Plant Stress Tolerance: Methods and Protocols€¦ · genes involved in signaling, transcription,...

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Chapter 3

Gene Regulation During Cold Stress Acclimation in Plants

Viswanathan Chinnusamy, Jian-Kang Zhu, and Ramanjulu Sunkar

Abstract

Cold stress adversely affects plant growth and development and thus limits crop productivity. Diverseplant species tolerate cold stress to a varying degree, which depends on reprogramming gene expres-sion to modify their physiology, metabolism, and growth. Cold signal in plants is transmittedto activate CBF-dependent (C-repeat/drought-responsive element binding factor-dependent) andCBF-independent transcriptional pathway, of which CBF-dependent pathway activates CBF regulon.CBF transcription factor genes are induced by the constitutively expressed ICE1 (inducer of CBFexpression 1) by binding to the CBF promoter. ICE1–CBF cold response pathway is conserved in diverseplant species. Transgenic analysis in different plant species revealed that cold tolerance can be significantlyenhanced by genetic engineering CBF pathway. Posttranscriptional regulation at pre-mRNA processingand export from nucleus plays a role in cold acclimation. Small noncoding RNAs, namely micro-RNAs(miRNAs) and small interfering RNAs (siRNAs), are emerging as key players of posttranscriptional genesilencing. Cold stress-regulated miRNAs have been identified in Arabidopsis and rice. In this chapter,recent advances on cold stress signaling and tolerance are highlighted.

Key words: Cold stress, second messengers, CBF regulon, CBF-independent regulation, ICE1,posttranscriptional gene regulation.

1. Introduction

Temperature profoundly influences the metabolism of organismsand thus is a key factor determining the growing season andgeographical distribution of plants. Cold stress can be classi-fied as chilling (<20◦C) and freezing (<0◦C) stress. Temperateplants have evolved a repertoire of adaptive mechanisms such asseed and bud dormancy, photoperiod sensitivity, vernalization,

R. Sunkar (ed.), Plant Stress Tolerance, Methods in Molecular Biology 639,DOI 10.1007/978-1-60761-702-0_3, © Springer Science+Business Media, LLC 2010

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40 Chinnusamy, Zhu, and Sunkar

supercooling (prevention of ice formation in xylem parenchymacells up to homogenous ice nucleation temperature, −40◦C), andcold acclimation. In cold acclimation, plants acquire freezing tol-erance on prior exposure to suboptimal, low, nonfreezing tem-peratures. The molecular basis of cold acclimation and acquiredfreezing tolerance in Arabidopsis and winter cereals has been stud-ied extensively. Plants modify their metabolism and growth toadapt to cold stress by reprogramming gene expression duringcold acclimation (1, 2). This chapter briefly covers cold stress sig-naling, transcriptional and posttranscriptional regulation of geneexpression in cold acclimation process, and the genetic engineer-ing of crops with enhanced cold tolerance.

2. Cold StressSensing

Thus far, the identity of stress sensor in plants is unknown. Thefluid mosaic physical state of the plasma membrane is vital forthe structure and function of cells, as well as to sense tempera-ture stress. The plasma membrane undergoes phase transitions,from a liquid crystalline to a rigid gel phase at low temperatureand to a fluid state at high temperature. Thus, a decrease in tem-perature can rapidly induce membrane rigidity at microdomains.Further, protein folding is influenced by temperature changes.Temperature-induced changes in the physical state of mem-branes and proteins are expected to change the metabolic reac-tions and thus the metabolite concentrations. Therefore, plantcells can sense cold stress through membrane rigidification, pro-tein/nucleic acid conformation, and/or metabolite concentration(a specific metabolite or redox status).

In alfalfa and Brassica napus, cold stress-induced plasmamembrane rigidification leads to actin cytoskeletal rearrangement,induction of Ca2+ channels, and increased cytosolic Ca2+ level.These events induce the expression of cold-responsive (COR)genes and cold acclimation. Further, a membrane rigidifier(DMSO) can induce COR genes even at 25◦C, whereas a mem-brane fluidizer (benzyl alcohol) prevents COR gene expressioneven at 0◦C (3, 4). Genetic evidence for plants sensing cold stressthrough membrane rigidification is from the study of the fad2mutant impaired in the oleic acid desaturase gene of Arabidop-sis. In wild-type Arabidopsis plants, diacylglycerol (DAG) kinaseis induced at 14◦C. The fad2 mutant (more saturated mem-brane) and transgenic Arabidopsis overexpressing linoleate desat-urase gene showed the expression of DAG kinase at 18 and 12◦C,respectively (5).

