stress. Interestingly, among all upregulated DEGs (differential expressed genes), the highest expression level was observed in ELIP1 (EARLY LIGHT-INDUCIBLE PROTEIN 1) which was upregulated 150-2000 folds above control under long-term cold stress in tea leaves. ELIPs are located in thylakoid membranes and are known to protect photosynthetic machinery from various environmental stresses in higher plants and have been reported to participate in the phytochrome signaling pathway (Rizza et al., 2011). Additionally, it was reported that the induction of ELIP1/2 expression is mediated via CRY1 (CRYPTOCHROME 1) in a blue light intensity-dependent manner (Kleine et al., 2007; Yang et al., 2017). During exposure to high irradiance, cry1 Arabidopsis mutants displayed inhibition of anthocianidin and flavonoids biosynthesis genes and phenylpropanoid genes, peroxidase genes, GST and ERD9 genes which are the components of various stress responses (Kleine et al., 2007). In our RNAseq study, the expression of CRY1 was also significantly increased under the 14 days of chilling stress and 3-day freezing temperature of tea plants. According to the previous data, CRY1 participates in the high temperature response in plants (Ma et al., 2016), suggesting that the mechanisms of temperature compensation might in principle be linked to the mechanisms of light perception (Gould et al., 2013), however the authors did not observe an accumulation of CRY transcripts under short-term cold stress in Arabidopsis.

Additionally, several new identified DEGs were upregulated under the long-term cold stress of tea plants which seems to be related to light sensing pathways. Among them, EID1 (EMPFINDLICHER IM DUNKELROTEN LICHT 1) -like F-box protein 3 is an F-box protein that related to red-light perception and functions as a negative regulator in phytochrome A (phyA)-specific light signaling. F-box proteins are components of SCF ubiquitin ligase complexes that target proteins for degradation in the proteasome, regulates photomorphogenesis and flowering in Arabidopsis (Marrocco et al., 2006). Similar to the previous finding (Ohta and Takaiwa, 2014), we identified DNAJ11 and DNAJ ERDJ3B-like in tea plant which encode co-chaperone components, stimulate Hsp70 ATPase activity, which is responsible for stabilizing the interaction of Hsp70 with client proteins. Knockout of these genes in Arabidopsis thaliana caused a decrease in photosynthetic efficiency, destabilization of PSII complexes and loss of control for balancing the redox reactions in chloroplasts (Chen et al., 2010). Also, FTIP 1/3 (FLOWERING LOCUS T-INTERACTING PROTEIN 1/3) was upregulated in tea plant under the long term cold stress. This gene is an essential regulator of FT encoding florigen and the regulator of photoperiodic control of flowering in plants. Loss of function of FTIP1 exhibits late flowering under long days, which is partly due to the compromised FT movement to the shoot apex (Liu et al., 2012). To summarise our results on tea RNAseq data, it can be suggested that the long term overlapping stress responses include the activation of several important genes of photo-perception which probably activate the phenylpropanoid pathway leading to the cell wall remodeling.

The cold signals perceived by plant cell-surface could be transmitted to the other cell compartments, such as the nucleus, where the expressions of many cold-responsive genes are activated (Zhu, 2016). An example of such mechanisms include Calcium (Ca²⁺) and IP₃ (inositol-1, 4, 5-triphosphate), which are two ubiquitous secondary messengers and play a crucial role in eliciting downstream cold-responsive signaling pathways (Knight et al., 1996; Virdi et al., 2015; Paknejad and Hite, 2018). Cold stress causes an activation of the IP₃-gated calcium channels, which then resulted in rapid induction of cytosolic calcium levels (Ca²⁺ spark) and subsequent upregulation of COR genes (Orvar et al., 2000; Sangwan et al., 2001). After sensing and interacting with Ca²⁺, calmodulin (CaM) undergoes conformational changes to activate CaM-binding transcription activator (CAMTA) factors to response to cold stress. The double mutations of camta1 and camta3 impaired cold tolerance of plants compared to wild type, suggesting their significant roles in the cold response. Many studies have also shown that the expression of CAMTA3/5 is significantly upregulated under cold stress, in which CAMTA3 further binds the CBF2 promoter to activate the expression of DREB1B and DREB1C genes. This establishes a link between calcium signals and cold acclimation in plants (Doherty et al., 2009; Eckardt, 2009; Kidokoro et al., 2017). Additionally, cold stress can stimulate the Ca²⁺ sensors calcineurin B-like protein (CBL), to interact with CBL-interacting serine/threonine-protein kinases (CIPKs), and ultimately increase the autophosphorylation and phosphorylation activity of CIPKs. The activation of CIPKs eventually resulted in an upregulation of CBFs genes to respond the cold stress in plants (Zhang et al., 2019). Overall, the above evidences indicated that Ca²⁺-CaM/CBL-CAMTA3/CIPK complex enables sensing and transduction of cold signaling of plants in response to cold stress through CBF-dependent pathways, despite their functions in tea plants need further investigations.

