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02 03 Chapter 3 04

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07 08 Gene Regulation During Cold Stress Acclimation in Plants 09

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11 Viswanathan Chinnusamy, Jian-Kang Zhu, and Ramanjulu Sunkar

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14 15 Abstract 16 17 Cold stress adversely affects plant growth and development and thus limits crop productivity. Diverse 18 plant species tolerate cold stress to a varying degree, which depends on reprogramming gene expres- 19 sion to modify their physiology, metabolism, and growth. Cold signal in plants is transmitted

20 to activate CBF-dependent (C-repeat/drought-responsive element binding factor-dependent) and

21 CBF-independent transcriptional pathway, of which CBF-dependent pathway activates CBF regulon. CBF transcription factor genes are induced by the constitutively expressed ICE1 (inducer of CBF 22 expression 1) by binding to the CBF promoter. ICE1–CBF cold response pathway is conserved in diverse 23 plant species. Transgenic analysis in different plant species revealed that cold tolerance can be significantly 24 enhanced by genetic engineering CBF pathway. Posttranscriptional regulation at pre-mRNA processing 25 and export from nucleus plays a role in cold acclimation. Small noncoding RNAs, namely micro-RNAs 26 (miRNAs) and small interfering RNAs (siRNAs), are emerging as key players of posttranscriptional gene 27 silencing. Cold stress-regulated miRNAs have been identified in Arabidopsis and rice. In this chapter,

28 recent advances on cold stress signaling and tolerance are highlighted.

29 Key words: Cold stress, second messengers, CBF regulon, CBF-independent regulation, ICE1, 30 posttranscriptional gene regulation. 31

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34 35 1. Introduction 36

37 38 Temperature profoundly influences the metabolism of organisms 39 and thus is a key factor determining the growing season and 40 geographical distribution of plants. Cold stress can be classi- 41 fied as chilling (<20◦C) and freezing (<0◦C) stress. Temperate 42 plants have evolved a repertoire of adaptive mechanisms such as 43 seed and bud dormancy, photoperiod sensitivity, vernalization, 44

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46 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 47

48 39 40 Chinnusamy, Zhu, and Sunkar

49 supercooling (prevention of ice formation in xylem parenchyma ◦ 50 cells up to homogenous ice nucleation temperature, −40 C), and

51 cold acclimation. In cold acclimation, plants acquire freezing tol- 52 erance on prior exposure to suboptimal, low, nonfreezing tem- 53 peratures. The molecular basis of cold acclimation and acquired

54 freezing tolerance in Arabidopsis and winter cereals has been stud- 55 ied extensively. Plants modify their metabolism and growth to 56 adapt to cold stress by reprogramming during 57 cold acclimation (1, 2). This chapter briefly covers cold stress sig-

58 naling, transcriptional and posttranscriptional regulation of gene 59 expression in cold acclimation process, and the genetic engineer- 60 ing of crops with enhanced cold tolerance.

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64 65 2. Cold Stress 66 Sensing 67

68 Thus far, the identity of stress sensor in plants is unknown. The 69 fluid mosaic physical state of the plasma membrane is vital for 70 the structure and function of cells, as well as to sense tempera- 71 ture stress. The plasma membrane undergoes phase transitions, 72 from a liquid crystalline to a rigid gel phase at low temperature 73 and to a fluid state at high temperature. Thus, a decrease in tem- 74 perature can rapidly induce membrane rigidity at microdomains. 75 Further, protein folding is influenced by temperature changes. 76 Temperature-induced changes in the physical state of mem- 77 branes and proteins are expected to change the metabolic reac- 78 tions and thus the metabolite concentrations. Therefore, plant 79 cells can sense cold stress through membrane rigidification, pro- 80 tein/nucleic acid conformation, and/or metabolite concentration 81 (a specific metabolite or redox status). 82 In alfalfa and Brassica napus, cold stress-induced plasma 83 membrane rigidification leads to actin cytoskeletal rearrangement, 2+ 2+ 84 induction of Ca channels, and increased cytosolic Ca level. 85 These events induce the expression of cold-responsive (COR) 86 genes and cold acclimation. Further, a membrane rigidifier ◦ 87 (DMSO) can induce COR genes even at 25 C, whereas a mem- 88 brane fluidizer (benzyl alcohol) prevents COR gene expression ◦ 89 even at 0 C (3, 4). Genetic evidence for plants sensing cold stress 90 through membrane rigidification is from the study of the fad2 91 mutant impaired in the oleic acid desaturase gene of Arabidop- 92 sis. In wild-type Arabidopsis plants, diacylglycerol (DAG) ◦ 93 is induced at 14 C. The fad2 mutant (more saturated mem- 94 brane) and transgenic Arabidopsis overexpressing linoleate desat- ◦ 95 urase gene showed the expression of DAG kinase at 18 and 12 C, 96 respectively (5). Cold Tolerance in Plants 41