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Cold Tolerance in Plants 41

3. SecondMessengersand Signaling

Cytosolic Ca2+levels act as second messenger of the cold stresssignal (6). Calcium may be imported into the cell or released fromintracellular calcium stores. Patch-clamp studies of cold-inducedpotential changes of the plasma membrane in Arabidopsis mes-ophyll protoplasts showed the cold-activatedcalcium-permeablechannel involved in the regulation of cytosolic Ca2+signatures(7). Membrane rigidification induced cytosolic Ca2+signatures;and COR gene expression was impaired by gadolinium, amechanosensitive Ca2+ channel blocker, which suggests theinvolvement of mechanosensitive Ca2+ channels in cold acclima-tion (4). Pharmocological studies implicated cyclic ADP-ribose-and inositol-1,4,5-triphosphate (IP3)-activated intracellularcalcium channels in COR gene expression (4). Calcium influxinto the cell appears to activate phospholipase C (PLC) and D(PLD), which produce IP3 and phosphatidic acid, respectively.IP3 can further amplify Ca2+signatures by activation of IP3-gatedcalcium channels (8). Genetic analysis revealed that loss-of-function mutants of FIERY1 (FRY1) inositol polyphosphate1-phosphatase show significantly higher and sustained levelsof IP3 instead of the transient increase observed in wild-typeplants. This situation leads to higher induction of COR genesand CBFs, the upstream transcription factors (9). In addition,the calcium exchanger 1 (cax1) mutant of Arabidopsis, which isdefective in a vacuolar Ca2+/H+ antiporter, exhibited enhancedexpression of C-repeat binding factor/dehydration responsiveelement binding (CBF/DREB) proteins and their target CORgenes (10). Therefore, cytosolic Ca2+ signatures are upstream ofthe expression of CBFs and COR genes in cold stress signaling.

Cold acclimation induces accumulation of ROS such asH2O2, both in chilling-tolerant Arabidopsis and chilling-sensitivemaize plants. ROS can act as a signaling molecule to reprogramtranscriptome probably through induction of Ca2+ signatures andactivation of mitogen-activated protein kinases (MAPKs) (11)and redox-responsive transcription factors. Arabidopsis frostbite1(fro1) mutant, which is defective in the mitochondrial Fe-S sub-unit of complex I (NADH dehydrogenase) of the electron trans-fer chain, shows a constitutively high accumulation of ROS.This high accumulation of ROS in fro1 results in reduced CORgene expression and hypersensitivity to freezing stress, probablybecause of desensitization of cells by the constitutively high ROSexpression (12).

Cold stress-induced second messenger signatures can bedecoded by different pathways. Calcium signatures are sensedby calcium sensor family proteins, namely calcium-dependent

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protein kinases (CDPKs), calmodulins (CaMs), and salt overlysensitive 3-like (SOS3-like) or calcineurin B-like (CBL) pro-teins. In a transient expression system in maize leaf protoplast, aconstitutively active form of an Arabidopsis CDPK (AtCDPK1)activated the expression of barley HVA1 ABA-responsive pro-moter::LUC reporter gene suggesting that AtCDPK is a posi-tive regulator in stress-induced gene transcription (13). Geneticand transgenic analyses implicated CDPKs as positive regulators,but a calmodulin, a SOS3-like or a CBL calcium binding protein,and a protein phosphatase 2C (AtPP2CA) are negative regulatorsof gene expression and cold tolerance in plants. Components ofMAPK cascades are induced or activated by cold and other abi-otic stresses. Genetic and transgenic analyses showed that MAPKsact as a converging point in abiotic stress signaling. ROS accu-mulation under these stresses might be sensed through a MAPKcascade (14). ROS activates the AtMEKK1/ANP1 (MAPKKK)–AtMKK2 (MAPKK)–AtMPK4/6 (MAPK) MAPK cascade, whichpositively regulates cold acclimation in plants (11). Many of thesephosphorylated proteins show activation or induction of geneexpression under multiple stress conditions, and genetic modi-fication results in alteration of multiple stress responses. Theseresults suggest that the proteins act as connecting nodes of stresssignal networks. Identification of the target proteins or transcrip-tion factors of protein kinase or phosphatase cascades will shedfurther light on stress signaling.