Under the long-term cold stress, we also observed the elevated expression of several genes, related to Ca²⁺-dependent signaling and protein phosphorylation in tea leaves. Among them, CNX1 (Calnexin) and calreticulin-like (CRT) which were reported to bind proteins on endoplasmic reticulum acting as molecular chaperones (Liu et al., 2017; Joshi et al., 2019); also, CIPK12 and CIPK6 which were reported to bind CBLs regulating Ca²⁺-signal response (Sardar et al., 2017; Czolpinska and Rurek, 2018; Bai et al., 2022). Additionally, several genes encoding the important components of membrane trafficking system and related to Ca²⁺-signaling were upregulated in tea plant under the long term cold stress, such as STRAP (SERINE-THREONINE KINASE RECEPTOR-ASSOCIATED PROTEIN), SAPK3 (SERINE/THREONINE-PROTEIN KINASE 3), leucine-rich repeat receptor-like protein kinase PXY1 (PHLOEM INTERCALATED WITH XYLEM-LIKE 1), INPP5A2 type I inositol polyphosphate 5-phosphatase 2, glycine-rich protein A3-like GRP (GLUTAMINE-RICH PROTEIN), indicating their important roles in response to long-term cold stress in tea plant. Similarly, in Populus, the calcium-dependent protein kinase 10 (CPK10) is upregulated under drought and frost and activates both drought- and frost-responsive genes to induce stress tolerance (Chen et al., 2013). In apple DEGs encoding protein phosphatases and serine/threonine-protein kinases were upregulated in response to different abiotic stresses (Li et al., 2019).

In addition, the protein phosphatase 2C (PP2C) was upregulated in tea plant under the long-term cold stress. PP2C are the key players in plant signal transduction processes, acting as the central components in ABA signal transduction and negative regulators of mitogen-activated protein kinase (MAPK) pathway (Rodriguez, 1998). Also, probable translation initiation factor eIF-2B (tif224) is increasingly expressed under 14-day-chilling and 3-day freezing stress in tea plant. This gene encodes a protein which is activated through phosphorylation by stress-sensing kinases, and leads to reduced levels of ternary complex required for initiation of mRNA translation under stress conditions (Wang et al., 2021). Our results confirmed that activation of Ca²⁺-signaling cascades is relevant to not only the short-term cold response but also for the long-term chilling and freezing-responses in tea plant.

Unlike Ca²⁺ and IP₃, the nitric oxide (NO), a gaseous signaling molecule in plants has gained much attention for its roles in cold tolerance. Cold acclimation induced a high expression level of nitratereductase 1 (NIA1) and stimulated the nitrate reductase (NR) activity, which was attributed to NR-dependent NO synthesis and eventually resulted in freezing response of plants (Zhao et al., 2009). Previous studies have suggested that NO could greatly induce the expression level of the S-adenosylmethionine synthetase (MfSAMS) gene in leaves of Medicago sativa subsp. falcata. Overexpression of MfSAMS in plant significantly improved cold tolerance of transgenic plants via up-regulating polyamine synthesis and oxidation (Guo et al., 2014). In vitro application of 0.02 mM NO could dramatically reduce the chilling injury index in tomato fruit by up-regulating the expression of LeCBF1, whereas NO inhibitors cause severe chilling injury (Zhao et al., 2011). Similarly, supplementing 500 uM NO in vitro caused the tea plants to significantly accumulate osmoregulation substances (e.g., soluble protein, soluble sugar, and proline) and activate superoxide dismutase and catalase. The expression levels of CsICE1 and CsCBF1 genes were up-regulated by exogenous NO, thereby alleviating the damage of cold to tea leaves under cold stress (Pan et al., 2016; Wang et al., 2021). However, it is unknown whether NO regulates CBF-dependent or -independent pathways in response to cold stress in tea plants, which needs further investigation.

