Convergence and Divergence: Signal Perception and Transduction Mechanisms of Cold Stress in Arabidopsis and Rice

Cold stress, including freezing stress and chilling stress, is one of the major environmental factors that limit the growth and productivity of plants. As a temperate dicot model plant species, Arabidopsis develops a capability to freezing tolerance through cold acclimation. The past decades have witnessed a deep understanding of mechanisms underlying cold stress signal perception, transduction, and freezing tolerance in Arabidopsis. In contrast, a monocot cereal model plant species derived from tropical and subtropical origins, rice, is very sensitive to chilling stress and has evolved a different mechanism for chilling stress signaling and response. In this review, the authors summarized the recent progress in our understanding of cold stress response mechanisms, highlighted the convergent and divergent mechanisms between Arabidopsis and rice plasma membrane cold stress perceptions, calcium signaling, phospholipid signaling, MAPK cascade signaling, ROS signaling, and ICE-CBF regulatory network, as well as light-regulated signal transduction system. Genetic engineering approaches of developing freezing tolerant Arabidopsis and chilling tolerant rice were also reviewed. Finally, the future perspective of cold stress signaling and tolerance in rice was proposed.


Introduction
Cold stress limits the geographical distribution, growth habits, and productivity of plants [1]. There are two types of cold stress, including chilling stress (0 to 15 • C) and freezing stress (below 0 • C). Many temperate plants, such as Arabidopsis, rapeseed, wheat, and rye, have acquired a complex network that senses and responds to freezing stress. Molecular mechanisms and regulation networks of plasma membrane cold perception, signaling and freezing stress tolerance of these plants have been well-reviewed [2][3][4][5][6][7][8][9]. On the other hand, rice (Oryza sativa L.), one of the most important cereal crops, is susceptible to chilling stress, especially in high elevation and high latitude temperate zones [10]. Many novel regulatory genes have been identified in chilling stress perception and signaling in rice in the past decade [11][12][13]. This allows us to compare and distinguish the similarities and differences of cold stress sensing and signaling mechanisms between Arabidopsis and rice [8,9].
Plants respond to the decreased temperatures through sophisticated processes at different levels. Initially, plant cells perceive cold stress signals through the plasma membrane (PM) rigidification, PM-bound G-protein associated receptors, or cold sensors, such as Ca 2+ influx channels, two-component histidine kinases, protein and nucleic acid conformations, or metabolite concentrations [6,14]. Then, the second messengers, including calcium ion, reactive oxygen species (ROS), and inositol phosphates, are generated. These second messengers further modulate the intracellular calcium (Ca 2+ ) level. Perturbation in cytosolic Figure 1. Plasma membrane-localized proteins perceive the cold stress signals in Arabidopsis and rice. (a) In Arabidopsis, freezing stress initiates the PM rigidification, PM-associated calcium channels MCA1/2, calcium sensor SYT1 and kinases including CRLK1, AtHK1/2/3, and CRPK1, as well as PM-localized PXL1, participate in primary cold stress sensing and perception. (b) In rice, chilling stress initiates the PM rigidification, many PM-associated proteins, including calcium channels ACA6, CNGC14/16, phospholipidase PLDα1, aquaporin proteins PIP2;1/PIP2;6, G-protein-associated cold sensor COLD1 and kinases GT4a and CPK17, participate in primary cold stress sensing and perception. However, a specific calcium channel for calcium influx is still not known.

