Plant Low-Temperature Stress: Signaling and Response

Abstract

Cold stress has always been a significant limitation for plant development and causes substantial decreases in crop yield. Some temperate plants, such as Arabidopsis, have the ability to carry out internal adjustment, which maintains and checks the metabolic machinery during cold temperatures. This cold acclimation process requires prior exposure to low, chilling temperatures to prevent damage during subsequent freezing stress and maintain the overall wellbeing of the plant despite the low-temperature conditions. In comparison, plants of tropical and subtropical origins, such as rice, are sensitive to chilling stress and respond differently to low-temperature stress. Plants have evolved various physiological, biochemical, and molecular mechanisms to sense and respond to low-temperature stress, including membrane modifications and cytoskeletal rearrangement. Moreover, the transient increase in cytosolic calcium level leads to the activation of many calcium-binding proteins and calcium-dependent protein kinases during low-temperature stress. Recently, mitogen-activated protein kinases have been found to regulate low-temperature signaling through ICE1. Besides, epigenetic control plays a crucial role during the cold stress response. This review primarily focuses on low-temperature stress experienced by plants and their strategies to overcome it. We have also reviewed recent progress and previous knowledge for a better understanding of plant cold stress response.

1. Introduction

Cold stress is among important abiotic stresses that substantially controls the crop production and geographical distribution of many plant species [1,2]. Low-temperature stress affects many aspects of plants, including germination, growth, development, and reproduction (Figure 1) [3,4,5,6]. Generally, plants encounter two forms of low-temperature stress i.e., chilling and freezing. For plants, chilling temperatures are low but positive temperatures (0–15 °C) that could vary with the plant’s tolerance level and variety. Chilling temperatures also depend upon air temperature and wind speed during the exposure. In contrast, at freezing temperature (below 0 °C), the plant starts to struggle against freezing injury [7].

Rice (Oryza sativa L.), is susceptible to chilling stress, especially in high elevation and high latitude temperate zones [8,9]. Chilling temperature for more than 4 days results in poor germination, retarded seeding growth, sometimes death of rice seedlings. Besides, some researchers also reported a large-scale epidemic of rice blast during chilling stress, resulting in a significant loss to rice production [10]. At chilling temperatures, sensitive plants are affected at all stages (from vegetative to reproductive stages) of their life. In chillling sensitive plants, chilling injury can be observed in the form of chlorosis, stiffness of seedlings, withering and often the death of seedlings. These phenotypes often work as indicators to differentiate chilling sensitive and tolerant varieties [9].

However, many temperate plants, such as Arabidopsis, rapeseed, wheat, and rye, have developed abilities that detect and respond to freezing stress via cold acclimation. The prerequisite for cold-acclimation is exposure to a low, non-freezing temperature that brings about coping ability during subsequent cold stress. The cold acclimation mechanism maintains the overall wellbeing of the plant despite the non-conducive low-temperature conditions [11].

Perennial plants, such as trees, detect the impending season by sensing changes in photoperiod and temperature throughout the year. Seasonal day length and temperature variation signals trigger transitions from active growth to dormant phases and frost sensitivity to cold hardiness in these plants. This shift is obligatory for woody plants to survive the winter in temperate climates. Compared to annual herbaceous plants, the morphological and physiological characteristics of overwintering plants help them synchronize with climatic rhythms resulting in their survival in harsh cold habitats [12]. Endogenous regulating systems such as photoreceptors and the circadian clock assess light changes and measure photoperiods. Even under optimal conditions, if day length goes below a certain range, growth cessation and development of apical buds are stimulated in trees. Simultaneous with the growth cessation, overwintering tissues increase freezing tolerance. Previous studies have suggested that either short-day or low temperatures can substantially enhance the freezing tolerance of trees. However, seasonal cold acclimation of woody perennials is predominantly induced by short-day during autumn, which is further assisted by low temperature. For example, under short-day conditions, hardwood trees such as Silver Birch and Populus spp. develop initial provisional freezing tolerance, which increases further upon subsequent exposure to low nonfreezing and freezing temperatures [13,14,15].

During low-temperature stress, multiple pathways such as protein and nucleic acid biosynthesis, respiratory, defence, and secondary metabolism are affected in these plants. Consistently, the expression level of different genes involved in carbohydrate metabolism, hormone biosynthesis, transcripts linked to membranes lipids and polysaccharides change due to low temperature stress [16]. Guy et al. (1985) first reported a group of genes contributing to freezing tolerance and named them cold-regulated (COR) genes. In Arabidopsis, COR genes include early dehydration-inducible (ERD) genes, low-temperature induced (LTI), responsive to desiccation (RD), etc. [17]. Since then, considerable progress has been made in our understanding of molecular mechanisms of cold stress response in plants.