Many plants have evolved sophisticated cold response mechanisms to survive in cold stress during long-term evolution (Thomashow, 1999; Kalberer et al., 2006). It is commonly acknowledged that the ICE1-CBF-COR transcriptional cascade is one of the key cold signaling pathways, which is highly conserved in tea and other flowering plants. Plant genomes contained two copies of the ICE gene. The ICE1 protein was found particularly abundant in the MYC-binding sites (CANNTG) of the CBF promoter (Chinnusamy et al., 2003). A handful of studies have shown that ice1 mutation blocked the expression of the CBF3 gene, whereas overexpression of ICE1 significantly increased the expression of CBF3 in transgenic plants (Tang et al., 2020). Besides, the ICE2 is considered a redundant duplicate of ICE1, which performs similar activities in plants in terms of cold responsiveness (Fursova et al., 2009; Kim et al., 2015). In plants, ICE typically regulates a large number of downstream genes in response to cold stress, of which CBFs serve as one of the most important targets (Vogel et al., 2005; Wang et al., 2012). CBFs act as the on/off switches of cold response. Their expression levels could be rapidly induced within 15 minutes under cold treatments, affecting the expression of over 4000 putative downstream target genes such as COR15a, COR47, and COR6.6 (Gilmour et al., 1998; Seki et al., 2001; Maruyama et al., 2004; Maruyama et al., 2009; Park et al., 2015; Zhao et al., 2016; Shi et al., 2018). In Arabidopsis, the CBF gene family is composed of three tandem genes located on chromosome IV and exhibits consistent expression patterns in response to cold stress. CBF1,3-overexpressed Arabidopsis plant had increased freezing tolerance, while cbfs mutants were vulnerable to freezing stress (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Medina et al., 1999). Unlike Arabidopsis, five CsCBF members have been identified in tea plants (Wang et al., 2019). In another investigation, six CsCBF genes were predicted (Hu et al., 2020). Interestingly, all the CsCBFs were strongly upregulated under cold stress, with the exception of CsCBF3 (TEA010806). Overexpression of CsCBF1 (GenBank EU563238), CsCBF2 (KC702795), CsCBF3 (EU857638), and CsCBF5 (TPIA CSS001387) in Arabidopsis and Nicotiana displayed an enhanced cold tolerance, with increased photosynthesis ability, high level of proline, sugar and ROS content, but reduced malondialdehyde under cold stress compared to wild type (Chang et al., 2012; Yin et al., 2016; Zhou et al., 2022a; Zhang et al., 2022b). CBF proteins can recognize C-repeat/dehydration- responsive motif (CCGAC, CRT/DRE) in the promoters of a subset of COR genes and activate the expression of COR genes. It was predicted that a total of 685 potential COR genes were regulated by CsCBF in tea plants, including circadian rhythms and hormone signaling genes (Wang et al., 2019). Although the overexpression of COR15A and CsCOR1 has no discernible effect on the survival of plants under cold stress, most COR genes greatly contribute to cold tolerance in plants (Jaglo-Ottosen et al., 1998; Li et al., 2010b). For example, the expression of RD29A was induced by CBF3, thereby improving the survival of frozen plants (Liu et al., 1998). Further, CBF1 occupies the clock genes LUX promoter. LUX is required for plants to survive in freezing stress (Chow et al., 2014). Interestingly, many previous studies also suggested that alternative splicing event is likely to drive the regulation complexity of CsCOR during cold acclimation (Li et al., 2020). It is possible that the alternative splicing of CsCOR plays an important role in cold acclimation of tea plants.