Cold Stress Signal Transduction Mechanisms in Arabidopsis and Rice
Following the cold stress perceptions, cold stress signal transduction events occur the cytosol and nucleus of plant cells. The second messengers, such as Ca 2+ and reactiv oxygen species (ROS), transmit the external cold signals to intracellular signaling system Figure 1. Plasma membrane-localized proteins perceive the cold stress signals in Arabidopsis and rice. (a) In Arabidopsis, freezing stress initiates the PM rigidification, PM-associated calcium channels MCA1/2, calcium sensor SYT1 and kinases including CRLK1, AtHK1/2/3, and CRPK1, as well as PM-localized PXL1, participate in primary cold stress sensing and perception. (b) In rice, chilling stress initiates the PM rigidification, many PM-associated proteins, including calcium channels ACA6, CNGC14/16, phospholipidase PLDα1, aquaporin proteins PIP2;1/PIP2;6, G-protein-associated cold sensor COLD1 and kinases GT4a and CPK17, participate in primary cold stress sensing and perception. However, a specific calcium channel for calcium influx is still not known.
In rice, there are several identified PM-localized kinases involved in cold stress perception. OsACA6, a PM Ca 2+ -ATPase, interacts with CaM-binding protein OsCaMBP1, calcium-dependent protein kinase (CDPK)-related kinase OsCRK2, and receptor-like kinase (RLK) OsRLK2 [33]. PM-localized OsCPK17 interacts with and phosphorylates the sucrosephosphate synthase OsSPS4 and aquaporin OsPIP2;1/OsPIP2;6, can enhance the cold stress tolerance in rice [34] (Figure 1b). CTB4a, a conserved leucine-rich repeat receptor-like kinase, interacts with a beta subunit of adenosine triphosphate (ATP) synthase AtpB and improves the yield under cold stress [35]. Interestingly, the protein level of phospholipase Dα1 (OsPLDα1) increases at one minute after cold treatment. It activates OsMPK6 and OsSIZ1, followed by the regulations of OsDREB1s expression in cold signaling [36] (Figure 1b). Therefore, there is much convergence of primary PM-located protein kinases in cold stress perceptions between Arabidopsis and rice.
Progress has been made in calcium signaling, phospholipid signaling, MAPK cascade sig naling, and ROS signaling in the past decades. Here, we compare the recent advances i signal transduction pathways of freezing stress in Arabidopsis and chilling stress in ric (Figures 2 and 3), highlighting the divergence and convergence in cold stress in both plan species.

Figure 2.
Putative model of cold stress signaling networks toward freezing stress tolerance in Arabidopsis. The cold-induced calcium signature in the cytosol is recognized by the calcium sensor proteins, including CaM, CDPK, CBL1/CIPK7, and CAMAT3, as well as the bZIP transcription factor SGB1 pathway. In addition, CRLK1-MEK1-MKK1/2-MPK4/6 cascade, ROS signaling, and phospholipid signaling work together to regulate cold stress signaling, and many ICE1-DREB transcription activators and repressors have been identified to regulate the COR gene expressions, finally leading to freezing tolerance in Arabidopsis.

Figure 2.
Putative model of cold stress signaling networks toward freezing stress tolerance in Arabidopsis. The coldinduced calcium signature in the cytosol is recognized by the calcium sensor proteins, including CaM, CDPK, CBL1/CIPK7, and CAMAT3, as well as the bZIP transcription factor SGB1 pathway. In addition, CRLK1-MEK1-MKK1/2-MPK4/6 cascade, ROS signaling, and phospholipid signaling work together to regulate cold stress signaling, and many ICE1-DREB transcription activators and repressors have been identified to regulate the COR gene expressions, finally leading to freezing tolerance in Arabidopsis.

Calcium Signaling
Calcium influx into the cytosol is an early event in cold stress [6,17]. This transien elevation in calcium concentration is also called intracellular calcium signature. Calcium influx is primarily sensed by the calcium sensor proteins, containing the helix-loop-helix domain with the EF-hand motif. In plants, calcium sensors include four major classes CaM/CaM-like protein (CML), calcium-dependent protein kinase (CDPK or CPK), cal cineurin B-like (CBL) protein, and CBL-interacting protein kinase (CIPK). In addition, a small annexin family has been identified as a calcium sensor to cold stress response in Arabidopsis.
In  There are at least four chilling stress signaling pathways in rice. MYB4-ROS-bZIP cascade is involved in the ROS signaling process. MKK6-MP3 cascade and phospholipid signaling work with calcium signaling concomitantly in chilling stress signaling in rice. ICE1-DREB transcriptional regulatory cascade is conserved in the Arabidopsis and rice. Furthermore, these pathways' upstream and downstream signal transducer proteins play cooperative and regulatory roles in cold stress tolerance in rice.