In this review article, we have summed up primary responses exhibited by the plant during low-temperature stress and included recent findings of plant cold stress research.

2. Perception of Cold Signal

2.1. Membrane Modification as a Signal of Low-Temperature Stress

The plasma membrane of an organism is similar to a safety net that protects and maintains the cell shape and discriminately exchanges proteins, signal molecules, gases, and other substances required for various physiological and developmental processes [18]. This transport is facilitated either by open spaces in the plasma membrane or different transport proteins, pumps and gated channels [19,20,21]. The major components of the plasma membrane include lipids, proteins, and carbohydrates. Cold stress initially causes injury to the plasma membrane by altering its physical and chemical properties [8]. The plasma membrane adopts unconventional ways to produce stress-inducible lipids through the endoplasmic reticulum in response to cold stress. Remarkably during cold stress, glycerolipids function as signal molecules besides protecting organellar membranes [22].

The lipid composition of lipid bilayers and their associated lipid head groups (esterified fatty acids form) vary at different growth temperatures [23]. Cold stress is not an exception in this case. For instance, during 1980, several groups reported that the high melting point fatty acids (hexadecanoic, trans-3-hexadecenoic and octadecanoic acid) and saturated fatty acids (hexadecanoic and octadecanoic acids) differ among higher plants during their response to chilling stress [24,25,26,27,28]. An excellent and comprehensive study by Norio Murata further sheds light on the role of phosphatidylglycerols during the chilling stress tolerance. It was demonstrated that there is a clear difference between chilling stress tolerant and sensitive plants for phosphatidylglycerol, dipalmitoyl plus the l-palmitoyl-2-(trans-3-hexadecenoyl) contents, which is around 3–19% in chilling resistant plants compared to 26–65% in the susceptible plants [29]. Additionally, the chilling sensitivity of plants depends on the degree of unsaturated fatty acids in the phosphotidylglycerol residues in the chloroplast membranes [30]. Chilling-resistant plants, such as spinach and Arabidopsis thaliana, contain more cis-unsaturated fatty acids, mediated by chloroplast-localized enzyme glycerol-3-phosphate acyltransferase, compared to chilling-sensitive plants like Nicotiana tabacum. Consistently, transformation with cDNA of glycerol-3-phosphate acyltransferase alters fatty acid unsaturation and chilling stress tolerance in several plants [30].

Moreover, lipid flippases of P-type ATPases can exchange lipid molecules between membrane leaflets [31]. In cold-stressed plants, following functional deformities in the plasma membrane, cytosolic pH declines to an extent where tonoplast starts crumpling [32,33]. Mechanosensitive Ca²⁺ channels are suggested to be involved in this breakdown of the vacuolar membrane [34]. Appealingly, cold-acclimated plants express tonoplast monosaccharide transporters (TMT), which increase the level of sugars and provide cellular homeostasis [35]. The flexibility of the chloroplast membrane is also compromised after a period of cold [36]. The process involves modification in acyl-lipid/CoA desaturase 1 gene, ensuring a decrease in fatty acid composition [37]. Additionally, a thylakoid localized lipoxygenases (LOX) reversibly communicates with thylakoid membrane lipids under dark, chilling conditions. LOXs are involved in the first step of the biosynthesis of oxylipin, one of the many stress inhibitors which increase plant tolerance to stresses [38]. Besides, a nonessential plastid-encoded ribosomal protein in tobacco, Rpl33, is required for cold stress tolerance and in the Rpl33 knockout plants the recovery from chilling stress is severely compromised [39]. These reports collectively indicate that most plants modify their membrane as a necessary physiological process to combat low-temperature stress.

Apart from the membrane modification, changes in the plant cytoskeleton are also implicated in cold stress sensing. The cold exposure for only a few minutes can affect the cytoplasmic architecture and organization of the cytoskeleton [40]. The cytoskeleton maintains the structural integrity by reorganizing or disassembling during cold stress exposure. Besides, the cold-induced depolymerization of the cytoskeleton is believed to be necessary to induce low temperature-responsive genes [40,41,42,43].