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98 3. Second

99 Messengers 100 and Signaling 2+ 101 Cytosolic Ca levels act as second messenger of the cold stress

102 signal (6). Calcium may be imported into the cell or released from

103 intracellular calcium stores. Patch-clamp studies of cold-induced

104 potential changes of the plasma membrane in Arabidopsis mes-

105 ophyll protoplasts showed the cold-activatedcalcium-permeable 2+ 106 channel involved in the regulation of cytosolic Ca signatures 2+ 107 (7). Membrane rigidification induced cytosolic Ca signatures;

108 and COR gene expression was impaired by gadolinium, a 2+ 109 mechanosensitive Ca channel blocker, which suggests the 2+ 110 involvement of mechanosensitive Ca channels in cold acclima-

111 tion (4). Pharmocological studies implicated cyclic ADP-ribose-

112 and inositol-1,4,5-triphosphate (IP3)-activated intracellular

113 calcium channels in COR gene expression (4). Calcium influx

114 into the cell appears to activate phospholipase C (PLC) and D

115 (PLD), which produce IP3 and phosphatidic acid, respectively. 2+ 116 IP3 can further amplify Ca signatures by activation of IP3-gated

117 calcium channels (8). Genetic analysis revealed that loss-of-

118 function mutants of FIERY1 (FRY1) inositol polyphosphate

119 1-phosphatase show significantly higher and sustained levels

120 of IP3 instead of the transient increase observed in wild-type

121 plants. This situation leads to higher induction of COR genes

122 and CBFs, the upstream transcription factors (9). In addition,

123 the calcium exchanger 1 (cax1)mutantofArabidopsis, which is 2+ + 124 defective in a vacuolar Ca /H antiporter, exhibited enhanced

125 expression of C-repeat binding factor/dehydration responsive

126 element binding (CBF/DREB) proteins and their target COR 2+ 127 genes (10). Therefore, cytosolic Ca signatures are upstream of

128 the expression of CBFs and COR genes in cold stress signaling.

129 Cold acclimation induces accumulation of ROS such as

130 H2O2, both in chilling-tolerant Arabidopsis and chilling-sensitive

131 maize plants. ROS can act as a signaling molecule to reprogram 2+ 132 transcriptome probably through induction of Ca signatures and

133 activation of mitogen-activated protein (MAPKs) (11)

134 and redox-responsive transcription factors. Arabidopsis frostbite1

135 (fro1) mutant, which is defective in the mitochondrial Fe-S sub-

136 unit of complex I (NADH dehydrogenase) of the electron trans-

137 fer chain, shows a constitutively high accumulation of ROS.

138 This high accumulation of ROS in fro1 results in reduced COR

139 gene expression and hypersensitivity to freezing stress, probably

140 because of desensitization of cells by the constitutively high ROS

141 expression (12). Cold stress-induced second messenger signatures can be 142 143 decoded by different pathways. Calcium signatures are sensed

144 by calcium sensor family proteins, namely calcium-dependent 42 Chinnusamy, Zhu, and Sunkar

145 protein kinases (CDPKs), calmodulins (CaMs), and salt overly 146 sensitive 3-like (SOS3-like) or calcineurin B-like (CBL) pro-

147 teins. In a transient expression system in maize leaf protoplast, a 148 constitutively active form of an Arabidopsis CDPK (AtCDPK1) 149 activated the expression of barley HVA1 ABA-responsive pro-

150 moter::LUC reporter gene suggesting that AtCDPK is a posi- 151 tive regulator in stress-induced gene transcription (13). Genetic 152 and transgenic analyses implicated CDPKs as positive regulators, 153 but a calmodulin, a SOS3-like or a CBL calcium binding protein,

154 and a protein phosphatase 2C (AtPP2CA) are negative regulators 155 of gene expression and cold tolerance in plants. Components of 156 MAPK cascades are induced or activated by cold and other abi-

157 otic stresses. Genetic and transgenic analyses showed that MAPKs 158 act as a converging point in abiotic stress signaling. ROS accu- 159 mulation under these stresses might be sensed through a MAPK

160 cascade (14). ROS activates the AtMEKK1/ANP1 (MAPKKK)– 161 AtMKK2 (MAPKK)–AtMPK4/6 (MAPK) MAPK cascade, which 162 positively regulates cold acclimation in plants (11). Many of these 163 phosphorylated proteins show activation or induction of gene

164 expression under multiple stress conditions, and genetic modi- 165 fication results in alteration of multiple stress responses. These 166 results suggest that the proteins act as connecting nodes of stress 167 signal networks. Identification of the target proteins or transcrip- 168 tion factors of or phosphatase cascades will shed 169 further light on stress signaling.