4. TranscriptionalRegulation

Chilling-tolerant plants reprogram their transcriptome inresponse to acclimation temperature. Cold-regulated genes con-stitute about 4–20% of the genome in Arabidopsis (15). Thepromoter region of many COR genes of Arabidopsis con-tains C-repeat (CRT)/DREs, initially identified in the promoterof responsive to dehydration 29A (RD29A/COR78/LTI78).As well, ABA-responsive elements are present in many cold-induced genes. Genetic screens using dehydration and cold stress-responsive promoter-driven LUCIFERASE (RD29A::LUC andCBF3::LUC) led to the isolation of mutants, which unraveledcold-responsive transcriptional networks.

4.1. CBF Regulonsand Cold Tolerance

Yeast one-hybrid screens to identify CRT/DRE binding pro-teins led to the identification of CRT/DREBs (CBFs/DREBs)in Arabidopsis. CBFs belong to the ethylene-responsive elementbinding factor/APETALA2 (ERF/AP2)-type transcription factorfamily. Arabidopsis encodes three CBF genes (CBF1/DREB1B,CBF2/DREB1C, and CBF3/DREB1A), which are inducedwithin a short period of exposure to cold stress. CBFs bind to

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CRT/DRE cis-elements in the promoters of COR genes andinduce their expression (16, 17). Ectopic expression of CBFsin transgenic Arabidopsis induced the expression of COR genesat warm temperatures and induced constitutive freezing toler-ance. These transgenic Arabidopsis plants were also tolerant tosalt and drought stresses (17–19). Microarray analysis of CBF-overexpressing transgenic plants identified several CBF targetgenes involved in signaling, transcription, osmolyte biosynthesis,ROS detoxification, membrane transport, hormone metabolism,and stress response (20, 21). Transgenic overexpression of Ara-bidopsis CBFs is sufficient to induce cold tolerance in diverse plantspecies (Table 3.1). Further, CBF homologs have been identified

Table 3. 1Abiotic stress tolerance of transgenic plants overexpressing CBFs

GeneTransgenicplant Stress tolerance of transgenic plants References

AtCBF1/2/3 Brassicanapus

Constitutive overexpression enhanced both basaland acquired freezing tolerance

(22)

AtCBF1 Tomato Constitutive overexpression enhanced oxidativestress tolerance under chilling stress; enhancedtolerance to water-deficit stress

(23, 24)

AtDREB1A/CBF3

Tobacco Transgenic plants expressing RD29A::DREB1Aexhibited enhanced chilling and droughttolerance

(25)

AtDREB1A/CBF3

Wheat Transgenic plants expressing RD29A pro-moter::AtDREB1A gene showed delayedwater stress symptoms

(26)

AtCBF3 Rice Constitutive overexpression resulted in enhancedtolerance to drought and high salinity and amarginal increase in chilling tolerance

(27)

AtDREB1A/CBF3

Maize RD29A::CBF3 transgenic plants are more toler-ant to cold, drought, and salinity

(28)

AtCBF1 Potato Constitutive or stress-inducible expression ofCBF1 or CBF3 but not CBF2 conferredimproved freezing tolerance to frost-sensitiveSolanum tuberosum

(29)

OsDREB1 Arabidopsis Overexpression in Arabidopsis induced targetCOR genes and conferred enhanced toleranceto freezing and drought stresses

(30)

OsDREB1A/B Rice Constitutive expression conferred improved tol-erance to cold, drought, and salinity

(31)

ZmDREB1 Arabidopsis Overexpression in Arabidopsis induced CORgenes and conferred tolerance to freezing anddrought

(32)

BnCBF5 andBnCBF 17

B. napus Overexpression led to increased constitutivefreezing tolerance, increased photochemicalefficiency and photosynthetic capacity

(33)

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from several chilling-tolerant and chilling-sensitive plant speciesand transgenic analysis confirmed their pivotal role in cold accli-mation (Table3.1).