2.2. Ca²⁺ Signaling in Response to Cold

Calcium (Ca²⁺) is a versatile signal transduction element, which acts as a secondary messenger and regulates several physiological processes [44,45]. Cytosolic Ca²⁺ concentration increases transiently due to different abiotic and biotic stimuli in the living organism, including plants [46]. These Ca²⁺ signatures are specific for each cue in terms of frequency, duration, amplitude, and location (Figure 2) [47]. The prolonged period of high cytosolic Ca²⁺ could cause reactive oxygen species (ROS) accumulation and metabolic dysfunctions leading to physiological damage to the cell [48]. The physical alterations in the plasma membrane during cold stress also change the Ca²⁺ concentration [1,49]. Cold-induced signals that curb intracellular Ca²⁺ sometimes lead to the initiation of a protein phosphorylation cascade, which starts several other signaling molecules apart from activating second messengers.

Calcium channels, including cyclic nucleotide-gated channels (CNGCs), nonspecific cation channels, contribute to Ca²⁺ fluxes in various stress responses [50]. In plants like Arabidopsis and rice, CNGCs form a large family of non-selective cation-conducting channels that are mostly localized at the plasma membrane. In response to cold stress, the expression levels of rice CNGCs have been shown to be differentially regulated, suggesting their role in cold stress response [51]. Consistently, the G-protein regulator Chilling Tolerance Divergence 1 (COLD1), in combination with Rice G-protein a Subunit 1 (RGA1), regulates the cold-induced influx of Ca²⁺ to confer cold sensing in rice [52]. However, it is unclear whether COLD1 acts as a Ca²⁺ channel or only modulates its activity.

The calcium channels also play an important role in the cold acclimation of Arabidopsis [53]. For example, Mid1-Complementing Activity 1 (MCA1) and MCA2 are mechanosensitive Ca²⁺ permeable channels contributing to cold-induced Ca²⁺ influx; although they are not the only ones transporting Ca²⁺ across the plasma membrane [54]. Recently Liu et al. (2021) reported that AtANN1, a Ca²⁺ permeable transporter and a member of the subfamily of phospholipid and Ca²⁺ binding proteins, participates in freezing tolerance by mediating the generation of Ca²⁺ signals [55].

The cytosolic Ca²⁺ signal is primarily transmitted through Ca²⁺ sensors. Upon sensing the elevation in Ca²⁺ concentration, these sensors change their phosphorylation status by binding to calcium in their specific motifs (EF-hands). To date, several calcium sensors such as calmodulin (CaM), calcineurin B-like proteins (CBLs), CBL-interacting protein kinases (CIPKs), calcium-dependent protein kinases (CDPKs) and phosphatases have been reported [56]. Two important cold-induced calcium sensors are discussed below.

2.2.1. Calmodulin (CaM) Involvement in Cold Stress Signaling

Calmodulins (CaM) are Ca²⁺ binding proteins that can sense the changes in the concentration of Ca²⁺ ions and function as a calcium buffer. CaMs transduce signals and maintain a homeostatic balance of Ca²⁺ to minimize its cytotoxic effects. Single plant species may have several isoforms of CaM, displaying a wide array of responses [57,58]. CaM has four functional EF-hands that cause conformational changes of CaM upon binding with Ca²⁺. After attaching to Ca²⁺, CaM activates several other protein targets involved in numerous cellular processes [58]. During environmental cues such as low temperature, rapid transcription of CaM proteins are reported in several plants, including Arabidopsis [59,60,61].

Calmodulin-binding transcription activators (CAMTA) proteins belong to a class of transcription factors conserved in eukaryotes and respond to calcium signals by binding to calmodulin. Arabidopsis contains six CAMTA genes [62] that act as a transcriptional regulator after binding to a specific (G/A/C)CGCG(C/G/T) cis-element. CAMTA proteins perceive an increase in Ca²⁺ level as a response to cold stress by interacting with various calmodulin proteins [59]. Doherty, et al. [63] first suggested the role of CaM signaling during cold stress. They showed that CAMTA3 functions as a positive regulator of the CBF2 transcription factor by binding to the cis-element (CM2 motif) of CBF2. Consistently in the camta1camta3 double mutant, the CBF gene family expression was reduced by 50% approximately [63]. Since then, several reports have suggested the involvement of the CAMTA during cold stress [64,65,66,67].