170 171 4. Transcriptional 172 Regulation 173 174 Chilling-tolerant plants reprogram their transcriptome in 175 response to acclimation temperature. Cold-regulated genes con- 176 stitute about 4–20% of the genome in Arabidopsis (15). The 177 promoter region of many COR genes of Arabidopsis con- 178 tains C-repeat (CRT)/DREs, initially identified in the promoter 179 of responsive to dehydration 29A (RD29A/COR78/LTI78). 180 As well, ABA-responsive elements are present in many cold- 181 induced genes. Genetic screens using dehydration and cold stress- 182 responsive promoter-driven LUCIFERASE (RD29A::LUC and 183 CBF3::LUC) led to the isolation of mutants, which unraveled 184 cold-responsive transcriptional networks. 185

186 4.1. CBF Regulons Yeast one-hybrid screens to identify CRT/DRE binding pro- 187 and Cold Tolerance teins led to the identification of CRT/DREBs (CBFs/DREBs) 188 in Arabidopsis. CBFs belong to the ethylene-responsive element 189 binding factor/APETALA2 (ERF/AP2)-type transcription factor 190 family. Arabidopsis encodes three CBF genes (CBF1/DREB1B, 191 CBF2/DREB1C,andCBF3/DREB1A), which are induced 192 within a short period of exposure to cold stress. CBFs bind to Cold Tolerance in Plants 43

193 CRT/DRE cis-elements in the promoters of COR genes and 194 induce their expression (16, 17). Ectopic expression of CBFs

195 in transgenic Arabidopsis induced the expression of COR genes 196 at warm temperatures and induced constitutive freezing toler- 197 ance. These transgenic Arabidopsis plants were also tolerant to

198 salt and drought stresses (17–19). Microarray analysis of CBF- 199 overexpressing transgenic plants identified several CBF target 200 genes involved in signaling, transcription, osmolyte biosynthesis, 201 ROS detoxification, membrane transport, hormone metabolism,

202 and stress response (20, 21). Transgenic overexpression of Ara- 203 bidopsis CBFs is sufficient to induce cold tolerance in diverse plant 204 species (Table 3.1). Further, CBF homologs have been identified

205 206 Table 3. 1 207 Abiotic stress tolerance of transgenic plants overexpressing CBFs 208 209 Transgenic 210 Gene plant Stress tolerance of transgenic plants References 211 AtCBF1/2/3 Brassica Constitutive overexpression enhanced both basal (22) 212 napus and acquired freezing tolerance 213 AtCBF1 Tomato Constitutive overexpression enhanced oxidative (23, 24) 214 stress tolerance under chilling stress; enhanced 215 tolerance to water-deficit stress 216 AtDREB1A/ Tobacco Transgenic plants expressing RD29A::DREB1A (25) 217 CBF3 exhibited enhanced chilling and drought 218 tolerance 219 AtDREB1A/ Wheat Transgenic plants expressing RD29A pro- (26) 220 CBF3 moter::AtDREB1A gene showed delayed 221 water stress symptoms

222 AtCBF3 Rice Constitutive overexpression resulted in enhanced (27) 223 tolerance to drought and high salinity and a 224 marginal increase in chilling tolerance 225 AtDREB1A/ Maize RD29A::CBF3 transgenic plants are more toler- (28) 226 CBF3 ant to cold, drought, and salinity 227 AtCBF1 Potato Constitutive or stress-inducible expression of (29) 228 CBF1 or CBF3 but not CBF2 conferred 229 improved freezing tolerance to frost-sensitive 230 Solanum tuberosum 231 OsDREB1 Arabidopsis Overexpression in Arabidopsis induced target (30) 232 COR genes and conferred enhanced tolerance

233 to freezing and drought stresses 234 OsDREB1A/B Rice Constitutive expression conferred improved tol- (31) 235 erance to cold, drought, and salinity 236 ZmDREB1 Arabidopsis Overexpression in Arabidopsis induced COR (32) 237 genes and conferred tolerance to freezing and drought 238 239 BnCBF5 and B. napus Overexpression led to increased constitutive (33) 240 BnCBF 17 freezing tolerance, increased photochemical efficiency and photosynthetic capacity 44 Chinnusamy, Zhu, and Sunkar

241 from several chilling-tolerant and chilling-sensitive plant species 242 and transgenic analysis confirmed their pivotal role in cold accli-

243 mation (Table3.1). 244 These evidences suggest that a CBF transcription network 245 plays a pivotal role in cold acclimation of evolutionarily diverse

246 plant species. Transcriptome analysis of transgenic tomato and 247 Arabidopsis plants overexpressing LeCBF1 and AtCBF3 revealed 248 that CBF regulons from freezing-tolerant and freezing-sensitive 249 plant species differ significantly (35).