These evidences suggest that a CBF transcription networkplays a pivotal role in cold acclimation of evolutionarily diverseplant species. Transcriptome analysis of transgenic tomato andArabidopsis plants overexpressing LeCBF1 and AtCBF3 revealedthat CBF regulons from freezing-tolerant and freezing-sensitiveplant species differ significantly (35).

Constitutive overexpression of CBFs under the transcriptionalcontrol of the 35S cauliflower mosaic virus promoter in transgenicplants resulted in severe growth retardation under normal growthconditions in diverse plant species such as Arabidopsis (18, 19,34, 36), B. napus (22), tomato (23, 24), potato (29), and rice(31). Inhibition of metabolism and change in growth-regulatinghormones appears to be important causes of the growth inhibi-tion of CBF-overexpressing plants. Reduction in the expressionof photosynthetic genes appears to reduce photosynthesis andgrowth under cold stress. Transgenic plants constitutively overex-pressing CBFs showed higher induction of the STZ/ZAT10 zincfinger transcription factor gene, which appears to repress genesinvolved in photosynthesis and carbohydrate metabolism and thusreduce the growth of these transgenic plants (21). Microarrayanalysis revealed that cold stress regulates several genes involvedin biosynthesis or signaling of hormones such as ABA, gib-berellic acid (GA), and auxin, which suggests the importanceof these hormones in coordinated regulation of cold toleranceand plant development (15). GA promotes important processesin plant growth and development, such as seed germination,growth through elongation, and floral transition. Growth retar-dation of transgenictomato plants constitutively overexpressingAtCBF1 was reversed by GA3 treatment (24). This finding sug-gested a link between CBFs and GA in cold stress-induced growthretardation. During cold stress, growth retardation appears to beregulated by CBFs through nuclear-localized DELLA proteins,which repress growth in Arabidopsis. GA stimulates the degrada-tion of DELLA proteins and promotes growth. CBFs enhancethe expression of GA-inactivating GA2-oxidases, and thus allowthe accumulation of the DELLA protein repressor of GA1-like3 (RGL3), which leads to dwarfism and late flowering. Further,mutant plants of DELLA genes encoding GA-insensitive [GAI]repressor of GA1-3 [RGA] were significantly less freezing toler-ant than were wild-type plants after cold acclimation. This find-ing suggests that DELLAs might contribute significantly to coldacclimation and freezing tolerance (37).

4.2. Regulators ofCBF Expression

Transcription of CBF genes is induced by cold stress. Hence,constitutive transcription factors present in the cell at normal

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growth temperatures may induce the expression of CBFs on acti-vation by cold stress. A systematic genetic analysis by CBF3::LUCbioluminescent genetic screening led to the identification of aconstitutively expressed and nuclear-localized transcription fac-tor, inducer of CBF expression 1 (ICE1) in Arabidopsis. ICE1encodes a MYC-type basic helix-loop-helix (bHLH) transcriptionfactor, can bind to MYC recognition elements in the CBF3 pro-moter, and induces the expression of CBF3 during cold acclima-tion. The ice1 mutant is defective in both chilling and freezingtolerance, whereas transgenic Arabidopsis overexpressing ICE1showed enhanced freezing tolerance (38). Transcriptome anal-ysis revealed the dominant ice1 mutant with impaired expres-sion of about 40% of cold-regulated genes, in particular 46% ofcold-regulated transcription factor genes (15). Therefore, ICE1is a master regulator that controls CBF and many other cold-responsive regulons. Overexpression analysis showed that ICE2(At1g12860, a homolog of ICE1) induces the expression ofCBF1 and confers enhanced freezing tolerance in Arabidop-sis after cold acclimation (39). In wheat, the ICE1 homologsTaICE141 and TaICE187 are constitutively expressed and acti-vate the wheat CBF group IV, which are associated with freezingtolerance. Overexpression of TaICE141 and TaICE187 in Ara-bidopsis enhanced CBF and CORgene expression and enhancedfreezing tolerance only after cold acclimation. This finding sug-gests that similar to Arabidopsis ICE1, wheat ICE1 also needs tobe activated by cold acclimation (40).