It is well recognized that both transcriptional regulation and post-translational modifications play important role in regulating the CBF cascade pathway. According to recent studies, the expression of CBFs and the stability and transcriptional activity of ICE1 are very important for cold tolerance. The ICE1/2 are constitutively expressed (Tang et al., 2020). Previous studies have suggested that the phosphorylation, ubiquitination and sumoylation of ICE1 greatly regulates CBF expression by changing its own protein stability and transcriptional activity ( Figure 2 , Shi et al., 2018; Ding et al., 2020). Indeed, ICE1 is ubiquitinated and degraded by the high expression of osmotically responsive gene 1 (HOS1, E3 ubiquitin ligase), leading to the instability of ICE1 protein and low expression of CBF (Dong et al., 2006; Park et al., 2011). Whereas cold-activated SUMO E3 ligase SIZ1 (SAP and Miz) -mediated sumoylation of ICE1 increases its stability, positively regulating the cold tolerance in plants (Miura et al., 2007). In addition, three protein kinases also mediated the post-translational modification of ICE1. Low temperature induces the open stomata 1 (OST1) kinase activity, which then interacts with ICE1 and HOS1, improving the ICE1 activity and suppressing HOS1-mediated ICE1 degradation (Ding et al., 2018). By contrast, the other two protein kinases Brassinosteroid-insensitive 2 (BIN2) and mitogen-activated protein kinase 3/6 (MPK3/6) interacts with, and phosphorylate ICE1, which promoted the degradation of ICE1 (Li et al., 2017a; Ye et al., 2019). Moreover, MPK6 attenuated the inhibitory effect of MYB15 on CBF expression to enhance freezing tolerance in Arabidopsis (Agarwal et al., 2006; Kim et al., 2017). Mechanistically, jasmonate-zim-domain protein 1/4 (JAZ1/4) can also inhibit the CBF translational activity by interacting with ICE1/2 in Arabidopsis (Hu et al., 2013a). The most recent study in tea plants found that CsWRKYs (CsWRKY29 and CsWRKY37) conferred plants cold tolerance, and CsWRKY4/CsOCP3 (OVEREXPRESSOR OF CATIONIC PEROXIDASE 3) interacted with CsICE1 and inhibited its transcriptional activation on CsCBF1/3, demonstrating the relevance of CsCBF cascade pathway on cold tolerance of tea plants (Peng et al., 2022; Zhao et al., 2022).

The expression of CBFs is regulated by several types of transcriptional activators or repressors involved in light signaling, phytohormones signaling, circadian rhythms and Ca²⁺ signaling ( Figure 2 ). Recent studies have shown that the PIFs (PIF3, 4, and 7), downstream genes of photoreceptor and thermosensor phyB, negatively regulates the expression of CBF and freezing tolerance of Arabidopsis (Leivar et al., 2008; Lee and Thomashow, 2012). Chilling stress initiates the formation of CBFs-PIF3-phyB complex which later serve to control the cold adaption (Jiang et al., 2020; Xu and Deng, 2020). The transcription factors (TFs) in hormone signaling maintain the homeostasis of CBF levels. For instance, the CBFs expression are repressed by ethylene insensitive 3 (EIN3) in ethylene pathway, but up-regulated by brassinazole-resistant 1/brassinosteroid insensitive 1-EMS-supressor 1 (BZR1/BES1) in brassinosteroids signaling (Shi et al., 2012; Li et al., 2017b). Circadian rhythms core genes are likely to antagonistically function to keep the rhythmic expression of CBF. Circadian clock associated 1/late elongated hypocotyl (CCA1/LHY) are shown to activate the expression of CBFs by binding to their promoters, while pseudo-response regulators (PRRs) inhibit the expression of CBFs (Nakamichi et al., 2009; Dong et al., 2011). In Arabidopsis, CCA1/LHY regulates cold-responsive DREB1 expression only under gradual decrease in temperature during the day, whereas rapid drop in the temperature can induce the cytosolic calcium levels and activate Ca²⁺ signaling (Kidokoro et al., 2017). Ca²⁺ signaling impairment prevents CsCBF expression but accumulates higher catechins under cold conditions, suggesting their potential correlations in response to cold stress of tea plants (Ding et al., 2019). The CsCAMTA2 (orthologous gene of CAMTA3 in Arabidopsis) was strongly up-regulated in tea plant, and the cis-element [(G/A/C)CGCG(C/G/T) or (A/C)CGTGT, CsCAMTA targeted] was observed in the promoter of CsCBF1 and CsCBF2 (Zhou et al., 2022b). There had been at least 8 Calmodulin-like (CBL) genes and 25 CIPK genes identified in tea plants, which were further divided into four and five subfamilies. Of them, four CsCBLs (CsCBL1/3/5/9) and nineteen CsCIPKs genes were significantly induced by cold stress. Studies have shown that CsCBL1 could interact with CsCIPK1/10b/12, while CsCBL9 was found to interact with CsCIPK1/10b/12/14b; hence, the Ca²⁺-CsCBL-CsCIPK module mediated cold stress signaling in tea plant was proposed (Li et al., 2019b; Ma et al., 2019; Wang et al., 2020). In Arabidopsis, it was reported that CAMTAs worked together to suppress the SA (salicylic acid) synthesis by targeting EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and to improve freezing tolerance (Kim et al., 2013).