250 Constitutive overexpression of CBFs under the transcriptional 251 control of the 35S cauliflower mosaic virus promoter in transgenic 252 plants resulted in severe growth retardation under normal growth

253 conditions in diverse plant species such as Arabidopsis (18, 19, 254 34, 36), B. napus (22), tomato (23, 24), potato (29), and rice 255 (31). Inhibition of metabolism and change in growth-regulating

256 hormones appears to be important causes of the growth inhibi- 257 tion of CBF-overexpressing plants. Reduction in the expression 258 of photosynthetic genes appears to reduce photosynthesis and 259 growth under cold stress. Transgenic plants constitutively overex-

260 pressing CBFs showed higher induction of the STZ/ZAT10 zinc 261 finger transcription factor gene, which appears to repress genes 262 involved in photosynthesis and carbohydrate metabolism and thus 263 reduce the growth of these transgenic plants (21). Microarray 264 analysis revealed that cold stress regulates several genes involved 265 in biosynthesis or signaling of hormones such as ABA, gib- 266 berellic acid (GA), and auxin, which suggests the importance 267 of these hormones in coordinated regulation of cold tolerance 268 and plant development (15). GA promotes important processes 269 in plant growth and development, such as seed germination, 270 growth through elongation, and floral transition. Growth retar- 271 dation of transgenictomato plants constitutively overexpressing 272 AtCBF1 was reversed by GA3 treatment (24). This finding sug- 273 gested a link between CBFs and GA in cold stress-induced growth 274 retardation. During cold stress, growth retardation appears to be 275 regulated by CBFs through nuclear-localized DELLA proteins, 276 which repress growth in Arabidopsis. GA stimulates the degrada- 277 tion of DELLA proteins and promotes growth. CBFs enhance 278 the expression of GA-inactivating GA2-oxidases, and thus allow 279 the accumulation of the DELLA protein repressor of GA1-like 280 3 (RGL3), which leads to dwarfism and late flowering. Further, 281 mutant plants of DELLA genes encoding GA-insensitive [GAI] 282 repressor of GA1-3 [RGA] were significantly less freezing toler- 283 ant than were wild-type plants after cold acclimation. This find- 284 ing suggests that DELLAs might contribute significantly to cold 285 acclimation and freezing tolerance (37). 286 287 4.2. Regulators of Transcription of CBF genes is induced by cold stress. Hence, 288 CBF Expression constitutive transcription factors present in the cell at normal Cold Tolerance in Plants 45

289 growth temperatures may induce the expression of CBFs on acti- 290 vation by cold stress. A systematic genetic analysis by CBF3::LUC

291 bioluminescent genetic screening led to the identification of a 292 constitutively expressed and nuclear-localized transcription fac- 293 tor, inducer of CBF expression 1 (ICE1) in Arabidopsis. ICE1

294 encodes a MYC-type basic helix-loop-helix (bHLH) transcription 295 factor, can bind to MYC recognition elements in the CBF3 pro- 296 moter, and induces the expression of CBF3 during cold acclima- 297 tion. The ice1 mutant is defective in both chilling and freezing

298 tolerance, whereas transgenic Arabidopsis overexpressing ICE1 299 showed enhanced freezing tolerance (38). Transcriptome anal- 300 ysis revealed the dominant ice1 mutant with impaired expres-

301 sion of about 40% of cold-regulated genes, in particular 46% of 302 cold-regulated transcription factor genes (15). Therefore, ICE1 303 is a master regulator that controls CBF and many other cold-

304 responsive regulons. Overexpression analysis showed that ICE2 305 (At1g12860, a homolog of ICE1) induces the expression of 306 CBF1 and confers enhanced freezing tolerance in Arabidop- 307 sis after cold acclimation (39). In wheat, the ICE1 homologs