ICE1 appears to negatively regulate the expression ofMYB15 (an R2R3-MYB family protein) in Arabidopsis. MYB15is an upstream transcription factor that negatively regulatesCBF expression. Transgenic Arabidopsis overexpressing MYB15showed reduced expression of CBFs and freezingtolerance,whereas myb15 T-DNA knockout mutants showed enhancedcold induction of CBFs and enhanced freezing tolerance. In ayeast two-hybrid system, ICE1 interacted with MYB15 (41).Further, the expression of MYB15 is increased in ice1 mutants(R236H and K393R) (41, 42). Thus, the ICE1–MYB15 inter-action appears to play a role in regulating CBF expression levelsduring cold acclimation (41).

Although ICE1 is expressed constitutively, only on expo-sure to low temperature does it induce transcription of the CBFand other cold stress-responsive genes (38, 40). Posttranslationalmodifications play a key role in regulating the activity of ICE1under cold stress. Cold stress activates ICE1 sumoylation (42)and negatively regulates ICE1 levels by targeted proteolysis (43).The Arabidopsis High expression of Osmotically responsive gene 1(HOS1) encodes a RING finger ubiquitin E3 ligase. The nuclearlocalization of HOS1 is enhanced by cold stress. HOS1 physicallyinteracts with ICE1 and targets ICE1 for polyubiquitination and

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proteolysis of ICE1 after 12 h of cold stress. Overexpression ofHOS1 in transgenic Arabidopsis results in a substantial reductionin level of ICE1 protein and that of its target genes, as well ashypersensitivity to freezing stress. Thus, HOS1 mediates ubiqui-tination of ICE1 and plays a critical role in maintaining the levelof ICE1 target genes in the cell during cold acclimation (43).Sumoylation of proteins prevents the proteasomal degradation oftarget proteins. The null mutant of Arabidopsis SUMO E3 ligase,SAZ1 (SAP and Miz1), exhibits reduced cold induction of CBFsand the target COR genes, as well as hypersensitivity to chillingand freezing stresses. SIZ1 catalyzes SUMO conjugation to K393of ICE1 during cold acclimation and thus reduces polyubiquiti-nation of ICE1. Mutation in a K393 residue of ICE1 impairs itsactivity (42). Hence, SIZ1-mediated sumoylation facilitates ICE1stability and activity, whereas HOS1 mediation reduces ICE1 pro-tein levels during cold acclimation.

Stomata play a crucial role in regulating photosynthesis andtranspiration. Recently, the scream-D dominant mutant and ice1mutant were found to be the same as R236H, which resultsin constitutive stomatal differentiation in the epidermis, andthe entire epidermis differentiates into stomata. Thus, ICE1 isrequired for controlled stomatal development. ICE1protein inter-acts and forms a dimer with other bHLH transcription fac-tors, SPEECHLESS (SPCH), MUTE, and FAMA, which regu-late stomatal development. ICE1 may act as a link between theformation of stomata and the plant response to environmentalcues (44).

Recently, members of the calmodulin binding transcriptionactivator (CAMTA) family proteins have been identified as tran-scriptional regulators of CBF2 expression. Cold-induced expres-sion of CBF2 was considerably lower in camta3 mutant as com-pared to WT plants. The CAMTA3 protein binds to conservedDNA motifs present in CBF2 promoter and regulates CBF2expression. The camta1/camta3 double mutant exhibited hyper-sensitivity to freezing stress as compared to WT plants. SinceCAMTA proteins can interact with calmodulins, cold-inducedcalcium signals may regulate CBFs expression through CAMTAproteins (45).

4.3. CBF1, CBF2, andCBF3 Play DifferentRoles in ColdAcclimation

Microarray analysis revealed that CBFs regulate about 12% of thecold-responsive transcriptome. Overexpression of CBFs enhancesosmolyte accumulation, reduces growth, and enhances abioticstress tolerance (Table 3.1). Constitutive overexpression stud-ies of transgenic Arabidopsis suggested that CBF1, CBF2, andCBF3 have redundant functional activities (36). However, theice1 mutant, impaired mainly in CBF3 but not CBF1 and CBF2,showed chilling and freezing hypersensitivity (38). Studies of thecbf2 T-DNA insertion mutant of Arabidopsis revealed that CBFs

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have different functions in cold acclimation. cbf2 null mutantsshowed increased expression of CBF1 and CBF3 and enhancedtolerance to freezing (with or without cold acclimation), dehy-dration, and salt stresses. Further, CBFs show a temporal differ-ence in expression, with the cold-induced expression of CBF1 andCBF3 preceding that of CBF2. These results suggest that CBF2negatively regulates CBF1 and CBF3 to optimize the expressionof downstream target genes (45). In potato (Solanum tubero-sum), overexpression of AtCBF2 failed to confer freezing toler-ance (29). Transgenic analysis of CBF1 and CBF3 RNAi linesrevealed that both CBF1 and CBF3 are required for the full setof CBF regulon expression and freezing tolerance (46).