Calcium Signaling
Calcium influx into the cytosol is an early event in cold stress [6,17]. This transient elevation in calcium concentration is also called intracellular calcium signature. Calcium influx is primarily sensed by the calcium sensor proteins, containing the helix-loop-helix domain with the EF-hand motif. In plants, calcium sensors include four major classes: CaM/CaM-like protein (CML), calcium-dependent protein kinase (CDPK or CPK), calcineurin B-like (CBL) protein, and CBL-interacting protein kinase (CIPK). In addition, a small annexin family has been identified as a calcium sensor to cold stress response in Arabidopsis.
In Arabidopsis, overexpression of AtCaM3 hinders the cold induction of RD29A and KIN1, and the AtCaM4 negatively regulates freezing tolerance by interacting with a CaMbinding protein PATL1 [37]. AtCBL1 interacts with AtCIPK7 and binds to the DREB core element of COR promoters to negatively regulate freezing tolerance [38]. CaM-binding transcription activator protein CAMTA3 binds to the conserved CG-1 element in the CBF2 promoter, regulating CBF2 expression in cold stress signaling [39]. A vacuolar Ca 2+ /H + antiporter AtCAX1 enhances the DREB1 transcription in cold acclimation response [40]. Recently, an AtOST1-AtANN1cascade was found to regulate calcium signaling in the CBF1-dependent manner to enhance freezing tolerance in Arabidopsis In rice, a CaM-like protein OsCML16 and its six putative targets have been identified to be involved in cold stress response in rice [42]. However, there is no report about the role of OsCaMs in cold stress signaling. OsCDPK7 enhances cold stress tolerance by the increased accumulation of a putative target gene rab16A [43,44]. OsCDPK13 enhances cold stress tolerance by activating a ubiquitin-like nuclear protein OsCRTintP1, calreticulin interacting protein 1 [45,46]. OsCPK24 interacts with and phosphorylates OsGrx10, a glutathione-dependent thioltransferase, in cold stress response [47]. Overexpression of OsCIPK3, a CBL-interacting protein kinase, improves cold stress tolerance [48]. It is worth mentioning that OsCIPK31 is strongly induced by cold and salt stress and interacts with AtCBL3, suggesting the convergence of CBL/CIPK pathways in cold stress signaling Arabidopsis and rice [49]. As described above, the CaM-associated signaling pathways in cold stress signaling wait for further confirmations in Arabidopsis and rice.