308 TaICE141 and TaICE187 are constitutively expressed and acti- 309 vate the wheat CBF group IV, which are associated with freezing 310 tolerance. Overexpression of TaICE141 and TaICE187 in Ara- 311 bidopsis enhanced CBF and CORgene expression and enhanced 312 freezing tolerance only after cold acclimation. This finding sug- 313 gests that similar to Arabidopsis ICE1, wheat ICE1 also needs to 314 be activated by cold acclimation (40). 315 ICE1 appears to negatively regulate the expression of 316 MYB15 (an R2R3-MYB family protein) in Arabidopsis.MYB15 317 is an upstream transcription factor that negatively regulates 318 CBF expression. Transgenic Arabidopsis overexpressing MYB15 319 showed reduced expression of CBFs and freezingtolerance, 320 whereas myb15 T-DNA knockout mutants showed enhanced 321 cold induction of CBFs and enhanced freezing tolerance. In a 322 yeast two-hybrid system, ICE1 interacted with MYB15 (41). 323 Further, the expression of MYB15 is increased in ice1 mutants 324 (R236H and K393R) (41, 42). Thus, the ICE1–MYB15 inter- 325 action appears to play a role in regulating CBF expression levels 326 during cold acclimation (41). 327 Although ICE1 is expressed constitutively, only on expo- 328 sure to low temperature does it induce transcription of the CBF 329 and other cold stress-responsive genes (38, 40). Posttranslational 330 modifications play a key role in regulating the activity of ICE1 331 under cold stress. Cold stress activates ICE1 sumoylation (42) 332 and negatively regulates ICE1 levels by targeted proteolysis (43). 333 The Arabidopsis High expression of Osmotically responsive gene 1 334 (HOS1) encodes a RING finger ubiquitin E3 ligase. The nuclear 335 localization of HOS1 is enhanced by cold stress. HOS1 physically 336 interacts with ICE1 and targets ICE1 for polyubiquitination and 46 Chinnusamy, Zhu, and Sunkar

337 proteolysis of ICE1 after 12 h of cold stress. Overexpression of 338 HOS1 in transgenic Arabidopsis results in a substantial reduction

339 in level of ICE1 protein and that of its target genes, as well as 340 hypersensitivity to freezing stress. Thus, HOS1 mediates ubiqui- 341 tination of ICE1 and plays a critical role in maintaining the level

342 of ICE1 target genes in the cell during cold acclimation (43). 343 Sumoylation of proteins prevents the proteasomal degradation of 344 target proteins. The null mutant of Arabidopsis SUMO E3 ligase, 345 SAZ1 (SAP and Miz1), exhibits reduced cold induction of CBFs 346 and the target COR genes, as well as hypersensitivity to chilling 347 and freezing stresses. SIZ1 catalyzes SUMO conjugation to K393 348 of ICE1 during cold acclimation and thus reduces polyubiquiti- 349 nation of ICE1. Mutation in a K393 residue of ICE1 impairs its 350 activity (42). Hence, SIZ1-mediated sumoylation facilitates ICE1 351 stability and activity, whereas HOS1 mediation reduces ICE1 pro- 352 tein levels during cold acclimation. 353 Stomata play a crucial role in regulating photosynthesis and 354 transpiration. Recently, the scream-D dominant mutant and ice1 355 mutant were found to be the same as R236H, which results 356 in constitutive stomatal differentiation in the epidermis, and 357 the entire epidermis differentiates into stomata. Thus, ICE1 is 358 required for controlled stomatal development. ICE1protein inter- 359 acts and forms a dimer with other bHLH transcription fac- 360 tors, SPEECHLESS (SPCH), MUTE, and FAMA, which regu- 361 late stomatal development. ICE1 may act as a link between the 362 formation of stomata and the plant response to environmental 363 cues (44). 364 Recently, members of the calmodulin binding transcription 365 activator (CAMTA) family proteins have been identified as tran- 366 scriptional regulators of CBF2 expression. Cold-induced expres- 367 sion of CBF2 was considerably lower in camta3 mutant as com- 368 pared to WT plants. The CAMTA3 protein binds to conserved 369 DNA motifs present in CBF2 promoter and regulates CBF2 370 expression. The camta1/camta3 double mutant exhibited hyper- 371 sensitivity to freezing stress as compared to WT plants. Since 372 CAMTA proteins can interact with calmodulins, cold-induced 373 calcium signals may regulate CBFs expression through CAMTA 374 proteins (45).

375

376 4.3. CBF1, CBF2, and Microarray analysis revealed that CBFs regulate about 12% of the 377 CBF3 Play Different cold-responsive transcriptome. Overexpression of CBFs enhances 378 RolesinCold osmolyte accumulation, reduces growth, and enhances abiotic 379 Acclimation stress tolerance (Table 3.1). Constitutive overexpression stud- 380 ies of transgenic Arabidopsis suggested that CBF1, CBF2, and 381 CBF3 have redundant functional activities (36). However, the 382 ice1 mutant, impaired mainly in CBF3 but not CBF1 and CBF2, 383 showed chilling and freezing hypersensitivity (38). Studies of the 384 cbf2 T-DNA insertion mutant of Arabidopsis revealed that CBFs Cold Tolerance in Plants 47

385 have different functions in cold acclimation. cbf2 null mutants 386 showed increased expression of CBF1 and CBF3 and enhanced

387 tolerance to freezing (with or without cold acclimation), dehy- 388 dration, and salt stresses. Further, CBFs show a temporal differ- 389 ence in expression, with the cold-induced expression of CBF1 and

390 CBF3 preceding that of CBF2. These results suggest that CBF2 391 negatively regulates CBF1 and CBF3 to optimize the expression 392 of downstream target genes (45). In potato (Solanum tubero- 393 sum), overexpression of AtCBF2 failed to confer freezing toler-

394 ance (29). Transgenic analysis of CBF1 and CBF3 RNAi lines 395 revealed that both CBF1 and CBF3 are required for the full set 396 of CBF regulon expression and freezing tolerance (46).