2.2.2. CBL-CIPK Module in Cold Stress Response

Calcineurin like (CBL) proteins (also SOS3-like Ca²⁺ binding proteins, SCaBLs) are a family of Ca²⁺ sensors identified in different plant species, including Arabidopsis [68]. These CBLs/SCaBPs do not possess enzymatic activity by themselves, however, CBLs are triggered by the change in Ca²⁺ signature and precisely interact with CIPK (CBL-interacting protein kinases) or SOS2-like protein kinase [69]. Increasing evidence suggests that the CBL-CIPK network plays a key role during cold stress response. In Arabidopsis, CBL1 participates in cold stress signaling along with CIPK7 [70]. Similarly, in Cassava (Manihot esculenta) MeCBL2 is induced by cold stress with MeCIPK7 suggesting their role in cold signal transduction [71]. In 6-week-old grapevine leaves, VvCBLs and VvCIPKs showed differential expression against various stress conditions. Heat stress down-regulated the expression level of VvCBL10, VvCBL11 and VvCBL12; however, their expression increased by salt, PEG and cold stress. Besides, VvCIPK34 transcript was increased during cold and heat stress, but decreased by salt and PEG treatments [72]. Similarly, in pea plants, exposure of NaCl, wounding and cold up-regulated PsCBL and PsCIPK, whereas drought and ABA did not display changes in transcript level of PsCBL and PsCIPK [73]. Abiotic treatments in canola also increased the transcript levels of BnaCBLs and BnaCIKPs. Six hours of cold stress up-regulated the expression of BnaCBL1, but BnaCBL10 took 24 h, whereas BnaCBL2, −3, −4 were down-regulated by cold exposure. Besides, a significant up-regulation of BnaCIPK3, −6, −12, −15, −23, −26 were found during cold, suggesting their participation in cold stress signaling [74]. Consistently, rice plants over-expressing CIPK genes displayed enhanced resistance to drought, salt, and cold stresses. OsCIPK03, OsCIPK12, and OsCIPK15 over-expressing plants showed an increase in resistance against abiotic stresses. Moreover, compared to wild-type plants, they accumulated a higher level of compatible solutes and proline [75]. Similarly, ectopic expression of Lepidium CIPK gene in N. tabacum resulted in an increased level of proline accumulation, cell membrane stability and cold stress tolerance [76,77]. These findings indicate that the CBL-CIPK module is a crucial signaling module engaged in cold stress signaling. However, the functions of several CIPK proteins remain elusive in Arabidopsis and other important crop plants. Further studies will help us to understand the specificity of CBL-CIPK signaling pathway, which could be utilized as a tool in engineering stress hardy plants.

3. Transcriptional Regulation: CBF and Regulation of Cold

Transcription factors (TFs) display a pivotal role in gene regulation by binding to cis-elements in the promoter region of the target gene. The concerned gene is either turned off/on via TF mediated regulation and brings about a particular outcome. There are approximately 58 families of different TFs that have been identified in plants [78]. The majority of the TFs belong to multigene families [79]. Structurally, TFs are multidomain proteins with DNA-binding domain and activation domain [79]. Individual members of the same family may respond differently to different stress conditions. However, a significant overlap of the gene-expression profiles suggests that some genes get induced by the same transcription factor [80]. Several transcription factors are reported to be involved in low-temperature stress responses such as C-repeat binding factors (CBFs), basic helix-loop-helix (bHLH), NAC, MYB, etc. [81,82,83,84]. In Arabidopsis, many transcription factor genes are induced during low-temperature stress [85].

The DREB/CBF-mediated cold-responsive pathway is well studied because of the upregulation of CBF1-3/DREB1A-C due to cold stress [7,86,87]. Overexpression of CBF/DREB induces cold tolerance in Arabidopsis and other plants as well [88,89,90]. Interestingly, CBF/DREB transcription factors lack the DRE element in their promoter region and are unable to regulate their own expression. But, some of the upstream regulators, such as ICE (inducer of CBF expression) [91,92], CAMTA3 (calmodulin-binding transcription activator 3) [63], ZAT12 [93], and EIN3 (Ethylene Insensitive 3) [94] regulate the expression of CBF/DREB. Among ICEs, ICE1 and ICE2 are the most upstream cold-inducible gene and consequently, they regulate downstream CBFs and CORs. Both ICE1 and ICE2 encode MYC-like basic helix-loop-helix (bHLH) transcription activator and binds to the promoter regions of CBF1-3 to regulate their expression positively [92,95,96]. CBF transcription factors have been characterized extensively by using transgenic plants that have the ectopic expression of CBF/DREB1 genes, RNAi or CRISPR/Cas9 editing [97,98,99,100]. Overexpression of CBF increases the expression of COR genes resulting in cold acclimation even at optimum temperatures [87,97,101]. The transcript level of ICE1 itself is not induced by cold, suggesting that ICE1 function is post-translationally regulated [91]. In recent years, several factors identified to regulate ICE1 expression, including HOS1 (high expression of osmotically responsive gene 1, SIZ1 (SAP and Miz), and OST1 (open stomata 1) [102,103,104]. HOS1 acts as a RING-type ubiquitin E3 ligase and is also reported to function as a chromatin remodeling factor. HOS1 negatively regulates the CBF expression by ubiquitinating and degrading ICE1 during low-temperature stress [102,105].