Besides CBF2, the C2H2 zinc finger transcription factorZAT12 negatively regulates the expression of CBF1, CBF2, andCBF3 during cold stress. Arabidopsis transgenic plants overex-pressing ZAT12 showed decreased expression of CBFs under coldstress (47). los2 mutant plants showed an enhanced and more sus-tained induction of ZAT10/STZ during cold stress and enhancedcold sensitivity. LOS2 encodes a bifunctional enolase that nega-tively regulates the expression of ZAT10 (48).

Transgenic Arabidopsis plants overexpressing AtMKK2showed constitutive expression of CBF2, which suggests that theCBF2 expression is probably positively regulated by a MAPK sig-naling cascade (11). Arabidopsis FIERY2 (FRY2), which encodesan RNApolymerase II C-terminal domain (CTD) phosphatase,appears to act as a negative regulator of CBFs and their targetCOR genes because the fry2 mutant showed enhanced expres-sion of CBFs and COR genesunder cold stress and ABA. Sincethe fry2 mutant is hypersensitive to freezing despite enhancedexpression of CBFs, FRY2 may positively regulate the expressionof certain genes critical for freezing tolerance (49). The main-tenance of an optimal level of CBFs at an appropriate time isnecessary, because constitutive overexpression affects growth anddevelopment significantly. Further, CBF expression is under thecontrol of a circadian clock. The maximal cold-induced increasein transcription of CBFs occurs when cold stress is imposed 4 hafter dawn. Transgenic plants overexpressing arrhythmic CCA1showed no temporal difference in the cold induction of CBFexpression (50).

4.4. CBF-IndependentRegulons

Genetic and transgenic analyses revealed that several classes oftranscription factors besides CBFs play an important role in coldacclimation. The eskimo1 (esk1) mutant of Arabidopsis was iden-tified through freezing tolerance genetic screening. The esk1mutant accumulated constitutively high levels of proline andexhibited constitutively freezing tolerance. ESK1 is constitutivelyexpressed and encodes the protein domain of unknown function(23). Transcriptome comparison of CBF2-overexpressing plants

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and esk1 mutants showed that different sets of genes are regulatedby CBF2 and ESK1. However, the mechanism of action of ESK1in freezing tolerance has yet to be revealed (51).

PRD29A::LUC reporter gene-based genetic screening led tothe identification of two constitutively expressed transcription fac-tors, HOS9 (a homeodomain protein) and HOS10 (an R2R3-type MYB), which are necessary for cold tolerance in Arabidop-sis. hos9 and hos10 mutants are less freezing tolerant than wild-type Arabidopsis (52, 53). Transcriptome analysis revealed distinctCBF and HOS9 regulons (52). HOS10 probably regulates ABA-dependent cold acclimation pathways, because HOS10 positivelyregulates NCED3 (9-cis-epoxycarotenoid dioxygenase) and thusABA accumulation during cold stress (53).

Gene expression analysis revealed several transcription fac-tors induced during cold acclimation. Transgenic analysis of cold-inducible transcription factors helped in validation of functions ofsome transcription factors in cold tolerance. Constitutive overex-pression of the soybean C2H2-type zinc finger protein SCOF1in Arabidopsis transgenic plants enhanced the expression of CORgenes and conferred constitutive freezing tolerance. SCOF1 inter-acts with soybean G-box binding factor 1 (SGBF1) and mayenhance the DNA binding activity of the SGBF1. SGBF1 isinduced by both cold and ABA (54). Overexpression of the cold-regulated rice transcription factors MYB4 (an R2R3-type MYB)and OsMYB3R-2 (an R1R2R3 MYB) enhanced freezing toleranceof Arabidopsis (55, 56).