Furthermore, the post-translational modification of CBFs is important in cold tolerance. For instance, cytosolic redox protein thioredoxin h2 interacts with CBF and reduces the transformation of oxidized CBF oligomers (inactive) to active monomers, whereby this structural switching and functional activation of CBFs confers the plant with cold tolerance (Lee et al., 2021). It is worth to note that epigenetic regulation, including DNA methylation, chromatin remodeling, and small RNA regulation, also extensively influences the cold tolerance of plants throughout the entire life (Park et al., 2018). A recent study showed that hundreds of cold-responsive genes, including CsCBF4 and CsUGT91Q2, were significantly demethylated during cold stress, indicating that DNA methylation is involved in cold response of tea plants (Tong et al., 2021). Besides, the histone deacetylases were also reported to participate in the cold stress response of tea plants. Low temperature reduced the transcription of HD2 type histone deacetylase in tea plant, indicating that chromatin remodeling mediated by histone modifications may regulate the expression of cold-responsive genes (Ma et al., 2013; Yuan et al., 2020). Degradome sequencing has identified 763 related cleavage target genes and miRNAs associated with cold stress tolerance. There were 74 and 91 differentially expressed microRNAs (miRNAs) identified from cold-tolerant ‘Yingshuang’ and cold-sensitive ‘Baiye 1’ cultivars, respectively. Of them, miR156, miR159, and miR396 showed distinct expression patterns among different cold-sensitive tea varieties under cold conditions (Zhang et al., 2014b). In addition, 14 circular RNAs have been identified to contribute to the chilling tolerance of tea plant (Huang et al., 2023).

The innovation of genomic and transcriptomic sequencing, together with functional genomics, have identified a total of 128 gene/families involved in cold response in tea plants ( Table 1 ). For example, the cold-responsive bZIP transcription factor CsbZIP6 and CsbZIP18 were experimentally evidenced to reduce the freezing tolerance of tea plants by ABA-independent and ABA-dependent pathway, respectively (Wang et al., 2017; Yao et al., 2020b). In addition, a total of 89 structural genes involving in sugar signaling, redox process, ascorbic acid metabolism, hormone signaling, carotenoid biosynthesis, terpenoid metabolism, Ca²⁺ signaling, osmoregulator, amino acid metabolism, fiber signaling, and light signaling, were also identified and characterized to be associated with cold tolerance of tea plants. Correspondingly, 59 sugar-related genes engaged in sugar metabolism, transportation and signaling are solidly stimulated, including the beta-amylase gene (CsBAM), disproportionating enzyme gene (CsDPE2), fructokinase gene (CsFRK), invertase gene (CsINV5), Suc-phosphate synthase gene (CsSPS) and raffinose synthase gene (CsRS2) (Yue et al., 2015). Sugar signaling and osmoregulator related genes regulate the cold tolerance of tea plants mainly through the osmotic-dependent pathway. Overexpression of Invertase 5 (CsINV5) enhanced the cold tolerance of transgenic Arabidopsis through up-regulating the transcription of HXK2 and P5CS1/2 (Qian et al., 2018). CsSWEET1a, CsSWEET16 and CsSWEET17 improved the freezing resistance of plants by promoting sugar transport across the plasma membrane (Wang et al., 2018; Yao et al., 2020a). The sequences of proline biosynthesis and degradation have been identified and available at NCBI, CsP5CS (pyrroline-5-carboxylate synthase, KJ143742.1), CsOAT (Ornithine-D-aminotransferase, KJ641844.1) and CsP5CR (pyrroline-5-carboxylate reductase, KY368574), CsP5CDH (pyrroline-5-carboxylate dehydrogenase, KY368572) and CsProDH (Pro-dehydrogenase, KY368573) included (Ban et al., 2017).