Moreover, SIZ1 mediated sumoylation of ICE1 controls the CBF expression by stabilizing ICE1 [106]. A transcription factor, MYB15 (a member of the R2R3-MYB family), also negatively regulates the expression of CBF genes in Arabidopsis [107]. MYB15 is expressed in the absence of cold stress and represses the expression of CBFs by binding to MYB recognition sites of their promoters [84,108]. Additionally, ZAT12, a C2H2 zinc finger transcription factor, also functions as a negative regulator of CBF. Arabidopsis plants overexpressing ZAT12 decrease the expression level of CBF under cold stress [109]. As discussed above, during cold stress several negative regulatory pathways work on the finetuning of CBF-dependent signaling. However, the physiological significance of repressive pathways still remains elusive. Probably, high levels of CBF could be harmful to plant growth and a balance is maintained in the form of these repressors. Indeed, CBF regulon plays an indispensable role in cold acclimation, though it is not the only player. These studies have significantly contributed to our understanding of CBF pathway and its significance during low-temperature stress. However, recently, the established ICE-CBF pathway has been challenged. Kidokoro et al. demonstrated that neither ice1-1 mutant nor ICE1 overexpression regulates the expression of DREB1A/ICE3 [110,111]. At first hand, the finding sends a shock wave among the cold stress community, but their results also showed that the promoter region DREB1A is hypermethylated due to another transgene, not ICE1 [110,111]. Although the ICE-CBF pathway is well-studied and established for cold stress signaling, it is eye-opening that suggests revalidating the pathway and findings associated with this signaling cascade.

4. Mitogen-Activated Protein Kinase: The Attenuator of ICE1

MAPKs (mitogen-activated protein kinases) play an indispensable role in integrating multiple intracellular signals transmitted by various stress-induced secondary messengers, thereby activating or deactivating proteins via post-translational phosphorylation of target [112]. Generally, the MAPK cascade consists of three kinases acting in a row and inactive MAPKKKs are activated by a stimulus or signal messenger. Once activated, MAPKKKs activate MAPKKs by phosphorylating it at conserved serine/threonine. Activated MAPKKs further activate MAPKs by phosphorylating MAPK. Recently, the involvement of MAPK signaling has been found to regulate the cold stress response via the ICE1 pathway in A. thaliana [113,114]. MPK3 and MPK6 phosphorylate ICE1, which inhibits CBF expression, whereas MEKK1-MKK1/2-MPK4 cascade enhances cold tolerance by acting antagonistically to MPK3/MPK6 (Figure 3) [114].

MPK3, MPK4, and MPK6 are induced within 30 min of the cold exposure indicating their involvement in cold stress response. Consistently, mpk3/mpk6 mutant display enhanced freezing tolerance; however, mpk3 and mpk6 inducible MKK5^DD displayed opposite results [113,114]. When subjected to cold MPK mediated phosphorylation leads to degradation of ICE1, resulting in the fine-tuning of ICE1 mediated CBF regulon [113,114]. Interestingly, earlier reports showed that OST1 mediated phosphorylation increased ICE1 stability [104]. Additionally, OST1 increases the stability of ICE1 by antagonizing HOS1 E3 ubiquitin ligase [102]. In comparison, MPK3/6 mediated degradation of ICE1 does not affect the HOS-ICE interaction and could be working differently [113]. These findings suggest a ying-yang mechanism in ICE1 stability by two different types of kinases. Deciphering the puzzle of MPK3/MPK6 mediated ICE1 degradation mechanism will further enhance our knowledge of ICE1 regulation and turnover. In contrast to MPK3/MPK6, MEKK1-MEKK1/2-MPK4 cascade works under calcium/calmodulin-regulated receptor-like kinase1 (CLRK1) during cold stress. CRLK1 and CRLK1 induced MPK4 inhibits MPK3/6 activation, thereby positively regulating freezing tolerance [114]. CRLKs induce downstream MEKK cascade, which further amplifies the downstream signal (Figure 3). The MPK4 mediated inhibition of MPK3/6 promotes the stability of ICE1, thereby modulating the cold-responsive transcription factor-like CAMTA and MYB [64,108]. These studies demonstrate that fine-tuning of cold stress signaling is an essential step, and sometimes a signaling component could work in both directions as an activator/suppressor. It also corroborates that calcium sensors such as CRLKs play an indispensable role in cold signal transduction.