397 Besides CBF2, the C2H2 zinc finger transcription factor 398 ZAT12 negatively regulates the expression of CBF1, CBF2, and 399 CBF3 during cold stress. Arabidopsis transgenic plants overex-

400 pressing ZAT12 showed decreased expression of CBFs under cold 401 stress (47). los2 mutant plants showed an enhanced and more sus- 402 tained induction of ZAT10/STZ during cold stress and enhanced 403 cold sensitivity. LOS2 encodes a bifunctional enolase that nega-

404 tively regulates the expression of ZAT10 (48). 405 Transgenic Arabidopsis plants overexpressing AtMKK2 406 showed constitutive expression of CBF2, which suggests that the 407 CBF2 expression is probably positively regulated by a MAPK sig- 408 naling cascade (11). Arabidopsis FIERY2 (FRY2), which encodes 409 an RNApolymerase II C-terminal domain (CTD) phosphatase, 410 appears to act as a negative regulator of CBFs and their target 411 COR genes because the fry2 mutant showed enhanced expres- 412 sion of CBFs and COR genesunder cold stress and ABA. Since 413 the fry2 mutant is hypersensitive to freezing despite enhanced 414 expression of CBFs, FRY2 may positively regulate the expression 415 of certain genes critical for freezing tolerance (49). The main- 416 tenance of an optimal level of CBFs at an appropriate time is 417 necessary, because constitutive overexpression affects growth and 418 development significantly. Further, CBF expression is under the 419 control of a circadian clock. The maximal cold-induced increase 420 in transcription of CBFs occurs when cold stress is imposed 4 h 421 after dawn. Transgenic plants overexpressing arrhythmic CCA1 422 showed no temporal difference in the cold induction of CBF 423 expression (50).

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425 4.4. CBF-Independent Genetic and transgenic analyses revealed that several classes of 426 Regulons transcription factors besides CBFs play an important role in cold 427 acclimation. The eskimo1 (esk1)mutantofArabidopsis was iden- 428 tified through freezing tolerance genetic screening. The esk1 429 mutant accumulated constitutively high levels of proline and 430 exhibited constitutively freezing tolerance. ESK1 is constitutively 431 expressed and encodes the protein domain of unknown function 432 (23). Transcriptome comparison of CBF2-overexpressing plants 48 Chinnusamy, Zhu, and Sunkar

433 and esk1 mutants showed that different sets of genes are regulated 434 by CBF2 and ESK1. However, the mechanism of action of ESK1

435 in freezing tolerance has yet to be revealed (51). 436 PRD29A::LUC reporter gene-based genetic screening led to 437 the identification of two constitutively expressed transcription fac-

438 tors, HOS9 (a homeodomain protein) and HOS10 (an R2R3- 439 type MYB), which are necessary for cold tolerance in Arabidop- 440 sis. hos9 and hos10 mutants are less freezing tolerant than wild- 441 type Arabidopsis (52, 53). Transcriptome analysis revealed distinct

442 CBF and HOS9 regulons (52). HOS10 probably regulates ABA- 443 dependent cold acclimation pathways, because HOS10 positively 444 regulates NCED3 (9-cis-epoxycarotenoid dioxygenase) and thus

445 ABA accumulation during cold stress (53). 446 Gene expression analysis revealed several transcription fac- 447 tors induced during cold acclimation. Transgenic analysis of cold-