Besides, the phenylpropanoid pathway serves as a rich source of metabolites in plants, as a starting point for the biosynthesis of lignin, flavonoids and coumarins (Fraser and Chapple, 2011; Hori et al., 2020; Oliveira et al., 2020). Recent studies showed upregulation of lignin biosynthesis genes along with downregulation in cellulose biosynthesis genes under osmotic stresses in tree species (Wildhagen et al., 2018; Chen et al., 2019; Hori et al., 2020). Additionally, an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expanding proteins, affecting the cell wall plasticity and reinforcement of the secondary wall with hemicellulose and lignin deposition to increase cell wall thickening were highlighted (Gall et al., 2015). In accordance with these data, our RNAseq data revealed many upregulated genes related to the cell wall remodeling and biosynthesis in tea plant (UDP-Arap, XTH30, AGPS1, BGLU, ENODL2, AXY4, UEL-1, PRP-F1, API, PPME, GALT6, GATL7, UXS2, UXS4, TBL32, GlcAT14A, XUT1, GAE3, 4CL, API, RRT1, rfbC, glucan endo-1,3-beta-glucosidase 7-like and 8-like, etc.) confirming the importance of this pathways in the long-term cold stress in tea plant. Additionally, elevated expression of beta-glucosidase (BGLU) that catalyzes intermediates for cell wall lignification synthesis was observed in tea. Also, several DEGs (RRT1, PPME, XTH, UXS2, UXS4, GAE3, XUT1) related to xyloglucan and pectin biosynthesis were upregulated in tea leaves under the long-term cold stress. Among them, RRT1 (RG-I RHAMNOSYLTRANSFERASE 1) is required for both cellular adhesion and cell wall plasticity (Takenaka et al., 2018). PPME, pectinesterase-like PMEs (PECTIN METHYLESTERASE INHIBITORs) maintains apoplastic Ca²⁺-homeostasis, controlling stomatal movements and in regulating the flexibility of the guard cell wall (Wu et al., 2018). Previous studies have also suggested that inhibiting the pectin methylesterase activity of tea plants, including Pectin Methylesterase Inhibitor 2 and 4 (CsPMEI2 and CsPMEI4), slightly reduces the cold tolerance of transgenic Arabidopsis (Li et al., 2021). XTHs (Xyloglucan endotransglucosylase/hydrolase) cuts and re-joins hemicellulose chains in Plant cell wall, contributing to wall assembly, affecting cellulose deposition (Wu et al., 2018). Additionally, more genes related to pectin biosynthesis were found upregulated under long-term cold stress in tea plant, namely UXS2/4 (UDP-GLUCURONIC ACID DECARBOXYLASE 2/4) and GAE3 (UDP-D-GLUCURONATE 4-EPIMERASE 3). These genes are required for the biosynthesis of heteroxylans and xyloglucans and for the side chains of pectin (Kuang et al., 2016; Borg et al., 2021).