Phospholipid Signaling
An increasing number of studies have shown that the metabolism of the membrane lipids plays an important role in the temperature stress response in plants. In Arabidopsis, in a few seconds after cold exposure, diacylglycerol kinase (DGK) is activated to converse diacylglycerol (DAG) into phosphatidic acid (PA), followed by a change in membrane fluidity [50]. Overexpression of a PM-bound phospholipase gene PLDδ enhances freezing tolerance in rice seedlings [51]. Suppressed expression of PLDα1 results in a significant increase in freezing tolerance [52]. Acyl-coenzyme A: diacylglycerol acyltransferase DGAT1 enhances freezing tolerance via CBF2 regulon and NADPH oxidase RbohD (respiratory burst oxidase homolog D)-dependent H 2 O 2 production in Arabidopsis [53]. The acylcoenzyme A-binding protein (ACBP) family has six members (AtACBP1-6) in Arabidopsis. Overexpression of AtACBP6 enhances freezing tolerance by activating PLDδ to decrease phosphatidylcholine (PC) levels and accumulate PA [54]. Overexpression of AtACBP1 increases freezing sensitivity via the expression of PLDα1 and PLDδ and maintains a membrane-associated PA pool [55]. Further, a temperature-induced lipid pathway has been demonstrated. The FAD2, FAD5 and ACT1 have been identified as the key enzymes in influencing fatty acid flux between the eukaryotic and prokaryotic pathways cold stress response in Arabidopsis [56].
In plants, glycerol-3-phosphate acyltransferase (GPAT) of chloroplasts is a key enzyme to catalyze transferring the acyl group of acyl-(acyl-carrier-protein) (ACP) into the sn-1 position of glycerol 3-phosphate in the first step of glycerolipid biosynthesis in chloroplasts. Ectopic overexpressing of AtGPAT in rice largely induces the unsaturation of fatty acids and chilling tolerance of photosynthesis under low temperature [57]. In rice, the ω-3 fatty acid (FA) desaturase (FAD8) mutant does not acclimate to cold stress [58]. OsPLDα1 increases the levels of PA that bind to OsMPK6 in cold signaling and tolerance [36]. Interestingly, comparative glycerolipidomics analysis of freezing stress (−6 • C and −12 • C) in Arabidopsis and chilling stress (4 • C and 10 • C) in rice has illustrated that Arabidopsis has a higher double bond index (DBI) and lower average acyl chain length (ACL) than rice under cold stress condition [59]. Accordingly, glycerolipid metabolism and signaling show great potentials in applying cold stress tolerance engineering in Arabidopsis and rice.

MAPK Cascade Signaling
In plants, the MAPK cascade consists of three sequentially phosphorylating and activating components, a MAP kinase kinase kinase (MEKK/MAPKKK), a MAP kinase kinase (MEK/MAPKK), and a MAP kinase (MPK/MAPK). MAPKs phosphorylate various downstream substrates, including transcription factors, protein kinases, phospholipases, and cytoskeleton-associated proteins, finally leading to the activation of specific gene expressions under stress conditions [60].
In rice, there is not identified complete MAPK pathway involved in cold stress signaling until now. Our previous study established that the OsMKK6-OsMPK3 cascade modulates chilling signaling and tolerance in rice [66]. OsMPK3 phosphorylates and stabilizes OsICE1, which directly transactivates the expression of OsTPP1, thereby positively regulating chilling tolerance [67]. Moreover, PA binds to OsMPK6 and mediates chilling stress signaling and tolerance [36]. Therefore, these results have demonstrated a divergence in MAPK signaling pathways and regulation network in cold stress response in Arabidopsis and rice.

ROS Signaling
Under cold stress, excess ROS is produced and brings about oxidative damage and cold stress response in plant cells. In Arabidopsis, AtMEKK1-AtMKK2-AtMPK4/AtMPK6 cascade regulates the ROS-scavenging enzymes to maintain redox homeostasis under cold stress [62]. Overexpression of a ROS-regulated C 2 H 2 zinc finger transcription factor AtZAT12 decreases the expressions of AtCBF1/2/3 genes under cold stress [68]. In addition, AtHAP5A, a heme-associated protein, positively modulates the freezing resistance by binding AtXTH21 and inhibits ROS accumulation under freezing stress [69]. Stromal and thylakoid-bound ascorbate peroxidases sAPX and tAPX trigger COR15A, PAL1, and CHS expressions under cold stress [70]. AtTrx-h2 regulates the expressions of COR genes under freezing stress [71].
H 2 O 2 levels are increased within 1.5 h of 10 • C stress in rice seedlings [72]. A subset of 121 early-response genes was upregulated during the initial 24 h of 10 • C stress [73]; Among them, four are transcription factor genes, including ROS-bZIP1 and asl/ocs-like element-containing genes. A hypothetical model of ROS-mediated regulon (ROS-bZIP-as1/ocs) is assembled independent of CBF/DREB-or ABA-mediated regulons in cold stress response [72,73]. Comparative metabolomics analysis of indica (9311) and japonica (Nipponbare) varieties revealed a ROS-dominated dynamic model involved in chilling stress adaptation and tolerance in rice [74]. Overexpression of OsZFP245 enhances cold stress tolerance by regulating proline levels and ROS-scavenging activities in rice seedlings [75]. Overexpressing OsAPX1 prevents the over-accumulation of H 2 O 2 and reduces lipid peroxidation in the spikelet tissues at the booting stage of rice [76]. Natural variation reveals that OsSAP16 controls low-temperature germination in rice [77]. Therefore, there exist specific and different pathways of ROS-mediated cold signaling in rice.