Some members of the abiotic, plant hormone, and pathogen-inducible ERF family play a crucial role in abiotic and bioticstress tolerance. The pepper ERF/AP2-type transcription factorCapsicum annuum pathogen and freezing tolerance-related pro-tein 1 (CaPF1) is induced by cold, osmotic stress, ethylene,and jasmonic acid. Transgenic Arabidopsis overexpressing CaPF1showed induction of pathogen-responsive as well as COR genesand exhibited enhanced tolerance to stress by freezing and topathogens (Pseudomonas syringae pv tomato DC3000) (57). Sim-ilarly, Triticum aestivum ERF1 (TaERF1] was induced by cold,drought salinity, ABA, ethylene, salicylic acid, and infection byBlumeria graminis f. sp. Tritici pathogen in wheat. TransgenicArabidopsis overexpressing TaERF1 exhibited enhanced toleranceto cold, salt, and drought stresses, as well as pathogens (58).Genes encoding the A-5 subgroup AP2 domain protein fromPhyscomitrella patens (PpDBF1) (59) and soybean (GmDREB3)(60) are cold induced, and overexpression of these genes con-ferred enhanced cold tolerance.

In wheat, wheat low-temperature-induced protein 19(WLIP19), encoding a basic-region leucine zipper protein,is induced by cold, drought, and ABA. WLIP19 activatesthe expression of COR genes in wheat. Transgenic tobacco

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overexpressing Wlip19 showed significant freezing tolerance.WLIP19 was found to interact and form a heterodimer withT.aestivum ocs-element bindingfactor 1 (TaOBF1), a bZIP tran-scription factor (61). The plant-specific transcription factor NAC(NAM, ATAF, and CUC) family plays a key role in stressresponse. Overexpression of cold stress-inducible rice SNAC2 intransgenic rice resulted in high cell membrane stability under coldstress. Microarray analysis showed upregulation of several stress-regulated genes in SNAC2-overexpressing plants (62). Theseresults suggest that several transcriptional networks operate dur-ing cold acclimation and cold stress tolerance of plants.

5. Posttranscrip-tional GeneRegulation

Posttranscriptional regulation at pre-mRNA processing, mRNAstability, and export from nucleus plays critical roles in cold accli-mation and cold tolerance (2).

5.1. Messenger RNAProcessing

Pre-mRNA processing and exports constitute important mecha-nisms of regulation of gene expression in eukaryotes. Pre-mRNAundergoes various nuclear processes such as the addition of a 5′methyl cap and poly(A) tail and intron splicing. Splicing is nec-essary to remove introns and to synthesize translationally com-petent mRNAs. Primary transcripts with more than one introncan undergo alternative splicing to produce functionally differ-ent proteins from a single gene. In plants, about 20% of genesundergo alternative splicing. Although most alternative splicingevents are uncharacterized in plants, but it appears to play animportant role in the regulation of photosynthesis, flowering,grain quality in cereals, and plant defense response. Recent stud-ies have implicated intron splicing in abiotic stress response. Inwheat, cold stress induction of two early cold-regulated (e-cor)genes coding for a ribokinase (7H8) and a C3H2C3 RING fin-ger protein (6G2) undergo stress-dependent splicing. Both ofthese genes are regulated by intron retention under cold stress,whereas 6G2 intron retention is also regulated by drought stress.However, homologs of these genes did not show stress-regulatedintron retention in Arabidopsis. Interestingly, barley homologs of7H8 and 6G2 showed stress-dependent intron retention undercold stress, whereas barley albino mutants defective in chloro-plast development failed to retain introns in these genes undercold stress (63). The Arabidopsis COR15A gene encoding achloroplast stromal protein with cryoprotective activity plays animportant role in conferring freezing tolerance to chloroplasts(64). The Arabidopsis stabilized1 (sta1) mutant is defective in the

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splicing of the cold-induced COR15A pre-mRNA and is hyper-sensitive to chilling, ABA, and salt stresses. STA1 encodes anuclear pre-mRNA splicing factor and is upregulated by coldstress. STA1 catalyzes splicing of COR15A, which is necessaryfor cold tolerance (65). Further, pre-mRNA of serine/arginine-rich (SR) proteins, which are involved in the regulation or execu-tion of mRNA splicing, undergo alternate splicing under cold andheat stresses in Arabidopsis (66). Further, in addition to a changein splicing pattern, expression levels of AtSR45a and AtSR30,SF2/ASF-like SR proteins, are also increased by high light andsalinity stresses in Arabidopsis (67). Thus, the stress-regulatedalternate splicing machinery may in turn change the splicing pat-tern of some of the stress-responsive genes.