448 inducible transcription factors helped in validation of functions of 449 some transcription factors in cold tolerance. Constitutive overex- 450 pression of the soybean C2H2-type zinc finger protein SCOF1 451 in Arabidopsis transgenic plants enhanced the expression of COR 452 genes and conferred constitutive freezing tolerance. SCOF1 inter- 453 acts with soybean G-box binding factor 1 (SGBF1) and may 454 enhance the DNA binding activity of the SGBF1. SGBF1 is 455 induced by both cold and ABA (54). Overexpression of the cold- 456 regulated rice transcription factors MYB4 (an R2R3-type MYB) 457 and OsMYB3R-2 (an R1R2R3 MYB) enhanced freezing tolerance 458 of Arabidopsis (55, 56). 459 Some members of the abiotic, plant hormone, and pathogen- 460 inducible ERF family play a crucial role in abiotic and biotic 461 stress tolerance. The pepper ERF/AP2-type transcription factor 462 Capsicum annuum pathogen and freezing tolerance-related pro- 463 tein 1 (CaPF1) is induced by cold, osmotic stress, ethylene, 464 and jasmonic acid. Transgenic Arabidopsis overexpressing CaPF1 465 showed induction of pathogen-responsive as well as COR genes 466 and exhibited enhanced tolerance to stress by freezing and to 467 pathogens (Pseudomonas syringae pv tomato DC3000) (57). Sim- 468 ilarly, Triticum aestivum ERF1 (TaERF1] was induced by cold, 469 drought salinity, ABA, ethylene, salicylic acid, and infection by 470 Blumeria graminis f. sp.Triticipathogen in wheat. Transgenic 471 Arabidopsis overexpressing TaERF1 exhibited enhanced tolerance 472 to cold, salt, and drought stresses, as well as pathogens (58). 473 Genes encoding the A-5 subgroup AP2 domain protein from 474 Physcomitrella patens (PpDBF1) (59) and soybean (GmDREB3) 475 (60) are cold induced, and overexpression of these genes con- 476 ferred enhanced cold tolerance. 477 In wheat, wheat low-temperature-induced protein 19 478 (WLIP19), encoding a basic-region leucine zipper protein, 479 is induced by cold, drought, and ABA. WLIP19 activates 480 the expression of COR genes in wheat. Transgenic tobacco Cold Tolerance in Plants 49

481 overexpressing Wlip19 showed significant freezing tolerance. 482 WLIP19 was found to interact and form a heterodimer with

483 T.aestivum ocs-element bindingfactor 1 (TaOBF1), a bZIP tran- 484 scription factor (61). The plant-specific transcription factor NAC 485 (NAM, ATAF, and CUC) family plays a key role in stress

486 response. Overexpression of cold stress-inducible rice SNAC2 in 487 transgenic rice resulted in high cell membrane stability under cold 488 stress. Microarray analysis showed upregulation of several stress- 489 regulated genes in SNAC2-overexpressing plants (62). These

490 results suggest that several transcriptional networks operate dur- 491 ing cold acclimation and cold stress tolerance of plants.

492

493

494 495 5. Posttranscrip- 496 tional Gene 497 Regulation 498 Posttranscriptional regulation at pre-mRNA processing, mRNA 499 stability, and export from nucleus plays critical roles in cold accli- 500 mation and cold tolerance (2).

501

502 5.1. Messenger RNA Pre-mRNA processing and exports constitute important mecha- 503 Processing nisms of regulation of gene expression in eukaryotes. Pre-mRNA  504 undergoes various nuclear processes such as the addition of a 5 505 methyl cap and poly(A) tail and intron splicing. Splicing is nec- 506 essary to remove introns and to synthesize translationally com- 507 petent mRNAs. Primary transcripts with more than one intron 508 can undergo alternative splicing to produce functionally differ- 509 ent proteins from a single gene. In plants, about 20% of genes 510 undergo alternative splicing. Although most alternative splicing 511 events are uncharacterized in plants, but it appears to play an 512 important role in the regulation of photosynthesis, flowering, 513 grain quality in cereals, and plant defense response. Recent stud- 514 ies have implicated intron splicing in abiotic stress response. In 515 wheat, cold stress induction of two early cold-regulated (e-cor) 516 genes coding for a (7H8) and a C3H2C3 RING fin- 517 ger protein (6G2) undergo stress-dependent splicing. Both of 518 these genes are regulated by intron retention under cold stress, 519 whereas 6G2 intron retention is also regulated by drought stress. 520 However, homologs of these genes did not show stress-regulated 521 intron retention in Arabidopsis. Interestingly, barley homologs of 522 7H8 and 6G2 showed stress-dependent intron retention under 523 cold stress, whereas barley albino mutants defective in chloro- 524 plast development failed to retain introns in these genes under 525 cold stress (63). The Arabidopsis COR15A gene encoding a 526 chloroplast stromal protein with cryoprotective activity plays an 527 important role in conferring freezing tolerance to chloroplasts 528 (64). The Arabidopsis stabilized1 (sta1) mutant is defective in the 50 Chinnusamy, Zhu, and Sunkar

529 splicing of the cold-induced COR15A pre-mRNA and is hyper- 530 sensitive to chilling, ABA, and salt stresses. STA1 encodes a

531 nuclear pre-mRNA splicing factor and is upregulated by cold 532 stress. STA1 catalyzes splicing of COR15A, which is necessary 533 for cold tolerance (65). Further, pre-mRNA of serine/arginine-

534 rich (SR) proteins, which are involved in the regulation or execu- 535 tion of mRNA splicing, undergo alternate splicing under cold and 536 heat stresses in Arabidopsis (66). Further, in addition to a change 537 in splicing pattern, expression levels of AtSR45a and AtSR30,

538 SF2/ASF-like SR proteins, are also increased by high light and 539 salinity stresses in Arabidopsis (67). Thus, the stress-regulated 540 alternate splicing machinery may in turn change the splicing pat-

541 tern of some of the stress-responsive genes.