ICE-CBF Transcriptional Cascade
ICE and CBF homologs are highly conserved in Arabidopsis and rice. In Arabidopsis, ICE-CBF transcriptional cascade has been established as the main regulatory response toolkit to cold signaling and freezing tolerance [78]. There are several identified positive and negative regulators, including HK2/3, CAMTA3, OST1, HOS1, and SIZ1/2 for ICE1, ICE1 for CBF3, MYB15 for CBFs, and SRF6 for COR [79][80][81]. ICE1 binds toe CBF3 promoter and induces the CBF3 expression in cold stress signaling [82]. Overexpression of ICE2 induces the expression of CBF1 and enhances freezing tolerance [83]. A RING-finger ubiquitin E3 ligase, HOS1, interacts with ICE1 and targets for polyubiquitylation and proteolysis of ICE1 after 12 h of cold stress [79]. SIZ1 catalyzes sumoylation of ICE1 during cold acclimation and thus reduces the ICE1 polyubiquitylation [80]. OsSFR6 enhances freezing tolerance in Arabidopsis [84]. However, there are no such reports of cold signaling in rice.
In rice, transcript levels of OsICE1 and OsICE2 remain constant during cold stress, indicating that they are posttranslationally modified under cold stress [88]. Ectopic overexpression of OsICE1 and OsICE2 in Arabidopsis imparts freezing tolerance by inducing CBF expressions [89]. OsDREB1A and OsDREB1B transcripts are induced within 40 min after cold stress. Overexpressing OsDREB1A increases chilling stress tolerance in rice [90]. Ectopic overexpression of OsDREB1A and OsDREB1B in Arabidopsis enhances the expressions of stress-inducible genes rd29a, cor15a, and rd17 [91]. In particular, OsMYBS3 represses the transcriptional level of the OsDREB1-dependent cold response pathway in rice [92]. OsROC1 enhances the chilling tolerance by activating OsDREB1 in rice [93]. These results have revealed the convergence in the ICE-CBF cascade and divergence in the regulation of the ICE-CBF cascade in cold stress signaling in both species. As expected, new kinases, phosphatases, and transcriptional regulatory factors upstream and downstream of DREBs will integrate the convergent and divergent networks of cold stress signaling in Arabidopsis and rice.

Light Modulates the Cold Stress Tolerance
Cross talk between light signaling and cold signaling has been elucidated in the model plant Arabidopsis and tomato and cereals species [13]. For example, in Arabidopsis, there are five phytochromes isoforms phyA-E. By using Arabidopsis phys mutants, Franklin and Whitelam confirmed that phyB and D prevent the realization of FR-induced cold tolerance at 16 • C [94,95]. Interestingly, phytochrome A and B function antagonistically to regulate cold stress via abscisic acid (ABA)-dependent jasmonate signaling in tomatoes [96]. Similar results have been obtained in wheat and barley [97]. In barley, the light-regulated signal transduction system was reported to connect the circadian clock, phospholipid signaling pathway, calcium signaling elements, and the downstream HvCBF3/4 genes expressions under cold stress [98].
In rice, phyB negatively regulates chilling tolerance. The phytochrome B-deficient mutant (phyB) shows the more stabilized chloroplast structure and higher unsaturated fatty acid (USFA) content in membrane lipids, thereby alleviating the chilling-induced photoinhibition. Moreover, expressions of genes associated with USFA syntheses such as OsFAD7 and OsFAD8 are higher in the phyB mutant than in the wild type, suggesting that OsFAD7 and OsFAD8 are the downstream genes of phyB [99]. On the other hand, phyB negatively regulates the OsDREB1 expression through interacting with OsPIL16 to reduce the membrane integrity under cold stress [100]. Therefore, phyB probably senses the red light signals and transduces the low-temperature signal together with a series of downstream genes, at least in part through changes in the composition and stability of cell membranes under cold stress. Nevertheless, there are many gaps to bridge between red light signal reception and cold stress response in plants.