5. The Role of MicroRNAs during the Cold Stress Response

MicroRNAs (miRNAs) are small non-coding RNAs that play a vital role in post-transcriptional gene regulation. In general, microRNAs size range from 20 to 22 nucleotides for animals and 20 to 24 nucleotides for plants. miRNAs act as negative regulators of translation by binding to complementary mRNA molecules and guiding degradation and/or translational repression of the target [115]. It is now well established that the miRNAs play a crucial role in almost every aspect of growth, development, response to biotic and abiotic factors, response to various environmental challenges by post-transcriptionally regulating the gene expression [116,117,118]. MicroRNA’s role during low-temperature response and other environmental cues has been well documented [119,120,121,122]. Several miRNAs have been identified for their involvement in low-temperature stress in Arabidopsis (see Megha et al., 2018 for more details, Supplementary Table S1). In various reports, several cold stress-responsive microRNAs have been reported in several plant species [117,122,123]. The up-regulation of several microRNAs viz. miR168, miR169, miR172, miR393, miR319, and miR408 during cold stress has been observed in different plant species [122,124]. Additionally, the heterologous expression of rice miR393a in switchgrass significantly increased cold tolerance through the regulation of auxin signaling [125]. Recently, 353 cold-regulated miRNAs were identified by comparing cold-tolerant and cold-sensitive varieties of Brassica. This study further suggested that miR166e, miR319, and Bra-novel-miR3936-5p may play essential roles during cold stress response [126]. Yang, et al. [127] also obtained 412 sugarcane miRNAs (261 known and 151 novel miRNAs) by deep sequencing. They reported 62 miRNAs were differentially regulated by cold and selected miR156 for functional analysis in tobacco. Transient overexpression of miR156 displays improved parameters of cold stress tolerance [127]. In the young spikes of common wheat, 192 conserved miRNAs and 9 novel miRNAs were identified [117]. A total of 34 conserved miRNAs and five novel miRNAs were differentially regulated between the cold-stressed and control samples. In another study, miRNA396b (ptr-miR396b) enhanced cold tolerance in transgenic lemon (Citrus limon) plants overexpressing ptr-miR396b as compared to wild-type. Ptr-miR396b guided the cleavage of the 1-aminocyclopropane-1-carboxylic acid oxidase (ACO, a rate-limiting enzyme in ethylene biosynthesis) and overexpressing lines displayed a reduction in ACO transcript levels and ethylene content compared with the WT. The low level of ethylene under cold stress could be because of ACO transcript reduction by Ptr-miR396b [128]. Moreover, ethylene targets the CBF pathway and acts as a negative regulatory signal in cold stress response [94]. Interestingly, a recent paper describes that a transcriptional repressor of auxin signaling, SLR/IAA14 protein, plays a crucial role in integrating miRNAs in auxin and cold stress response [129].

Collectively, these reports indicate that miRNAs play a crucial role during cold stress response by attenuating the gene expression and degrading the target. mRNA targets degraded by miRNAs could be deleterious and energy-consuming if translated during cold stress.

6. Antioxidants during the Cold Stress Response

Reactive Oxygen Species (ROS) are many chemically reactive compounds produced during oxygen metabolism. Examples of ROS include peroxide, superoxide, hydroxyl radical & singlet oxygen [130]. Adverse conditions like various environmental stresses (high light intensity, metals, high toxic levels of oxygen and freezing temperature) and biotic stresses like infections bring about oxidative injuries due to the overproduction of ROS [131,132]. Their overabundance oxidizes several types of molecules like proteins, lipids, etc. [133]. Additionally, ROS disrupts the double-layer membrane integrity by disabling its receptors and enzymes. ROS also interferes with DNA structure and its repair mechanism [134]. To restore redox homeostasis, plant activates several genes encoding defensive enzymes such as glutathione peroxidase, glutathione and ascorbate. In addition, enzymes of the ascorbate-glutathione cycle (viz. ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase), fat-soluble vitamins like tocopherol and carotenoid predominantly assist in hunting ROS [135,136,137,138,139]. Since cold stress accompanies low to freezing temperatures that vary with geographical distribution, hence plants too exhibit the variety of antioxidant mechanisms depending upon the severity of chilling stress. Chloroplasts are the worst affected by peroxides, which is a crucial feature during chilling temperature with high light stress [140,141,142]. Chloroplast ascorbate peroxidase is a foreground enzyme during cold and high light stress [143,144,145]. Its reduced activity demonstrates an overall fall in photosynthesis and an increase of free radicals (peroxides and non-functional proteins) due to oxidation [146]. This causes lipid peroxidation of organelle membranes and other lipid inclusions besides disrupting metabolic pathways (the reductive carbon pathway and electron transport) and stomatal conductance [132,147]. Meanwhile, recent advances in research provide insight on the interaction of chloroplast and mitochondria in maintaining hydrogen peroxide equilibrium through malate shuttle, in addition to inducing programmed cell death (Figure 4) [148]. Hence, we can state that oxidative stress is an unavoidable side effect that follows low-temperature stress [149,150,151].