Cell walls remodeling proteins contain hydroxyproline-rich O-glycoproteins (HRGPs), which is classified into extensins (EXTs), arabinogalactan-proteins (AGPs) and Hyp/Pro-rich proteins (H/PRPs) (Cassab and Varner, 1988; Basu et al., 2015; Ajayi et al., 2021). According to our results, a set of genes involved in H/PRPs and AGPs metabolism (AGPS1, UEL-1, API, GALT6, GATL7, GlcAT14A, ENODL2, PRP-F1, etc.) were highly upregulated in tea plant suggesting that glycosylation of HRGPs is an important responsive mechanism under the long-term stress. Additionally, some genes (e.g., TBL27/32) related to O-acetylation of polysaccharides were upregulated under long-term cold in tea plant which is consistent with some earlier findings (Sun et al., 2020). O-Acetylation of polysaccharides change the physicochemical properties and acetyl-substituents inhibit the enzymatic degradation of wall polymers (Gall et al., 2015) suggesting the important role of the both processes for the long-term stress responses of tea plant. Thus, the increasing the cell wall plasticity, thickness and hydrophobicity by lignin biosynthesis, glycosylation of HRGPs, o-acetylation of polysaccharides, pectin biosynthesis and branching, xyloglucan and arabinogalactan biosynthesis can serve as important mechanisms of long-term cold responses in tea plant.

A well-known effect of abiotic stress in plants is the production of ROS, which can eventually oxidize lipids, proteins, and DNA, and thereby trigger the cell death (Akula and Ravishankar, 2011; Bartwal et al., 2013; Estravis-Barcala et al., 2020). Redox process and ABA metabolism regulated cold tolerance mainly through scavenging reactive oxygen species. For example, Glycosyltransferase CsUGT91Q2, CsUGT78A14, and CsUGT71A59 confer cold resistance to tea plant by improving the ROS clearance ability (Zhao et al., 2019a; Zhao et al., 2019b; Zhao et al., 2021). According to our recent results, in tea plant several upregulated DEGs related to lipid metabolism were upregulated under long term cold stress. For examples, the homologs of SEC14, an important regulators of phospholipid metabolism (Campos and Schaaf, 2017), EDR2, a negative regulator of cell death (Vorwerk et al., 2007), and genes encoding remorin-like (REMs) proteins accumulated in lipid rafts and physically interact with receptor-like kinases (Cai et al., 2020), probable phospholipid hydroperoxide glutathione peroxidase (PHGPX) participates in scavenging of lipid hydroperoxide (Jain and Bhatla, 2014), endoplasmic reticulum oxidoreductin-1-like (ERO1) participating in protein folding under oxidative stress (Matsusaki et al., 2019), probable carboxylesterase 11 (CXE11) which is involved in the catabolism of volatile esters such as butyl and hexyl acetate and activation of MeJA signaling (Cao et al., 2019), luminal-binding protein genes (BIP5-like) which increase in anti-oxidative defenses under water stress in transgenic tabacco and soybean (Valente et al., 2009). These results suggest the lipid stabilization against ROS can be an important mechanism of the long-term cold and freezing responses in tea plant.

Similarly, cold-induced enzyme or hormone pathway genes also affect the cold tolerance of tea plants. According to the recent studies, hormone-signaling pathways are consistently up-regulated under cold stress, which are involving in those of JA, brassinosteroids (BRs), and ABA (Wisniewski et al., 2014; Zheng et al., 2022). Interestingly, auxin signal transduction is activated in the opposite pattern with ethylene transduction it some tree species (Estravis-Barcala et al., 2020). In tea plant several new upregulated DEGs involved in hormone signaling were upregulated under the long-term cold stress (GID1C-like, LOG3-like, ILR1-like6, TTL1, TTL3, and 2g29380). These genes are related to the abovementioned signaling pathways. For example, GID1 (GA INSENSITIVE DWARF1) can bind negative regulators of GA responses called DELLA proteins (Hauvermale et al., 2014). LOG is a cytokinin-activating enzyme plays a pivotal role in regulating cytokinin activity (Kuroha et al., 2009). ILR1 (IAA-LEUCINE RESISTANT 1) regulates the rates of amido-IAA hydrolysis resulting in activation of auxin signaling (Sanchez Carranza et al., 2016). TTL1 (TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 1) regulates the transcript levels of several dehydration-responsive genes, such as CBF2, ERD1 (early response to dehydration 1), ERD3, and COR15a (Rosado et al., 2006; Lakhssassi et al., 2012). These results indicate a complex transcriptional landscape in response to abiotic stress, and in particular they show highly variable interactions between different hormone signal transduction pathways.