Downstream Response Pathways in the Arabidopsis Freezing Stress and Rice Chilling Stress
The development of genetically engineered plants by the repression and overexpression of selected genes seems to be a viable option to hasten the breeding of cold stress tolerance-improved crop plants. Genetic engineering of cold stress tolerance was reviewed in crop and horticultural plants in 2011 [101]. The recent progress in freezing tolerant transgenic Arabidopsis and chilling tolerant transgenic rice has been updated (Tables 1 and 2). In Arabidopsis, engineering of freezing stress tolerance have been obtained through protein kinases and their associated proteins such as AtLCBK1 [102], AtBZR1 [103], AtCRF4 [104], ARR22 [105], and AtTCF1 [106]. Many transcription factors, such as bZIP, ZFP, WRKY, MYB, NAC, GT, DEAR, AP2, AREB, and ERF, have been applied to improve freezing stress tolerance. Furthermore, AtACBP6 [107] and AtLCBK1 [102] have been identified as positive regulators of freezing tolerance. Based on the four signaling pathways above, these engineered genes function downstream of phospholipid signaling, ROS signaling, and CBF regulon under freezing stress response (Table 1). Thus, these regulatory and functional proteins can adjust the balance of dehydration, detoxification, and metabolism under freezing stress in Arabidopsis, increasing the widespread distribution and adaptation to temperature stress in natural habitats. lipase-like regulators Phospholipid Negative [113] In rice, a transgenic approach has been adopted to improve cold stress tolerance. We update the engineering cold stress-tolerant rice since 2011 (Table 2). Based on the functions of these genes in cold stress tolerance in rice, they can be divided into positive regulatory genes and negative regulatory genes. Positive regulatory genes include OsCAF1B [114] [129] and OsMYB2 [130]. In addition, negative regulatory genes include Os-MYB30 [131], OsbZIP52 [132], OsSPX1 [129] and OsWRKY45 [133]. Based on the four signaling pathways above, these engineered genes function downstream of ABA signaling, ROS signaling, and CBF regulon under chilling stress response in rice (Table 2). Therefore, these genes are responsible for ROS homeostasis, proton transport, pectin degradation, and trehalose biosynthesis under cold stress in rice.

Conclusions and Perspectives
This review analyzes the convergence in cold sensors PM-bound calcium channels and receptor-like protein kinases in cold stress sensing in Arabidopsis and rice. Identifying these proteins in calcium signaling and MAPK cascades will be helpful to strengthen the cold-induced perception mechanisms in Arabidopsis and rice. On the other hand, there is convergence in calcium signaling, MAPK cascade signaling, ICE-CBF transcriptional pathways, and divergence in phospholipid signaling and ROS signaling. These signaling pathways have evolved into distinctive and integrative networks in cold stress responses in both species.
In the future, regulon engineering will be a novel strategy by using a master regulatory switch to cause transcriptional changes in the cold response of rice [140]. An efficient engineering approach will be to generate constitutively active mutant genes, for example, by deleting the inhibitory domains of transcription factors or changing phosphorylation mimicking/depriving status in the signal transducers such as MAPK, MAPKK, or receptor kinases. Meanwhile, to prevent constitutive overexpression of cold-responsive genes from consuming extra energy and producing undesirable traits, tissue-specific and cold stressinducible promoters will be beneficial for the genetic improvement of cold tolerance in rice. Finally, a multi-genes site-specific assembly system will be developed. Elite constructs with