7. Contribution of Phytohormones during the Cold Stress Response

Phytohormones are well known for the regulation of growth and development in plants. Being small, they are capable of moving to a distant location from the place of their biosynthesis and carry out the appropriate function in exceptionally low concentrations [152]. Abscisic acid (ABA) is a bonafide stress hormone that plays key functions during stress [153]. Several reports suggest that ABA concentration elevates against stress in conjunction with the regulation of several genes [154,155,156]. During stress conditions, carotenoids are converted into bioactive ABA by de novo biosynthesis pathway. ABA biosynthesis is increased during cold stress, which improves plant ability to resist during unfavorable cold conditions [157,158]. Consistently, mutants of ABA biosynthetic genes display impaired cold acclimation response, whereas exogenous application of ABA improves the cold tolerance [159,160,161]. ABA biosynthesis and catabolic genes display their regulation in organ dependent manner during cold stress [162]. Plants subjected to cold stress induce a number of ABA-responsive genes [163,164]. Several COR genes that are induced during cold stress are also induced by ABA, indicating the involvement of ABA signal transduction in cold stress responses [165]. These observations suggest that ABA is an essential phytohormone participating in cold stress response.

Several findings suggest that ABA does not affect the CBF expression and believed to mediate the cold response through the CBF-independent pathway [166,167]. In support to this hypothesis, a set of ABA-independent, cold- and drought-induced gene contains Dehydration-Responsive/C-Repeat Element/Low-temperature Responsive Element (DRE/ CTR/LTRE) cis-acting element in their promoter regions [168,169,170]. However, some recent findings indicate that ABA-mediated cold response could be mediated by CBF-dependent pathway as well [171]. For instance, both ABA and low temperature induce the expression of transcription factor MYB96 [96]. MYB96 interacts with HHP1-3 (HEPTAHELICAL PROTEIN) and these HHP proteins interact with ICE1, ICE2, and CAMTA [96,172]. Altogether, it shows the possibility of ABA-mediated low-temperature stress response in CBF-dependent pathway [171].

Another phytohormone, auxin (indole-3-acetic acid, IAA) has been established to control virtually all aspects of plant development and response. Emerging evidences indicate the involvement of auxin in regulation of plant growth and development under high as well as low temperature stresses [173,174,175,176,177,178,179]. GH3 genes encode auxin-conjugating enzyme and modulate endogenous levels of active auxin through negative feedback regulation. In rice, overexpression of OsGH3-2 increased cold tolerance and reduced free IAA content, alleviated oxidative damage, and decreased membrane permeability [180]. Low temperature-induced root growth inhibition of Arabidopsis is also reported as a response of reduced auxin accumulation [181]. Recently, the involvement of auxin has been shown to preserve the root stem cells at a quiescent status under chilling stress [182]. Phytohormones cytokinin signal transduction is mediated by the two-component system (TCS), which is perceived by histidine kinase receptors and regulate the phosphorylation of response regulators [183]. Involvement of the Arabidopsis cytokinin TCS during cold stress signaling and response has been shown in various reports [181,184]. Moreover, Cytokinin Response Factors (CRF2) and CRF3 are involved in lateral root formation and initiation during cold stress whereas, CRF4 gets induced during cold and contributes to freezing tolerance [185,186].