Long-term cold stress down-regulated CsLOX expression while short period of low temperatures induced the expression of CsLOX1, 6 and 7, which highlights the role of JA in triggering and regulating cold tolerance of tea plants (Zhu et al., 2018). E3 ligase gene MIEL1 inhibited the accumulation of anthocyanin in apple by degrading MdMYB1 protein (An et al., 2017). Similar to the function of MdMIEL1, overexpression of the CsIEL1 gene in Arabidopsis decreased anthocyanin level during cold stress, which is possibly caused by the degradation of positive regulator through 26S-proteasome-mediated ubiquitination pathway (Xing et al., 2021).

In addition, transcriptome analysis shows only 12% of cold-responsive genes are dependent on the CBF regulons in Arabidopsis thaliana, indicating the presence of the other low-temperature regulation pathways (Fowler and Thomashow, 2002). Indeed, several previous studies have showed that many cold-inducible genes, including Alpha-tubulin (CaTUA), dehydrin (CsDHN1, 2), spermine synthase (CsSPMS), fatty acid desaturase (CsSAD), H1 histone (CsHis), CsbZIP and CsHSF function in cold response of tea plants in a CBF-independent pathway (Paul et al., 2012; Paul and Kumar, 2013; Wang et al., 2014; Zhu et al., 2015; Ding et al., 2016). Many phytohormone (auxin, cytokinins, ABA, gibberellins, JA, ethylene and brassinosteroids) responsive genes are intimately linked to the CBF-independent regulon under cold acclimation (Zhao et al., 2014; Joshi et al., 2016; Wani et al., 2016). With the development of transcriptomics and genomics, many CBF-independent transcriptional regulation factors involved in cold adaptions would be identified.

At present, the primary method of reducing cold stress in tea production is to breed cold-resistant tea plants and optimize cultivation conditions, most likely in a greenhouse or with a protective film. Applying exogenous substances, on the other hand, are the simplest, most convenient, and most effective method (Zhang et al., 2022b). In general, 93 metabolites changed significantly under cold stress, such as catechin, flavonoid, ABA and JA (Hao et al., 2018). These results corresponded to those in which low temperature increased accumulation of flavan-3-ols and proanthocyanidins, indicating that phytohormones and secondary metabolites may contribute to cold regulation in tea plants (Zhang et al., 2014a). Indeed, treatment with plant growth regulators spermidine (0.025 mM, 0.05 mM, 0.1 mM) alleviates damages caused by cold stress in pollen tubes of tea varieties (Cetinbas-Genc et al., 2020). Exogenous application of ABA not only induces CsCOR1 expression but also rapidly close stomata to reduce water loss, thereby ABA effectively alleviates chilling damage to plant, consistent with the changes in proline content (Li et al., 2010a; Hong et al., 2017). As efficient elicitor, exogenous methyl jasmonate application induces the expression of CsMYBs, and thus effectively promotes ROS scavenging and anthocyanin biosynthesis to alleviate cold stress damage (Han et al., 2022). Melatonin treatment alleviates cold stress on tea plant by improving biosynthesis antioxidant enzyme and antioxidant defense and redox homeostasis (Li et al., 2018a; Li et al., 2018b; Li et al., 2019a). Additionally, supplementation of γ-aminobutyric acid also contributes to the improvement of cold tolerance of tea plants, as exogenous application of CaCl_2, which has the same effect on cold tolerance. (Huang et al., 2015; Zhu et al., 2019). Airborne nerolidol and nerolidol glucoside exposure were also found to enhance cold stress tolerance of the tea plant through accumulating CsCBF1 and CsUGT91Q2 expression (Zhao et al., 2019b). However, the mechanism by which exogenous substances orchestrate cold tolerance has yet to be determined.