5.2. Small RNAs Small noncoding RNAs, namely micro-RNAs (miRNAs) andsmall interfering RNAs (siRNAs), act as ubiquitous repressors ofgene expression in animals and plants. Small RNAs are incor-porated into the argonaute (AGO) family of proteins contain-ing the RNA-induced silencing complex (RISC) or RNA-inducedtranscriptional silencing (RITS) complex. The RISC-containingmiRNA/siRNA induces posttranscriptional gene silencing bycleavage of mRNA and translational repression. Transcriptionalgene silencing is mainly mediated by siRNAs. Cold stress-upregulated and -downregulated miRNAs have been identified inArabidopsis. Abiotic stress-induced or -upregulated small RNAscan downregulate their target genes, which are likely negativeregulators and/or determinants of the stress response. In con-trast, stress-downregulated small RNAs can upregulate their tar-get mRNAs, which are likely positive regulators and/or determi-nants of stress tolerance (68).

Accumulation of ROS is induced by abiotic stresses. Super-oxide dismutases catalyze conversion of the superoxide radicalinto H2O2, which is then detoxified by ascorbate peroxidase.The miR398 expression is reduced and that of its target genesCSD1 and CSD2 enhanced under oxidative stress in Arabidopsis.Under normal conditions, miR398 targets the CSD mRNAs forcleavage, and thus stress-induced reduction in miR398 expres-sion results in accumulation of CSD transcripts. Because miR398and its target sequence on the CSD mRNAs are conservedacross plant species, miR398 appears to play a ubiquitous rolein ROS detoxification under abiotic stresses (69). siRNAs derivedfrom double-stranded RNAs (dsRNAs) formed from the mRNAsencoded by a natural cis–antisense gene pair are called naturalantisense transcript-derived siRNAs (nat-siRNAs). One of thenat-siRNAs derived from a cis–nat pair of SRO5 and P5CDH(�1-pyrroline-5-carboxylate dehydrogenase) regulates oxidativestress and osmolyte accumulation under salt stress in Arabidopsis.Salt stress-induced expression of SRO5 leads to SRO5-P5CDH

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dsRNA, which is then processed by DCL2, RDR6, SGS3, andDNA-dependent RNA polymerase IV (NRPD1A) to generate a24-nt nat-siRNA. The 24-nt nat-siRNA targets the cleavage ofP5CDH and thus accumulation of proline. Oxidative stress alsoinduces the expression of SRO5 and the 24-nt SRO5-P5CDHnat-siRNA and decreases P5CDH transcript levels (70, 71). Theseresults suggest that small RNAs play a key role in gene regulationin the cold and other abiotic stress response of Arabidopsis.

6. Conclusionsand Perspectives

Significant progress has been made to unravel the molecular basisof cold acclimation in model plant Arabidopsis and winter cereals.During cold acclimation, plants reprogram their gene expressionthrough transcriptional, posttranscriptional, and posttranslationalmechanisms. The ICE1–CBF transcriptional cascade plays crucialrole in cold acclimation in diverse plant species. Transgenic analy-sis revealed that genetic engineering of CBF pathway can improvecold tolerance across plant species. Recently, several componentsof CBF-independent transcriptional pathway of cold acclimationhave been identified. Besides transcriptional regulation, plantsemploy diverse posttranscriptional regulatory mechanisms to reg-ulate their gene expression during cold acclimation. Several coldstress-regulated miRNAs have been identified in Arabidopsis andrice. Characterization of cold-regulated miRNAs will help under-stand the role of posttranscriptional regulation of mRNA stabil-ity in cold stress response of plants. Cold-induced transcriptomediffers significantly among leaf, root, and reproductive (pollen)tissues. Most of the mechanisms of cold acclimation were studiedin vegetative stages of Arabidopsis. Further studies on transcrip-tional networks in reproductive tissues will identify key regulatorsof cold tolerance. Epigenetic processes play a key role in the regu-lation of plant development and stress responses. Further studieson the function of epigenetic processes, such as DNA methylationand chromatin modifications, and epigenetic stress memory willbe necessary.

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