In addition to ABA, auxin and cytokinin other phytohormones are also involved in cold stress response. For example, after low-temperature stress, the accumulation of ethylene has been reported in several plant species including Arabidopsis, tomato, winter rye, etc. [187,188,189]. Ethylene mediates physiological, developmental and stress responses through the activation of Ethylene Response Factors (ERFs) transcription factors [190]. Recently, ERF105 has been shown to operate in conjunction with the CBF pathway and play a key role during cold acclimation and freezing tolerance [191]. Additionally, high ethylene content increases freezing tolerance in non-acclimated Arabidopsis plants. Consistently, the exogenous application of ACC also enhances the survival rate during freezing stress [192]. Moreover, increased freezing response by ethylene overproducing mutant eto1-3 further supports the role of ethylene during freezing stress [192]. In contrast, Shi et al. (2012) showed that ethylene negatively regulates the freezing tolerance in Arabidopsis, accounting for significant sensitivity in ethylene overproducer mutants. They further showed that the ethylene insensitive mutant’s etr1-1, ein4-1, ein2-5, ein3-1, and ein3eil1 display enhanced endurance during freezing conditions [94]. These contrast results indicate that the role of ethylene further needs to be investigated for a better understanding of its role during low-temperature stress.

Brassinosteroids (BRs) are another class of phytohormones that play key role in plant development and defense [193,194,195,196]. Several reports suggest that the BRs are involved during cold stress response [197,198]. Consistently, exogenous application of BRs enhance the chilling tolerance in several plant species [197,198,199,200]. BRs regulate the expression of several COR genes and CBF regulon, thereby control the freezing tolerance [200]. These results clearly indicate that apart from growth regulation, BRs also enhance plant tolerance against freezing stress. Besides, other phytohormones such as Gibberellic acid (GA), Salicylic acid (SA), and Jasmonic acid (JA) also contribute significantly to cold stress response [171]. Additionally, the hormonal crosstalk among phytohormones has created hindrance to decipher the effect of individual phytohormone during the cold stress response. Together, these observations indicate the involvement of phytohormones during cold stress response. However, additional studies are required to identify the molecular components that integrate phytohormones and cold stress response.

8. Epigenetic Regulation

Plants show changes in chromatin modifications such as DNA and histone methylation when exposed to various stresses. Consistently, cold stress alters the expression of several genes due to changes in the chromatin structure [201,202]. Recent reports suggest that epigenetic regulators, such as histone deacetylases, contribute to the transcriptional regulation of COR genes. In particular histone deacetylase (HDACs), the enzyme that catalyzes the removal of an acetyl group from histones, regulate the cold stress response. For example, histone deacetylase HDA6 plays essential role during cold acclimation and freezing tolerance of Arabidopsis [203]. Another deacetylase of Arabidopsis, a WD40-repeat protein high expression of osmotically responsive genes 15 (HOS15) repress cold stress tolerance via histone deacetylation [204,205]. In response to cold stress, HOS15 acts as a positive regulator of cold stress by mediating the degradation of histone deacetylase 2C (HD2C) and changing the chromatin structure from repressive to active state thereby facilitating the expression of CBF mediated COR genes [206]. In maize, global deacetylation of histones H3 and H4 is observed due to cold induced up-regulation of HDACs. Maize HDACs regulate the cold induced DREB1 expression by altering the histone modification and changing chromatin structure at both for gene and specific site [207]. At the same time, active chromatin mark, H3K9ac, is reported be enriched at selective tandem repeats in heterochromatin of maize following cold stress [208]. Recently, Kindren et al. (2018) discovered a lncRNA, SVALKA, in Arabidopsis genome at cold-sensitive region that regulates the freezing tolerance by affecting the CBF1 expression [209]. Although much details are required to fully understand the epigenetic mechanism during cold stress response these finding clearly indicate that epigenetic regulation is indispensable for plant in response to cold stress.

9. Conclusions and Future Perspective

Plants display a plethora of responses to low-temperature stress, such as transcriptomic reprogramming, membrane modification, etc. Low-temperature responsive genes produce protective proteins, such as COR-polypeptides (Cold regulated proteins), antifreeze proteins (AFPs) and some of them even shield lipids. The major determinant for cells to survive during cold stress is their ability to tolerate dehydration and withstand repeated dehydrative/rehydrative cycles. In the plasma membrane, the extent of injury depends on lipid composition and the manifestation of specific cryoprotectants. The production of cryoprotectants is determined by the degree of cold shock and the kind of protection needed for the plant’s survival. Besides, microRNA regulation is also an indispensable response during cold stress. It would be interesting to further elucidate the regulation of miRNA itself by studying their promoter and identifying the overlap in key scis-elements compared to protein-coding genes or discovering different factors that control their expression. With a globally changing climate, the immediate challenge in crop production is to identify and integrate the missing links of the molecular basis of tolerance. The integration of knowledge in the breeding program of susceptible crops is expected to enhance their tolerance to stress and increase crop production.