542

543 5.2. Small RNAs Small noncoding RNAs, namely micro-RNAs (miRNAs) and 544 small interfering RNAs (siRNAs), act as ubiquitous repressors of 545 gene expression in animals and plants. Small RNAs are incor- 546 porated into the (AGO) family of proteins contain- 547 ing the RNA-induced silencing complex (RISC) or RNA-induced 548 transcriptional silencing (RITS) complex. The RISC-containing 549 miRNA/siRNA induces posttranscriptional gene silencing by 550 cleavage of mRNA and translational repression. Transcriptional 551 gene silencing is mainly mediated by siRNAs. Cold stress- 552 upregulated and -downregulated miRNAs have been identified in 553 Arabidopsis. Abiotic stress-induced or -upregulated small RNAs 554 can downregulate their target genes, which are likely negative 555 regulators and/or determinants of the stress response. In con- 556 trast, stress-downregulated small RNAs can upregulate their tar- 557 get mRNAs, which are likely positive regulators and/or determi- 558 nants of stress tolerance (68). 559 Accumulation of ROS is induced by abiotic stresses. Super- 560 oxide dismutases catalyze conversion of the superoxide radical 561 into H2O2, which is then detoxified by ascorbate peroxidase. 562 The miR398 expression is reduced and that of its target genes 563 CSD1 and CSD2 enhanced under oxidative stress in Arabidopsis. 564 Under normal conditions, miR398 targets the CSD mRNAs for 565 cleavage, and thus stress-induced reduction in miR398 expres- 566 sion results in accumulation of CSD transcripts. Because miR398 567 and its target sequence on the CSD mRNAs are conserved 568 across plant species, miR398 appears to play a ubiquitous role 569 in ROS detoxification under abiotic stresses (69). siRNAs derived 570 from double-stranded RNAs (dsRNAs) formed from the mRNAs 571 encoded by a natural cis–antisense gene pair are called natural 572 antisense transcript-derived siRNAs (nat-siRNAs). One of the 573 nat-siRNAs derived from a cis–nat pair of SRO5 and P5CDH 574 ( 1-pyrroline-5-carboxylate dehydrogenase) regulates oxidative 575 stress and osmolyte accumulation under salt stress in Arabidopsis. 576 Salt stress-induced expression of SRO5 leads to SRO5-P5CDH Cold Tolerance in Plants 51

577 dsRNA, which is then processed by DCL2, RDR6, SGS3, and 578 DNA-dependent RNA IV (NRPD1A) to generate a

579 24-nt nat-siRNA. The 24-nt nat-siRNA targets the cleavage of 580 P5CDH and thus accumulation of proline. Oxidative stress also 581 induces the expression of SRO5 and the 24-nt SRO5-P5CDH

582 nat-siRNA and decreases P5CDH transcript levels (70, 71). These 583 results suggest that small RNAs play a key role in gene regulation 584 in the cold and other abiotic stress response of Arabidopsis.

585

586

587

588 6. Conclusions

589 and Perspectives

590

591 Significant progress has been made to unravel the molecular basis

592 of cold acclimation in model plant Arabidopsis and winter cereals.

593 During cold acclimation, plants reprogram their gene expression

594 through transcriptional, posttranscriptional, and posttranslational

595 mechanisms. The ICE1–CBF transcriptional cascade plays crucial

596 role in cold acclimation in diverse plant species. Transgenic analy-

597 sis revealed that genetic engineering of CBF pathway can improve

598 cold tolerance across plant species. Recently, several components

599 of CBF-independent transcriptional pathway of cold acclimation

600 have been identified. Besides transcriptional regulation, plants

601 employ diverse posttranscriptional regulatory mechanisms to reg-

602 ulate their gene expression during cold acclimation. Several cold

603 stress-regulated miRNAs have been identified in Arabidopsis and

604 rice. Characterization of cold-regulated miRNAs will help under-

605 stand the role of posttranscriptional regulation of mRNA stabil-

606 ity in cold stress response of plants. Cold-induced transcriptome

607 differs significantly among leaf, root, and reproductive (pollen)

608 tissues. Most of the mechanisms of cold acclimation were studied

609 in vegetative stages of Arabidopsis. Further studies on transcrip-

610 tional networks in reproductive tissues will identify key regulators

611 of cold tolerance. Epigenetic processes play a key role in the regu-

612 lation of plant development and stress responses. Further studies

613 on the function of epigenetic processes, such as DNA

614 and modifications, and epigenetic stress memory will

615 be necessary.

616 617 References 618

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